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A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

  1. Jesusa Capera
  2. Mireia Pérez-Verdaguer
  3. Roberta Peruzzo
  4. María Navarro-Pérez
  5. Juan Martínez-Pinna
  6. Armando Alberola-Die
  7. Andrés Morales
  8. Luigi Leanza
  9. Ildiko Szabó  Is a corresponding author
  10. Antonio Felipe  Is a corresponding author
  1. Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, Spain
  2. Department of Biology, University of Padova, Italy
  3. Dept de Fisiología, Genética y Microbiología, Universidad de Alicante, Spain
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Cite this article as: eLife 2021;10:e69099 doi: 10.7554/eLife.69099

Abstract

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CBD), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CBDless), we demonstrate here that while the Kv1.3–Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CBDless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin–Kv1.3 axis during cell survival and apoptosis.

Introduction

The voltage-gated potassium channel Kv1.3 is present at the plasma membrane of different cell types, mostly neurons and leukocytes (Cahalan and Chandy, 2009; Martínez-Mármol et al., 2016; Solé et al., 2016). Kv1.3 participates in cell proliferation, activation, and apoptosis (Pérez-Verdaguer et al., 2016b). Thus, altered expression of the channel is linked to different pathologies such as autoimmune diseases and cancer (Rus et al., 2005; Vallejo-Gracia et al., 2013; Serrano-Novillo et al., 2019; Szabo et al., 2021). Kv1.3 is efficiently expressed on the cell surface, which depends on multiple forward trafficking signatures located at the C-terminal domain of the channel (Martínez-Mármol et al., 2013). Moreover, different ancillary interactions modulate the trafficking of Kv1.3 (Capera et al., 2019). For example, the Kv1.5 channel and the regulatory KCNE4 subunit retain Kv1.3 at the endoplasmic reticulum (ER), negatively modulating the channel surface expression (Vicente et al., 2006; Vicente et al., 2008; Solé et al., 2009). In addition, the scaffolding protein caveolin 1 (Cav1) controls Kv1.3 spatial localization in raft microdomains, which is important for signaling and cell physiology (Pérez-Verdaguer et al., 2016a; Pérez-Verdaguer et al., 2018). Therefore, by controlling Kv1.3 surface expression and localization, oligomeric associations fine-tune physiological events.

Caveolin (Cav) is the main structural component of caveolae, a specialized form of membrane lipid raft with a characteristic omega-shaped structure. Caveolae are abundant at the plasma membrane of highly differentiated cells, such as adipocytes, pneumocytes, and muscle cells, but they are not abundant in central neurons and lymphocytes. Cav1, the main isoform in non-muscle tissues, forms large oligomers with high affinity for cholesterol and sphingolipids. In addition, Cav1 can recruit different proteins into caveolar and non-caveolar rafts through its Cav scaffolding domain (CSD). Therefore, rafts, acting as signaling platforms, initiate signaling pathways, participate in vesicular transport, and contribute to cholesterol homeostasis (Simons and Ikonen, 1997; Razani et al., 2002; Parton et al., 2006). In addition, these microdomains are essential for immunological synapses during the immune response (Rao et al., 2004), as well as for the insulin modulation of adipocyte physiology (Pérez-Verdaguer et al., 2018).

In this context, the role of Cav1 in cancer progression raises an intense debate. Cav1 acts either as a tumor suppressor or as an oncogene, depending on the cancer type and the clinical stage of the disease. For instance, low Cav1 expression is associated with low survival in stromal breast cancer cells, whereas high expression of Cav1 indicates poor prognosis in invasive breast cancer cells (Qian et al., 2011). Thus, Cav1 regulates different oncogenic properties, such as neoplastic transformation, apoptosis resistance, migration, invasiveness, and angiogenesis (Quest et al., 2008; Qian et al., 2019), likely depending on the cell type and/or Cav1 interactions with specific partners. Reciprocal regulation between Cav1 and the cell oxidative state regulates cell survival and stress-dependent responses (Wang et al., 2017). In addition, Cav1 also plays non-caveolar functions (Volonte et al., 2016). Cav1 localizes not only to the plasma membrane, but also to mitochondria, where it participates in the regulation of cell bioenergetics and apoptosis and, consequently, in cancer progression (Nwosu et al., 2016). Cav1 upregulation promotes apoptosis resistance and provides a metabolic advantage to cancerous cells (Wang et al., 2017). In fact, Cav1 inhibits Bax-dependent cell death, helping cancer cells to escape chemotherapy (Zou et al., 2012; Shiroto et al., 2014). In contrast, Cav1 knockdown has been reported to cause hyperpolarization of the inner mitochondrial membrane (IMM) potential (∆ψm) and to alter the lipid composition of the IMM, leading cells to apoptosis (Bosch et al., 2011).

Kv1.3, which is present in lipid rafts (Bock et al., 2003) and associates with Cav1, participates in apoptosis and chemotherapy resistance through its mitochondrial localization (Szabó et al., 2008; Leanza et al., 2012). Mitochondrial Kv1.3 (mitoKv1.3) mediates the pro-apoptotic effects of Bax. Bax blocks Kv1.3 causing the hyperpolarization of the IMM and a subsequent reactive oxygen species (ROS) production. These events lead to the opening of the permeability transition pore (PTP) and to consequent IMM depolarization, which is followed by the release of cytochrome c and the triggering of the intrinsic apoptotic cascade. Sustained PTP opening leads to the loss of mitochondrial integrity and respiration and induces swelling (Szabó et al., 2008; Szabò et al., 2011). The effect of Bax, which is often downregulated in cancer cells, can be mimicked by mitochondria-targeted Kv1.3 inhibitors (Leanza et al., 2012). For this reason, mitoKv1.3 has become a potential target for chemotherapy and a solution for overcoming chemotherapeutic resistance (Leanza et al., 2012; Leanza et al., 2017; Szabo et al., 2021).

Because the Kv1.3 and Cav1 interaction in lipid rafts has an enormous influence on cell physiology (Vicente et al., 2006; Pérez-Verdaguer et al., 2016a; Pérez-Verdaguer et al., 2018), we analyzed the functional link between Kv1.3 and Cav1 in the regulation of intrinsic apoptosis. We demonstrated that the interaction of Kv1.3 with Cav1 is important for the plasma membrane targeting of the channel. On the other hand, once in the mitochondria, the Kv1.3–caveolin axis functions as an anti-apoptotic mechanism protecting the cells from Kv1.3-mediated cell death. Our data increases the understanding of the heterogeneity of cancer by consolidating the roles of Kv1.3 and Cav1 as targets for anti-cancer therapies.

Results

Interaction with caveolin-1 governs the spatial localization of Kv1.3

We have previously described that the interaction between Kv1.3 and Cav1 results in the localization of the channel to lipid rafts and caveolae (Pérez-Verdaguer et al., 2016a; Pérez-Verdaguer et al., 2018). Kv1.3 contains a caveolin-binding domain (CBD) situated on the N-terminal end of the channel. Molecular simulations of Kv1.3 identified this CBD as an α-helix in an exposed orientation of the tetrameric structure (Figure 1A). The sequence for the CBD (ɸxxxxɸxxɸ, where ɸ is an aromatic residue and x is an unspecified amino acid) of Kv1.3 is FQRQVWLLF. To further study the nature of the Kv1.3–Cav1 interaction, we abrogated the CBD by replacing the aromatic residues with Ala or Gly (Kv1.3 CBDless, Figure 1A). Deletion of the CBD motif of Kv1.3 caused the loss of Kv1.3–Cav1 association, as demonstrated by the absence of co-IP and Förster resonance energy transfer (FRET) (Figure 1B–D). Analogous CBDs are located at the N-terminus of HCN channels, and some point mutations of this motif are sufficient to alter Cav1 binding (Barbuti et al., 2012). Therefore, we analyzed whether this also applied for Kv1.3 (Figure 1—figure supplement 1). Any substitution of aromatic residues in the CBD decreased the colocalization with Cav 1 (Figure 1—figure supplement 1A–C). However, similar to HCN4, only the disruption of the last pair of aromatic amino acids (W171G/F174A) greatly impaired the association with Cav 1 (Figure 1—figure supplement 1D).

Figure 1 with 3 supplements see all
The caveolin-binding domain (CBD) of Kv1.3 mediates the interaction with Cav1 targeting the channel to lipid raft microdomains.

(A) Ribbon representation of a Kv1.3 tetramer. For clarity, the transmembrane domain structures are highlighted with different colors in one monomer, with the CBD in red. Both the cytoplasmic (bottom view) and the side (side view) planes are shown. Note the exposed orientation of the CBD at the proximal cytoplasmic N-terminal domain. A zoomed in image is provided for detail. Aminoacids are identified with letters and positions. Lateral chains are colored by element (C, gray; N, blue; O, red). The consensus sequence of the CBD is provided. The amino acid sequence shows the CBD of wild type (WT) Kv1.3. The Kv1.3 CBD mutant (CBDless) contains amino acid substitutions (in red) to abrogate the CBD. (B) Kv1.3–Cav1 coimmunoprecipitation assay. HEK 293 Cav cells were cotransfected with Cav1 and Kv1.3YFP WT or Kv1.3YFP CBDless. Total cell lysates were immunoprecipitated with Cav1 (IP: Cav1). IP−, absence of Cav1 antibody. SM, starting materials. Samples were immunoblotted (IB) against Cav1 or Kv1.3. (C) Representative images from a Förster resonance energy transfer (FRET) experiment on cell unroofing preparations (CUPs). HEK 293 Cav cells were cotransfected with Kv1.3YFP WT+Cav1 Cerulean (Cav1 Cer) and Kv1.3YFP CBDless+Cav1 Cer. From left to right: acceptor (Kv1.3 YFP) and donor (Cav1 Cer) prebleach and postbleach images. Square insets indicate the bleached zone. Line graphs at the right show changes in donor (cerulean) and acceptor (yellow) fluorescence after bleaching. (D) FRET efficiency (%). Values are the mean ± SE (n > 25). **p<0.01, ***p<0.001 (Student’s t-test). YFP+Cer were used as negative control. Positive controls were Kv1.3 YFP WT+Kv1.3 Cer WT. (E, F) Purification of detergent-resistant membrane fractions (lipid rafts). HEK293 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBDless and samples subjected to sucrose-density gradients (1–12 from low [top of tube] to high [bottom of tube] density fractions, respectively). Clathrin was used as a non-raft marker, and flotillin and caveolin as lipid raft markers.

In addition, the absence of the Cav1 interaction displaced Kv1.3 from lipid raft microdomains (Figure 1E,F). Although the CBD lies next to the T1 (Kv tetramerization domain), its abolition did not prevent tetramerization of the channel: (1) Kv1.3 CBDless formed tetramers that were observed by FRET (Figure 1—figure supplement 2A,B) and (2) Kv1.3 CBDless formed oligomeric structures observed with nondenaturing polyacrylamide gel electrophoresis (Figure 1—figure supplement 2C). However, the biophysical properties of the plasma membrane Kv1.3 CBDless were affected (Figure 1—figure supplement 3) as assessed in Xenopus oocytes. Two-electrode voltage-clamp recordings in oocytes microinjected with Kv1.3 WT or Kv1.3 CBDless (Figure 1—figure supplement 3A) indicated that Kv1.3 CBDless exhibited less current intensity, with a −20 mV hyperpolarized shift in the steady-state activation, compared to Kv1.3 WT (Figure 1—figure supplement 3C–G). In addition, the characteristic C-type inactivation of Kv1.3 was accelerated in Kv1.3 CBDless (Figure 1—figure supplement 3H,I) also exhibiting a slightly augmented cumulative inactivation (Figure 1—figure supplement 3J–L). Finally, both Kv1.3 channels, WT and CBDless, hyperpolarized the membrane potential (Figure 1—figure supplement 3M), but the CBDless mutant decreased the input resistance of the oocyte cell membrane (Figure 1—figure supplement 3N).

Although Kv1.3 CBDless was functional, an impaired Cav1 interaction altered the membrane distribution of the channel by excluding it from lipid raft structures (Figure 1E,F) and reduced the current density (Figure 1—figure supplement 3), which could be the consequence of reduced surface abundance (Martínez-Mármol et al., 2013). Therefore, we analyzed the targeting of Kv1.3 CBDless to membranes other than the plasma membrane in HEK 293 cells. The CBD motif disruption caused a notable intracellular retention (at the ER, Golgi, and mitochondria as shown below), which reduced the surface expression of the channel (Figure 2A–D); these data were further confirmed by biotinylation assays (Figure 2E). Kv1.3 usually appears as glycosylated and non-glycosylated protein forms (Figure 2F). Glycosylation studies, in the presence of tunicamycin, indicated an altered glycosylation for Kv1.3 CBDless, which mostly affected the larger glycosylated band (Figure 2F,G). Low levels of N-glycosylation, a reduced half-life (Figure 2H) and a decrease in surface expression, concomitant with a punctuated intracellular pattern for Kv1.3 CBDless (Figure 2A–C), suggested an altered maturation and stability of the channel.

The integrity of the CBD domain is involved in the surface expression of Kv1.3.

HEK 293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBDless. (A–C) Representative confocal images show colocalization of Kv1.3YFP WT and Kv1.3YFP CBDless with (A) plasma membrane (Mb), (B) Golgi, and (C) endoplasmic reticulum (ER). Green panels, Kv1.3; red panels, subcellular marker; merge panels show colocalization in yellow. The scale bar is 10 μm. ER (pDsRed-ER) and Mb (Akt-PH-pDsRed) were used as ER and Mb markers, respectively, and were cotransfected with the channel. Golgi was stained with an anti-cis-Golgi antibody (GM130). (D) Colocalization analysis (Pearson’s coefficient) between channel and subcellular markers. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBDless. Data are the mean ± SE (n > 30 cells) **p<0.01; ***p<0.001 vs Kv1.3 WT (Student’s t-test). (E) Cell surface biotinylation analysis of the surface expression of Kv1.3. HEK 293 Cav- cells were cotransfected with Kv1.3YFP WT and Kv1.3YFP CBDless in the presence (+) or the absence (−) of Cav1. SM, starting materials. PD, pull-down (biotinylated proteins). Samples were immunoblotted (IB) for Kv1.3, clathrin (negative control), and Cav1 (caveolin). (F) Cells transfected with Kv1.3YFP WT or Kv1.3YFP CBDless were treated with (+) or without (−) 0.5 μg/ml tunicamycin for 24 hr to inhibit N-glycosylation. Total cell lysates were immunoblotted against Kv1.3 (anti-GFP) and β-actin. (G) Relative Kv1.3 glycosylation. The percentage of glycosylated (G) and nonglycosylated (NG) forms was calculated from data in (F). Data are the mean ± SE of 4 independent experiments. (H) Kv1.3 protein stability. Cells transfected with Kv1.3YFP WT and Kv1.3YFP CBDless for 24 hr were further treated for 0, 6, and 12 hr with 100 μg/ml cycloheximide. Total protein extracts were separated by SDS–PAGE and immunoblotted using Kv1.3 and β-actin antibodies. Kv1.3 expression was corrected using β-actin levels and relativized by initial values at 0 hr. A.U, arbitrary units. Data are the mean ± SE of three independent experiments. **p<0.01 (two-way ANOVA). (I) Representative confocal images of Kv1.3YFP WT (Ia, Ib) or Kv1.3YFP CBDless (Ic, Id) in the presence (Ib, Id) or in the absence (Ia, Ic) of 5 μg/ml brefeldin A (+Befeldrin A) for 4 hr. The scale bar represents 10 μm.

N-glycosylation is an elaborated process that occurs at the ER and continues along the Golgi cisternae. To test the route of Kv1.3 CBDless within the cells, we disrupted ER–Golgi traffic with brefeldin A (BFA) and determined the channel distribution. As we previously described (Martínez-Mármol et al., 2013), BFA blocked the Kv1.3 WT trafficking and, interestingly, Kv1.3 CBDless exhibited a similar uniform ER distribution (Figure 2I).

Kv1.3 CBDless accumulates in mitochondria and alters mitochondrial morphology and function

As mentioned above, the plasma membrane channel participates in cell proliferation, whereas mitoKv1.3 overexpression facilitates apoptosis (Szabó et al., 2008). We showed that the Cav1 interaction is essential for plasma membrane targeting of the channel and that CBD disruption triggered a punctate intracellular phenotype. Therefore, we wondered whether this scenario affected the mitochondrial localization of the channel, especially because targeting mechanisms for mitoKv1.3 are unknown. While Kv1.3 does not display a classical N-terminal mitochondria-targeting pre-sequence, membrane-permeant mitochondriotropic channel antagonists have unequivocally demonstrated a pivotal role for mitoKv1.3 in the death of primary human lymphocytic leukemia cells as well as of B16F10 melanoma cells (Leanza et al., 2012; Leanza et al., 2017). Thus, we used melanoma cells in addition to HEK 293 cells, to decipher a general mechanism. Similar to HEK 293 cells, Kv1.3 CBDless exhibited less cell surface abundance than Kv1.3 WT in B16F10 melanoma cells (Figure 3—figure supplement 1). Interestingly, the expression of Kv1.3 CBDless decreased at the plasma membrane whereas augmented in the mitochondria of both HEK 293 cells (Figure 3A–D) and B16F10 melanoma cells (Figure 3E,F). Therefore, a deficient caveolin interaction, impairing surface expression, partially redirected Kv1.3 to mitochondria (Figure 3B), where it triggered mitochondrial network fragmentation (Figure 4A). Morphometric analysis of Kv1.3 CBDless HEK 293 cells indicated that mitochondria were smaller and more round-shaped than those observed in Kv1.3 WT cells (Figure 4B–D). Correlative transmission electron microscopy in both B16F10 melanoma (Figure 4E,F) and HEK 293 cells (Figure 4G) showed that only the mitochondria in cells expressing Kv1.3 CBDless lost cristae and became more round. In addition, mitochondrial functionality was also severely altered. Indeed, mitochondria of Kv1.3 CBDless cells were significantly depolarized (Figure 5A), and respiration was greatly impaired. The basal oxygen consumption rate (OCR) was dramatically lower in cells expressing Kv1.3 CBDless than in those expressing the WT channel and non-transfected cells (Figure 5B,C). ATP-linked respiration and the nonmitochondrial respiration, measured in the presence of oligomycin (ATPase synthase inhibitor) and antimycin (complex III blocker), respectively, were not affected. However, the maximal respiration rate, the reserve capacity and the proton leaking were significantly reduced, indicating an important loss of mitochondrial function (Figure 5B,C). Altogether, these data further confirmed the localization and role of both WT and CBDless Kv1.3 in mitochondria and pointed to their differential effects on mitochondrial physiology.

Figure 3 with 1 supplement see all
Kv1.3 CBDless targets to mitochondria.

HEK 293 and B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBDless. (A) Subcellular fractionation isolating mitochondrial (Mit) and membrane (Mb) fractions from HEK 293 cells. Samples were immunoblotted for GFP (Kv1.3), Na+/K+ ATPase (membrane marker) or VDAC (mitochondrial marker). (B) Relative membrane (Mb) mitochondrial (Mit) Kv1.3 expression. Kv1.3 abundance in (A) was normalized to Na+/K+ ATPase (Mb) and VDAC (Mit) expression and relativized to the Kv1.3 WT. Data are the mean ± SE (n = 4). *p<0.05 (Student’s t-test). (C) Representative confocal images of (Ca-Cc) Kv1.3YFP WT and (Cd-Cf) Kv1.3YFP CBDless (green) and mitochondria (pmitoRFP in red) from HEK 293 cells. (Cc, Cf) Merge shows colocalization in yellow. Insets magnify white squares for detail. (D) Quantification of colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 30). *p<0.05 (Student’s t-test). (E) Representative confocal images of (Ea–Ec) Kv1.3YFP WT and (Ed-Ef) Kv1.3YFP CBDless (green) and mitochondria (mitotracker in red) in B16F10 melanoma cells. (Ec, Ef) Merge shows colocalization in yellow. (F) Quantification of colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n = 12). *p<0.05 (Student’s t-test). Scale bar represents 10 μm.

Kv1.3 CBDless targets mitochondria altering mitochondrial morphology.

HEK 293 and B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBDless. (A) Representative confocal images of HEK-293 cells cotransfected with (Aa–Ad) Kv1.3YFP WT, (Ae–Ah) Kv1.3YFP CBDless (green) and pmitoRFP (red). Images were processed (tubeness (Ac, Ag) and skeleton (Ad, Ah)) to perform morphometric analysis (B–D) of mitochondria in Kv1.3 positive cells. Scale bar represents 10 μm. (B) The form factor (arbitrary units, A.U.) describes the particle shape complexity and is computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. (C) Average area of particles detected on the binary image. (D) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. Data are the mean ± SE (n > 30). ***p<0.001 (Student’s t-test). (E, F) Electron micrograph of B16F10 melanoma cells transfected with (Ea–Ec) Kv1.3YFP WT or (Fa–Fc) Kv1.3YFP CBDless. Cells were observed via confocal microscopy 3 days after transfection (Ea, Fa). Scale bar represents 40 μm. Next, cells were fixed and analyzed by correlative electron microscopy (Eb–Ec, Fb–Fc). (Eb, Fb) Transfected cells positive for Kv1.3 YFP (in green) from white squares in Ea and Fa. (Ec, Fc) Untransfected cells negative for Kv1.3 YFP from white squares in Ea and Fa. (G) Correlative electron micrograph of HEK 293 cells transfected with (Ga, Gb) Kv1.3YFP WT or (Gc, Gd) Kv1.3YFP CBDless. (Ga, Gc) Transfected cells positive for Kv1.3 YFP. (Gb, Gd) Untransfected cells negative for Kv1.3 YFP. Note the lack of mitochondrial cristae and the presence of swollen mitochondria in Kv1.3YFP CBDless transfected cells (Gc). Images are representative of three independent experiments. Scale bar represents 500 nm.

Kv1.3 CBDless severely impairs mitochondrial function.

HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBDless. Non-transfected cells were used as a control. (A) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Positive transfected cells (separated by sorting) were incubated with TMRM and analyzed by confocal microscopy. Data are the mean ± SE (n = 3). ***p<0.001 (one-way ANOVA). A.U, arbitrary units. (B) The oxygen consumption rate (OCR) of HEK293 cells transfected with Kv1.3YFP WT or Kv1.3YFP CBDless in the presence of 2 μg/ml oligomycin (ATPase synthase inhibitor), 200 nM FCCP (respiratory chain uncoupler), 1 μM antimycin (complex III blocker). (C) Normalized OCR parameters (%) extracted from (B). Data are the mean ± SE (n = 3). *p<0.05; **p<0.01; ***p<0.001 (one-way ANOVA). Black bar/circle, non-transfected control cells; Gray bar/circle, Kv1.3 WT; white bar/circle, Kv1.3 CBDless.

The disruption of mitochondrial morphology and the loss of mitochondrial function increased cell sensitivity toward apoptotic stimuli. HEK 293 (Figure 6A) and B16F10 melanoma cells (Figure 6—figure supplement 1) were treated with different pro-apoptotic compounds. Annexin V assays indicated that, in both cells types, Kv1.3 CBDless cells exhibited an elevated levels of apoptosis (Figure 6A, Figure 6—figure supplement 1), even in the absence of apoptosis-inducing agents. In this scenario, we analyzed whether the intracellular accumulation of Kv1.3, independently of CBDless mutation (Figure 2D), would affect the physiology of intracellular organelles leading to apoptosis. We took advantage of the Kv1.3 (YMVIii) mutant, which is highly ER retained (Martínez-Mármol et al., 2013). Concomitantly with an ER retention, Kv1.3 (YMVIii) accumulated in mitochondria (Figure 6—figure supplement 2A,B), which would warrant a nice control. Unlike Kv1.3 CBDless, Kv1.3 (YMVIii) associated with Cav 1 (Figure 6—figure supplement 2C), but triggered no relevant apoptosis (Figure 6—figure supplement 2D). The effect was specific to mitochondria because there were no differences in ER-stress markers, such as GRP78, XBP1, ATF4, and the eIF2αpS51/eIF2α ratio, between Kv1.3 WT and Kv1.3 CBDless (Figure 6—figure supplement 3).

Figure 6 with 3 supplements see all
Kv1.3 CBDless sensitizes cells to apoptosis.

HEK-293 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBDless. (A) Flow cytometry analysis evaluating apoptosis by Annexin V staining. Cells were cultured for 24 hr in the absence (control) or the presence of different pro-apoptotic compounds (0.5 μM staurosporine [STS], 10 μM ceramide, and 5 μM etoposide). Transfected cells were sorted and the % of Annexin V-positive cells was calculated. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBDless. Data are the mean ± SE (n = 3). **p<0.01 (Student’s t-test). (B–K) Electron micrographs showing ultrastructural features of HEK-293 cells transfected with Kv1.3YFP WT (B, C) or Kv1.3YFP CBDless (D–K). Kv1.3 was immunolabeled with 18 nm diameter gold particles. Arrowheads show Kv1.3. (B) Normal appearance of organelles in a Kv1.3YFP WT-transfected cell. (C) Expression of Kv1.3YFP WT at the inner mitochondrial membrane. (D) Kv1.3YFP CBDless triggered either severe (a) or mild (b) apoptotic cell phenotypes in cells. (E) Kv1.3 CBDless at the mitochondrial membrane. Note the absence of mitochondrial cristae. (F–H) Localization of Kv1.3 CBDless in cells affected with a mild apoptotic phenotype. (F) Kv1.3 CBDless at the ER. (G) Kv1.3 CBDless at the Golgi apparatus. (H) Kv1.3 CBDless at the mitochondrial membrane. (I–K) Localization of Kv1.3 CBDless in cells affected with a severe apoptotic phenotype. (I) Notable accumulation of Kv1.3 CBDless in membranes surrounding lysosomes or autolysosomes. (J) Intense staining of Kv1.3 CBDless at multivesicular bodies. (K) Kv1.3 CBDless in vacuole containing membrane whorls. Bars represent 500 nm.

Apoptosis was also clearly visible by electron microscopy (Figure 6D–K). Cells that expressed Kv1.3 WT triggered no morphological changes and maintained mitochondria with sufficient cristae (Figure 6B,C), while Kv1.3 CBDless cells generated two apoptotic phenotypes: some cells were severely affected and full of apoptotic bodies, whereas others showed milder effects (Figure 6D). Electron micrographs with immunogold labeling further supported the accumulation of Kv1.3 CBDless in intracellular organelles, such as the ER, Golgi, or mitochondria (Figure 6F–H, respectively). Cells displaying severe effects concentrated Kv1.3 CBDless in multilamellar bodies, apoptotic phagocytic structures, and close to membranous whorls, which are characteristic of cell death (Figure 6I–K, respectively) and are consistent with the punctate pattern observed in confocal studies (Figures 2 and 3).

Cav1-mitoKv1.3 functional link alleviates the pro-apoptotic activity of mitochondrial Kv1.3

As mentioned above, the physiological role of Kv1.3 in cell survival is complex because the channel exerts known, and apparently opposite, dual roles. However, the finding reported here using Kv1.3 CBDless may explain this duality. Functional Kv1.3 CBDless, which is unable to interact with Cav1, was trafficking to mitochondria rather than to the cell surface. Therefore, it is tempting to speculate that either the imbalance between plasma membrane Kv1.3 and mitoKv1.3 is the factor that contributes to apoptosis, or the lack of association with caveolin in mitochondria is what makes the difference. In any case, interaction with caveolin would be at the onset of both scenarios. Therefore, because caveolin exerts anti-apoptotic effects (Shiroto et al., 2014; Schilling et al., 2018), we wondered whether Cav1, by association with the channel, may modulate the role of Kv1.3 in mitochondria. Similar to Kv1.3, Cav1 is also present in mitochondria (Figure 7A), and both proteins are located in close proximity (Figure 7B,C). To evaluate the effects of the Cav1 interaction on mitoKv1.3, we used several different and complementary cell models. Jurkat T lymphocytes are normally deficient in caveolin (Jurkat Cav−), but express endogenous Kv1.3 (Szabò et al., 2005Figure 7D). However, we selected a clone of Jurkat cells, which did express Cav1 (Jurkat Cav+). In this context, evidence indicates that some leukocytes, that do not initially express caveolin, may express the protein under certain states of activation (Hatanaka et al., 1998). In Jurkat Cav−, no differences were observed in apoptosis when Kv1.3 WT or Kv1.3 CBDless were overexpressed (Figure 7D,E). However, Kv1.3 WT cells exhibited less apoptosis than cells expressing Kv1.3 CBDless in Jurkat Cav+ (Figure 7E). Next, we used 3T3-L1 preadipocytes, which endogenously express both Cav1 and Kv1.3 (Pérez-Verdaguer et al., 2018). By knocking-down Cav1 expression (Cav), 3T3-L1 cells became more sensitive to apoptosis (Figure 7F,G), recapitulating the Kv1.3 CBDless effects observed in HEK 293 and B16F10 melanoma cells. Our data indicated that altering the functional crosstalk between Cav1 and Kv1.3 plays a crucial role in determining the sensitivity of cells to apoptosis.

Caveolin modulates the pro-apoptotic activity of Kv1.3.

(A) Subcellular fractionation was used to isolate mitochondrial (Mit) and plasma membrane (Mb) fractions in HEK-293 cells transfected with Kv1.3YFP WT. Samples were immunoblotted for GFP (Kv1.3), Caveolin, Na+/K+ ATPase, and VDAC. (B, C) Electron micrographs showing HEK-293 cells transfected with Kv1.3YFP WT. Kv1.3 was immunolabeled with 18 nm gold particles (black arrowheads) and Cav1 with 12 nm gold particles (white arrowhead). The square inset in (B) indicates the zoomed in region in (C). Scale bars represent 500 nm. (D) Regular human Jurkat T lymphocytes express Kv1.3 and a negligible amount of endogenous Cav1 (Cav). In addition, a Jurkat cell line with notable expression of Cav1 was selected (Cav+). (E) Jurkat cells (Cav and Cav+) were electroporated with Kv1.3YFP WT or Kv1.3YFP CBDless. After 24 hr, apoptosis was assessed by Annexin V staining with flow cytometry. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBDless. (F) Mouse 3T3-L1 and 3T3-L1 Cav- preadipocytes were analyzed for the expression of endogenous Cav1 and Kv1.3. β-actin was used as a loading control. (G) Flow cytometric analysis quantifying apoptosis by Annexin V on 3T3-L1 (gray bar) and 3T3-L1 Cav- (white bar) preadipocytes. Note that the amount of Cav1 exerted notable effects on the Kv1.3-related apoptosis in native 3T3-L1 cells. Values are the mean ± SE of 3–6 independent experiments. *p<0.05; **p<0.01; ***p<0.01 (one-way ANOVA).

Finally, we isolated CD4+ T lymphocytes from blood of human donors. As expected, human T lymphocytes, which express membrane lipid raft integral proteins such as flotillin, express endogenously Kv1.3, but not Cav 1 (Figure 8A). Confocal experiments, performed in Kv1.3 WT and CBDless transfected cells, indicated that both Kv1.3 channels shared similar plasma membrane (Mb) and mitochondrial (mito) colocalization (Figure 8B–E). Morphometric analysis of Kv1.3 WT and CBDless T cells indicated that mitochondria were similarly affected by the expression of either channel (Figure 9). Thus, both channels triggered an increase in mitochondrial length and form factor, whereas mitochondrial area diminished in CD4+ lymphocytes. The expression of Kv1.3 WT and CBDless decreased also the mitochondrial membrane potential (Figure 10A), which is concomitant to similar levels of apoptosis (Figure 10B). Interestingly, the introduction of external Cav 1 in primary CD4+ cells (Cav+) partially counteracted apoptosis solely in cells expressing Kv1.3 CBDless. Our data from primary CD4+ human T lymphocytes showed that the presence of Cav 1, which interacts with Kv1.3 WT but not CBDless, partially protected cells against apoptosis and further confirmed what obtained with Jurkat T cells and 3T3-L1 cells.

Kv1.3 colocalizes with plasma membrane and mitochondria in primary human T lymphocytes.

CD4+ lymphocytes were isolated from human blood as indicated in Materials and methods. (A) Representative western blot from HEK 293 cells and T lymphocytes samples from two independent human donors showing differential protein expression of Kv1.3, Flotillin, and Cav1. β-Actin was a loading control. (B) Representative confocal images of Kv1.3 colocalization in plasma membrane (Mb) from Kv1.3YFP WT and Kv1.3YFP CBDless-transfected cells. WGA stained plasma membrane. (C) Representative confocal images of Kv1.3 colocalization in mitochondria (mito) from Kv1.3YFP WT and Kv1.3YFP CBDless expressing cells. MitoTracker was used for mitochondrial staining. Scale bar represents 10 μm. Quantification of Kv1.3/Mb (D) and Kv1.3/mito (E) colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20), Student’s t-test. Gray bars, Kv1.3YFP WT cells; white bars, Kv1.3YFP CBDless cells.

The expression of Kv1.3YFP WT and Kv1.3YFP CBDless in T lymphocytes alters the mitochondrial morphology.

Human CD4+ T lymphocytes were transfected with Kv1.3YFP WT or Kv1.3YFP CBDless. (A–D) Representative confocal images of non- transfected T cells. (E–H) T lymphocytes transfected with Kv1.3YFP WT. (I–L) T lymphocytes transfected with Kv1.3YFP CBDless. (A, E, and I) Kv1.3YFP (green); (B, F, and J) MitoTracker (red). Images were processed (tubeness (C, G, and K) and skeleton (D, H, and L)) to perform morphometric analysis (M, N, and O) of mitochondria. Scale bar represents 10 μm. (M) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. (N) The form factor (arbitrary units, A.U.) describes the particle shape complexity and is computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. (O) Average area of particles detected on the binary image. Data are the mean ± SE (n > 20). ***p<0.001 (Student’s t-test).

Caveolin-1 protects from apoptosis when associated with Kv1.3 in primary human T lymphocytes.

Human CD4+ T lymphocytes were transfected with Kv1.3YFP WT or Kv1.3YFP CBDless and the mitochondrial membrane potential (TMRM) and apoptosis were measured. YFP-transfected cells were used as a control. (A) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Cells were incubated with TMRM and analyzed by flow cytometry. A.U, arbitrary units. (B) T cells were electroporated with Kv1.3YFP WT or Kv1.3YFP CBDless with (Cav+) or without (Cav−) Cav1 Cerulean. After 24 hr, transfected cells were sorted and apoptosis was assessed by Annexin V staining with flow cytometry. The level of apoptosis in arbitrary units (A.U.) was measured in each group by resting the value of basal apoptosis in cells transfected with YFP in the presence (Cav−) or the absence (Cav−) of Cav1. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBDless. Cav−, regular CD4+ cells without Cav 1; Cav+, T cells transfected with Cav1 Cer. Data are the mean ± SE (n = 5–7). *p<0.05 (one-way ANOVA).

Discussion

Kv1.3 plays important but apparently contrasting roles in the cell physiology because Kv1.3 in the plasma membrane supports proliferation, while mitoKv1.3 sensitizes cells to apoptosis. Furthermore, caveolin is also involved in both pro- and anti-apoptotic events participating in the regulation of cell survival and in cancer protection. Our results indicate that the interaction of Kv1.3 with Cav1 has important physiological consequences for controlling apoptosis. Cav1 association, via the N-terminal located CBD of Kv1.3, drives the channel to the plasma membrane. Altering the CBD impairs membrane targeting, promoting the Kv1.3 intracellular retention and mitochondrial accumulation. In this way, we were able to distinguish the effects of intracellular/mitochondrial Kv1.3 from those of the plasma membrane channel. Once inside mitochondria, the CBDless channel facilitates apoptosis. In the presence of Kv1.3, either the depletion of caveolin or the lack of Cav1 binding favors apoptosis, suggesting that the Cav1 functional interaction with the channel facilitates apoptotic resistance. Therefore, we identified that Cav1 associates with Kv1.3, thereby modulating the pro-apoptotic effects of mitochondrial Kv1.3 channels. This observation is key for the understanding how these two proteins can reciprocally regulate their role in cancer progression, with significant implications for anti-cancer therapy.

Acute inhibition of mitoKv1.3 transient hyperpolarizes IMM inducing ROS release. Subsequent PTP opening leads to loss of mitochondrial integrity, IMM depolarization, swelling, and cytochrome c release. Overexpression of mitochondrial K+ transporting pathways drives the influx of depolarizing K+ into the matrix triggering mitochondrial depolarization as well as changes in ultrastructure (Paggio et al., 2019). Thus, acute changes in the mitochondrial membrane potential, upon block of the channel, induce ROS and PTP opening, while overexpression of mitochondria-located Kv1.3, that is more prominent in the case of CBDless mutant or in the absence of Cav (for both WT and CBDless Kv1.3), causes sustained depolarization that sensitizes cells to apoptotic stimuli. PTP opening further depolarizes IMM and reduces respiration due to swelling and loss of cytochrome c. Here we show that overexpression of WT or CBDless Kv1.3 equally depolarized mitochondria and caused apoptosis in primary T cells, which lack endogenous Cav. Interestingly, while overexpression of Kv1.3 promotes apoptosis, the channel deficiency renders the cells resistant to apoptosis (Szabó et al., 2008). Thus, similarly to the ATP-dependent K+ channel of pancreatic β cells (Miki et al., 1998), pharmacological block of Kv1.3 does not yield the same physiological effects of channel downregulation, as the block triggers a series of signaling events that cannot be induced in cells lacking Kv1.3.

Cav interacts with the CBD of target signaling proteins through its CSD. Mutating the CSD of Cav1 modulates migration of cancerous cells by affecting interaction with signaling partners (Okada et al., 2019). Kv1.3, by interaction with Cav1, targets to lipid raft microdomains. These observations are consistent with our previous data, showing that the presence of Kv1.3 in lipid rafts was dependent on Cav1 expression (Pérez-Verdaguer et al., 2016a). Evidence supports that CBDs should contain solvent exposed aromatic residues (Byrne et al., 2012). Our data support this claim because, in our molecular simulation, the Kv1.3 CBD is exposed and located close to transmembrane domains, which favor physical interactions with Cav1. Aromatic residues are involved in protein folding. Therefore, because of the proximity of the CBD to the channel tetramerization domain (T1), we assessed Kv1.3 CBDless functionality. Kv1.3 CBDless forms functional tetramers but with altered electrophysiological properties. The main feature is the reduction of current intensity, probably due to impaired surface expression. In fact, we have previously shown that the absence of Cav1 expression reduces the half-life, cell surface expression, and current amplitude of Kv1.3 (Pérez-Verdaguer et al., 2016a). Kv1.3 CBDless showed a hyperpolarizing shift in the steady-state activation and altered inactivation kinetics. These observations are not surprising because the lipid composition of the plasma membrane modulates the activity, kinetics, and voltage-dependence of ion channels (Levitan et al., 2010; Brini et al., 2018; Poveda et al., 2017; Zakany et al., 2019). Furthermore, the biophysical properties of Kv1.3 CBDless might be due to multiple additional factors, including changes in membrane lipid composition that occur during apoptosis (Tepper et al., 2000). Altogether, our data would support that Kv1.3 surface expression and thus the current density are notably dependent on its Cav1 interaction.

As mentioned above, the lack of interaction between Kv1.3 CBDless and Cav1 drastically impaired the cell surface targeting of the channel. However, Kv1.3 CBDless kept targeting to mitochondria, and this occurred before Golgi processing, given that the mitochondria-targeted channel is not glycosylated. This fact raises up an interesting scenario because we separated the effects of mitochondrial Kv1.3 from those of the plasma membrane channel. Expression of Kv1.3 CBDless notably sensitized the cells to apoptosis, most likely due to the preferential localization of this mutated channel to the mitochondria. Indeed, by itself, mitochondrial Kv1.3 CBDless dramatically altered mitochondrial morphology (i.e., lower cristae density) and function, triggering apoptosis. In addition, expression of Kv1.3 CBDless caused mitochondrial network fragmentation, something typically observed in apoptotic cells, which show a high fission-to-fusion ratio (Xie et al., 2018). In general, mitochondrial fission regulation couples mitochondrial dynamics/morphology to the cellular energetic state (Giacomello et al., 2020). Fission, induced by AMPK-mediated phosphorylation (AMP), senses adapting mitochondrial function and dynamics. AMPK facilitates mitochondrial fission downstream of mitochondrial dysfunction caused, for example, by an impaired mitochondrial respiration. This mechanism facilitates apoptosis in cells with severely dysfunctional mitochondria (Toyama et al., 2016). Apoptosis can be triggered by the permeabilization of the outer mitochondrial membrane and the prolonged depolarization of the IMM (dissipation of ∆ψm in IMM), which causes the release of pro-apoptotic factors from the mitochondrial intermembrane space to the cytosol (Zorova et al., 2018). We can therefore explain the ability of Kv1.3 CBDless triggered apoptosis by the observation that mitochondria were significantly depolarized and mitochondrial respiration was highly impaired, suggesting that cells were undergoing apoptosis. We propose that the absence of Cav1 interaction with mitoKv1.3 favors the pro-apoptotic effects of Kv1.3. The results obtained in human CD4+ lymphocytes, Jurkat T cells, and 3T3-L1 preadipocytes support this hypothesis. In T-lymphocytes, which lack of endogenous Cav1 expression, no differences in apoptosis between cells expressing Kv1.3 WT and Kv1.3 CBDless were found. Similarly, silencing of Cav1 expression in 3T3-L1 preadipocytes increased the apoptosis. However, the presence of Cav1 exhibited anti-apoptotic properties but solely in cells expressing the wild-type channel.

In summary, our data indicate that the physiological role of Kv1.3 is highly dependent on its interaction with Cav1. The Kv1.3 interaction with Cav1 can drive the channel either to the membrane and support proliferation or to mitochondria. MitoKv1.3 sensitizes cells to apoptosis, in agreement with our previous observation (Szabó et al., 2008), and possibly, mitochondrial Kv1.3 interaction with Cav1 modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms would fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Although a direct interaction of these proteins in mitochondria has not been confirmed, and warrants further investigation, evidence suggests that apoptosis is dependent on mitoKv1.3–caveolin functional axis. Our work has important implications not only in the understanding of Kv1.3-dependent cancer progression but possibly also in metabolic diseases, where Cav1 and Kv1.3 both play an important role. Our data would suggest an essential role for the caveolin–Kv1.3 axis during tumorigenesis and apoptosis.

Materials and methods

Expression plasmids and site-directed mutagenesis

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T.C. Holmes (University of California, Irvine, CA) provided the rat Kv1.3 in a pRcCMV construct. The channel was subcloned into pEYFP-C1 and pECerulean-C1 (Clontech). All Kv1.3 mutants were generated in the pEYFP-Kv1.3 channel using a QuikChange site-directed mutagenesis kit (Agilent Technologies). pEYFP-Kv1.3CBDless was subcloned into pECerulean-C1. For oocyte injection, Kv1.3 and Kv1.3CBDless were subcloned into pcDNA3 and were placed under the control of a T7 promoter and then the cRNA was synthetized. J.R. Martens (University of Florida Medical School) provided the rat Caveolin 1 (Cav1) in pECerulean-C1. Cav1 was cloned into pcDNA3. The plasma membrane marker Akt-PH-pDsRed (pDsRed-tagged pleckstrin homology (PH) domain of Akt) was a kind gift of F. Viana (Universidad Miguel Hernández, Spain). The ER marker (pDsRed-ER) was obtained from Clontech. The mitochondrial marker (pmitoRFP) was constructed by fusing the mitochondrial transit sequence of the human isovaleryl coenzyme A dehydrogenase to the N-terminus of RFP (pDsRed1-N1, Clontech). Constructs were verified by sequencing.

Cell culture and drugs

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HEK 293 cells (ATCC) were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin (Gibco). In some experiments, a HEK 293 Cav (lentiviral depletion of Cav1) was used (Pérez-Verdaguer et al., 2016a). B16F10 cells (ATCC) were grown in minimum essential media (Thermo Fisher Scientific) supplemented with 10 mM HEPES buffer (pH 7.4), 10% FBS, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 1% nonessential amino acids (100× solution; Thermo Fisher Scientific). Transient transfection was performed following the manufacturer’s instructions using Lipotransfectin (Attendbio), for HEK 293 cells, or TransIT-LT1 Transfection Reagent (Mirus), for B16F10 cells. Transfections were performed when cells were nearly 80% confluent. Jurkat human T lymphocytes (ATCC) were cultured in RPMI media containing 10% FBS and transfected using the Gene Pulser II Electroporation system and YFP-expressing cells were sorted using a FACSAria FUSION (BD Bioscience) instrument. 3T3-L1 preadipocytes (ATCC) were cultured in DMEM containing 10% NCS in a 7% CO2 atmosphere. All drugs were dissolved in dimethyl sulfoxide (DMSO) and diluted in DMEM. The final concentration of DMSO was <0.5% in all assays. All cell lines were routinely tested to be mycoplasma free.

Isolation of T-cell subsets, cell culture, and T-cell blast generation

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Human CD4+ T-cell subsets were isolated from peripheral whole blood using negative selection Rosette Sep kit from STEMCELL Technologies. Human T lymphocytes were cultured at 37°C, 5% CO2, in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, 1% glutamine, 1% penicillin–streptomycin (Gibco), 1× Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 10 mM HEPES (Life Technologies), and 50 U/ml IL-2 (Bionova). To generate T-cell blasts, the Dynabeads Human T-Activator CD3/CD28 for T-cell expansion and activation kit (Life Technologies) was used following manufacturer’s instructions. Human T-cell blasts were used after 6–7 days of expansion protocol. No IL-2 was supplemented in media the day before an experiment. In some experiments, pEYFP-Kv1.3 WT, pEYFP-Kv1.3CBDless, and pECerulean-Cav1 were electroporated into human CD4+ lymphocytes as abovementioned.

The protocol was reviewed and approved by the Ethics Committee of the Universitat de Barcelona and the Banc de Sang i Teixits de Catalunya (BST). Institutional Review Board (IRB00003099). All procedures followed the rules of the Declaration of Helsinki Guidelines. All donors signed a written informed consent, and samples were totally anonymous and untraceable.

Raft isolation

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Low-density, Triton-insoluble complexes were isolated as previously described (Pérez-Verdaguer et al., 2016a). Briefly, after three washes in phosphate-buffered saline (PBS), cells were homogenized in 1 ml of 1% Triton X-100 MBS (150 mM NaCl, 25 mM 2-morpholinoethanesulfonic acid 1-hydrate, pH 6.5) supplemented with 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride to inhibit proteases. Sucrose in MBS was added to a final concentration of 40%. A 5–30% linear sucrose gradient was layered on top and further centrifuged (39,000 rpm) for 20–22 hr at 4°C in a Beckman SW41Ti swinging rotor. Gradient fractions (1 ml) were collected from the top and analyzed by western blot.

Purification of mitochondria

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Mitochondria from HEK 293 cells were purified by differential centrifugation (adapted from Wieckowski et al., 2009). Briefly, 80% confluent cells were trypsinized and washed twice with PBS without Ca2+ and centrifuged at 600 × g for 10 min. Cells were homogenized in initial buffer 1 (225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA, 30 mM Tris, pH 7.4) and centrifuged again at 600 × g for 10 min to remove unlysed cells and nuclei. The supernatant was centrifuged at 7000 × g 10 min. The mitochondria-containing pellet was suspended in initial buffer 2 (225 mM mannitol, 75 mM sucrose, 30 mM Tris, pH 7.4) and centrifuged again at 7000 × g. The suspension of the pellet was repeated and centrifuged at 10,000 × g to obtain a purified mitochondrial fraction. The supernatant from the first 7000 × g centrifugation was then centrifuged at 20,000 × g for 30 min and the pellet contained the membranous fraction. Mitochondrial and membranous fractions were suspended in 50 μl of initial buffer 2. All centrifugations were performed at 4 °C.

Protein extraction, coimmunoprecipitation, biotinylation of cell surface proteins, and western blot analysis

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Cells were washed in cold PBS, lysed on ice with NHG solution (1% Triton X-100, 10% glycerol, 50 mM HEPES pH 7.2, 150 mM NaCl) supplemented with 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride to inhibit proteases. Homogenates were centrifuged at 16,000 × g for 15 min, and the protein content was measured using the Bio-Rad Protein Assay. For immunoprecipitation, samples were precleared with 30 μl of protein A-sepharose beads for 2 hr at 4°C with gentle mixing and the beads were then removed by centrifugation at 1000 × g for 30 s at 4°C as part of the coimmunoprecipitation procedures. Meanwhile, 50 μl of protein A-sepharose beads were incubated in 500 μl of NGH in the presence or in the absence of an anti-caveolin antibody (4 ng/μg protein) at 4°C with gentle agitation and washed three times to obtain antibody-bound A-sepharose beads. The precleared samples were then incubated overnight at 4°C with antibody-bound A-sepharose beads. Finally, supernatants were removed by centrifugation at 1000 × g for 30 s at 4°C, and beads were washed four times with NHG and resuspended in 100 μl of Laemmli SDS buffer.

Three oocytes were placed into a 1.5 ml Eppendorf tube and homogenized by pipetting in 100 μl of homogenization buffer (20 mM Tris–HCl pH 7.6, 0.1 M NaCl, 1% Triton X-100) with protease inhibitor cocktail. Homogenates were incubated for 20 min at 4°C to solubilize membrane proteins and centrifuged at 10,000 × g for 2 min at 4°C. Supernatants were transferred to a new tube, and protein content was determined using the Bio-Rad Protein Assay (Bio-Rad).

Cell surface biotinylation was determined with the Pierce Cell Surface Protein Isolation Kit (Pierce) following manufacturer’s instructions. Briefly, cell surface proteins were labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-biotin; Pierce). Then, cells were treated with lysis buffer, and clear supernatant was reacted with immobilized NeutrAvidin gel slurry in columns (Pierce) to isolate surface proteins. Protein samples (50 μg), raft fractions (50 μl), mitochondria and membranous fractions (50 μl), and immunoprecipitates were boiled in Laemmli SDS loading buffer and separated by 10% SDS–PAGE. Next, samples were transferred to PVDF membranes (Immobilon-P, Millipore) and blocked with 5% dry milk-supplemented with 0.05% Tween 20 in PBS. The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+/K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam). Finally, membranes were washed with 0.05% Tween 20 in PBS and incubated with horseradish peroxidase conjugated secondary antibodies (Bio-Rad).

Immunocytochemistry and confocal imaging

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Cells seeded on poly-d-lysine-treated coverslips were used 24 hr later for transfection. Cells were washed with PBS and fixed (only HEK 293 cells) with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). To detect cis-golgi, cells were permeabilized by incubating with 0.1% Triton X-100 for 10 min. After a 60 min in blocking solution (10% goat serum [Gibco], 5% nonfat dry milk, PBS), cells were treated with a mouse anti-GM130 antibody (1/1000, BD Transduction Laboratories) antibody in 10% goat serum and 0.05% Triton X-100 and were again incubated for 1 hr. After three washes, preparations were incubated for 45 min with an Alexa-Fluor-660 conjugated antibody (1:200; Molecular Probes), washed, and mounted in Mowiol (Calbiochem). All procedures were performed at RT. All images were acquired with a Leica TCS SP2 AOBS microscope. Colocalization analysis was performed with ImageJ (National Institutes of Health, Bethesda, MD) following Sastre et al., 2019, and the morphometric analysis of mitochondria was performed following Strack and Usachev, 2017.

In CD4+ human T lymphocytes, 500 nM MitoTracker (Thermo Fisher Scientific) was used to visualize mitochondria according to manufacturer’s instructions. For membrane surface labeling, Wheat Germ Agglutinin-Alexa555 (WGA, Invitrogen) was used. Cells were washed with PBS at 4°C and stained with a dilution of WGA (1/1500) in RPMI supplemented with 30 mM Hepes for 5 min at 4°C. Next, cells were washed twice and fixed with 4% paraformaldehyde for 10 min. Finally, cells were washed and mounted in Mowiol (Calbiochem). Confocal images were acquired with a Zeiss 880 confocal microscope.

Cell unroofing preparations (CUPs) and Förster resonance energy transfer (FRET)

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CUPs were obtained via osmotic shock as previously described (Oliveras et al., 2020). Briefly, cells were cooled on ice for 5 min and washed twice with PBS. Next, cells were incubated for 5 min in 1:3 diluted KHMgE (70 mM KCl, 30 mM HEPES, 5 mM MgCl2, 3 mM EGTA, pH 7.5) and were gently washed with nondiluted KHMgE to induce the hypotonic shock. Broken cells were removed from the coverslip by pipetting up and down. After two washes with KHMgE buffer, only membrane sheets remained attached. CUPs were fixed with fresh 4% paraformaldehyde for 10 min at RT and mounted in Mowiol mounting media.

FRET was performed using the acceptor photobleaching configuration. Samples were imaged with a Leica SP2 confocal microscope. Images were acquired before and after YFP bleaching using a 63× oil immersion objective at a zoom setting of 4. Excitation was performed via the 458 and 514 nm lines using an Ar laser, and 465–510 and 525–560 bandpass emission filters were used. FRET efficiency (FRETeff) was calculated using the following equation:

(FDafterFDbefore)/FDbeforex100

where FDafter: donor fluorescence (Cerulean) after and FDbefore before acceptor (YFP) bleach. Analysis was performed using ImageJ.

Transmission electron microscopy and correlative transmission electron microscopy

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Cells were transfected and, after 24 hr, fixed with 4% PFA and 0.1% glutaraldehyde at RT for 1 hr followed by a treatment with 2% PFA for 30 min. High-pressure freeze cryofixation with liquid N2 and cryosubstitution, Lowicryl resine embedding, polymerization of blocks, and ultrathin sections (60 nm) were performed in collaboration with Unitat de criomicroscòpia electrònica (CCiT, University of Barcelona). Samples were mounted over Formvar-coated grilles, and sections were finally contrasted with uranyl acetate 2% for 15 min. Immunolabeling was performed with the primary antibodies anti-Kv1.3 (Neuromab, 1:30) and anti-Caveolin 1 (1:70, Abcam). Secondary antibodies were conjugated to 12 and 18 nm gold particles as indicated. Samples were imaged using a Tecnai Spirit 120kV microscope. Correlative transmission electron microscopy was performed by the Microscopy Facility of the Department of Biology, University of Padova, as described in Leanza et al., 2017.

Cell death assays, mitochondrial membrane potential measurements, and oxygen consumption rate (OCR) measurements

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For the evaluation of apoptosis, plated cells were treated for 18 or 24 hr with the indicated drugs (0.5 μM staurosporine; 10 μM ceramide; 5 μM etoposide) in DMEM without serum and phenol red. After treatment, cells were washed with PBS and suspended in FACS buffer (10 mM HEPES, 140 Mm NaCl, 2.5 mM CaCl2, pH 7.4) containing Annexin V APC and DAPI for 15 min in the dark. Samples were immediately analyzed using either a Gallios flow cytometer (HEK 293 cells) or a microscope (B16F10 cells). To measure mitochondrial membrane potential, cells were incubated with 20 nM tetramethyl rhodamine methyl ester (TMRM) in DMEM without serum and phenol red. Next, samples were diluted up to 5 nM TMRM with more DMEM and analyzed by flow cytometry (FACSCanto II, Becton Dickinson).

Respiration was measured using an XF24 Extracellular Flux Analyzer (Seahorse, Bioscience). HEK 293 cells, with >60% transfection efficiency, were seeded at 1.5 × 104 cells/well in 100 µl of DMEM. After 24 hr, the medium was replaced with 670 µl/well of high-glucose DMEM without serum and sodium bicarbonate and supplemented with 10 mM sodium pyruvate and 2 mM l-glutamine. The OCR was measured upon the addition of oligomycin to block ATP synthase (2 µg/ml), FCCP uncoupler (200 nM), and Antimycin A to inhibit complex III (1 µM). All chemicals were added to 70 µl of DMEM. Positive YFP fluorescence was used to monitor cell transfection before OCR measurements by using a Leica microscope (not shown).

Molecular modeling

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Kv1.3 was modeled using high-resolution templates of remote or close homologs available from the Protein Data Bank (PDB; http://www.rcsb.org/pdb) as previously described (Martínez-Mármol et al., 2013; Solé et al., 2019). Transmembrane domains and the N-terminus (except for the first 49 amino acids [aa]) were modeled with the Kv1.2 potassium channel (PDB code 2R9R). The C terminus and the remaining 49 aa from the N-terminus were modeled with 3HGF (nucleotide-binding domain of the reticulocyte-binding protein Py235) and 1PXE (zinc-binding domain from neural zinc finger factor-1) structures, respectively. The procedure was defined by the i-Tasser online server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/), and sequence alignments were executed using CLUSTALW from the European Bioinformatics Institute site (http://www.ebi.ac.uk). The homology modeling was performed using the Swiss-Model Protein Modeling Server on the ExPASy Molecular Biology website (http://kr.expasy.org/) under the Project Mode. The final molecular graphic representations were created using PyMOL v1.4.1 (http://www.pymol.org/) (Martínez-Mármol et al., 2013; Solé et al., 2019).

Oocyte preparation, microinjection, and electrophysiological recordings

Request a detailed protocol

Animal handling was carried out in accordance with the guidelines for the care and use of experimental animals adopted by the E.U (RD214/1997). Adult female Xenopus laevis (Harlan Interfauna Ibérica) were immersed in cold 0.17% ethyl 3‐aminobenzoate methanesulfonate for 20 min, and a piece of ovary was drawn out aseptically. Fully grown immature oocytes, stages V and VI, were isolated and their surrounding layers were removed manually. Cells were kept at 15–16°C in a modified Barth’s solution (88 mM NaCl, 1 mM KCl, 2.40 mM NaHCO3, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 10 mM HEPES [pH 7.4], 100 U/ml penicillin, and 0.1 mg/ml streptomycin) until use. Oocytes were microinjected with 100 nL of cRNA from Kv1.3 WT or Kv1.3CBDless pCDNA3.

Membrane current recordings were performed at 21–25°C, 16–72 hr after injection using a high-compliance two-microelectrode voltage-clamp system (TurboTEC-10CD npi, Tamm). The recording methodology has been described in detail elsewhere (Morales et al., 1995; Olivera-Bravo et al., 2007). Briefly, oocytes were placed in a 150 μL recording chamber and continuously superfused with normal frog Ringer’s solution (115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.0). The membrane potential was held at −100 mV and 20 mV depolarizing voltage steps were applied to +40 mV for a duration of 2.5 s. Membrane currents were low-pass filtered at 30–200 Hz and recorded on a PC, after sampling (Digidata 1200, Molecular Devices, San Jose, CA) at fivefold the filter frequency, using the WCP v.3.2.8 package developed by J. Dempster (Strathclyde Electrophysiology Software, University of Strathclyde, UK).

Statistics

The results are expressed as the mean ± SE. Student’s t-test, one-way ANOVA, and Tukey’s post hoc test and two-way ANOVA were used for statistical analysis (GraphPad PRISM v5.01). p<0.05 was considered statistically significant.

Data availability

All data generated or analysed during this study are publicly available on Dryad at https://doi.org/10.5061/dryad.mcvdnck13.

The following data sets were generated
    1. Felipe
    (2021) Dryad Digital Repository
    Data from: A novel mitochondrial Kv1.3-caveolin axis controls cell survival and apoptosis.
    https://doi.org/10.5061/dryad.mcvdnck13

References

  1. Book
    1. Strack S
    2. Usachev YM
    (2017)
    Techniques to Investigate Mitochondrial Function in Neurons
    Springer Science.

Decision letter

  1. Baron Chanda
    Reviewing Editor; Washington University in St. Louis, United States
  2. Kenton J Swartz
    Senior Editor; National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
  3. Shrinivasan Raghuraman
    Reviewer; University of Utah, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Kv 1.3 channels either cause cell proliferation or apoptosis depending on their sub-cellular localization. In this study, Capera et al. show that the association of Kv 1.3 channels with caveolin is critical for localization to plasma membrane and that the disruption of this interaction causes mitochondrial targeting and triggers apoptosis.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "A novel mitochondrial Kv1.3-caveolin axis controls cell survival and apoptosis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Kv 1.3 channels are involved in cell proliferation of leukocytes and are also known to promote apoptosis in tumor cells. To understand the mechanisms that contribute to these seemingly opposing roles of Kv1.3, this study examines the contribution of interaction between caveolin 1 (Cav1) and Kv1.3 channels on subcellular distribution and functional properties. Multiple lines of evidence suggest that the interaction between Kv1.3 and Cav1 in mitochondria prevents Kv1.3 mediated cell-death. However, the reviewers have raised a number of substantive concerns. Although many of these concerns can be addressed by adding new experiments, it seems unlikely that the revised manuscript can be resubmitted within the next couple of months. We are happy to give due consideration to the revised version of this manuscript if you decide to submit it to eLife again but it will be treated as new submission. Please do not hesitate to contact us if you have questions.

Reviewer #1:

This manuscript described the effects of the ablation of the Caveolin 1 (Cav1) interaction site in Kv1.3. on subcellular distribution, functional properties and effects on apoptosis. The main conclusion of the paper is that the interaction between Kv1.3 and Cav1 plays a crucial role not only in the proper plasma membrane localization of Kv1.3, resulting in an enrichment of mitochondrial localization, but that the interaction with Cav1 at the mitochondria also modulates the induction of apoptosis. Such an effect would justify the proposal of a Cav1-Kv1.3 axis, and would be a very relevant finding adding to the emerging importance of both Cav1 and Kv1.3 in physiology and pathology, especially in cancer.

The main concern is the possibility that an "overload" of the mitochondria with either wild type or CBDless Kv1.3 is responsible for the observed effects.

1. Whereas the enrichment in mitochondrial Kv1.3 in CBDless channels leaves little doubt, to what extent that interaction at the mitochondria is important for apoptosis is less clear. Kv1.3 wild type overexpression induces an increase of apoptosis on its own. If Cav1 binding-defective mutants are more abundant at the mitochondria, this alone could explain the increase in apoptosis described. The effects reported in both Jurkat and 3T3-L1 could in principle be also explained if the excess of Kv1.3 is equally targeted to mitochondria in Jurkat cells regardless of CBD due to the low Cav1 level, and if knockdown of Cav1 reduces PM Kv1.3 in preadipocytes and therefore increases mitoKv1.3 (at least it seems to alter the ratio between mature and non-glycosylated channel, Figure 7F).

2. As the authors state, it appears that the reduced PM activity of Kv1.3CBDless is due to trafficking defects (less targeting to the membrane and shorter half-life). Actually, the mutant is activated at less depolarized potentials, suggesting a more favorable transition to an open state. Inhibition of mitoKv1.3 by Bax or pharmacological agents triggers apoptosis in several cell types. It is therefore counterintuitive, or at least it is not directly explained in the manuscript, that overexpression of a variant of the channel, which is in fact "more active" than the wild type induces more apoptosis, unless the effect is different from the physiological role of mitoKv1.3.

3. It would be extremely interesting to know what the effect of mitochondria-targeted Kv1.3 inhibitors like the ones described previously by some of the authors have on cells overexpressing Kv1.3 (wild type and CBDless), and if such agents can counteract the effect of Cav1 knockdown. One would expect that such treatment would be protective rather than apoptosis-inducing.

Reviewer #2:

The comparison of WT-Kv1.3 and the CBDless Kv1.3 mutant (166-FQRQVWLLF-174 to 166-AQRQVGLLA-174) is the central focus of this paper. In an earlier paper (Sci Rep 2016;6:22453), the authors reported that the CBDless Kv1.3 mutant did not interact with caveolin-1 and exhibited reduced lipid raft portioning. In the present paper, the authors report that the CBDless Kv1.3 mutant preferentially accumulates in mitochondria, resulting in altered mitochondrial physiology and cell survival.

CBDless Kv1.3 mutant

• In Xenopus oocytes, the CBDless Kv1.3 mutant produced roughly 1/4th the current amplitude of WT Kv1.3. The authors conclude that the reduced current density is the consequence of reduced surface abundance. However, reduced current density could also be due to decreased production of the CBDless Kv1.3 mutant. A Western blot comparing Kv1.3 protein expression in Xenopus oocytes injected with WT Kv1.3 versus the CBDless mutant could exclude this possibility.

• In Xenopus oocytes, the CBDless triple mutation in the N-terminus alters voltage-dependence of activation and accelerates C-type inactivation (which involves changes in the outermost selectivity filter; J Gen Physiol 141: 151-160; Nat Struct Mol Biol. 2017; 24: 857-865). Clearly, the CBDless mutation impacts regions of Kv1.3 distant from the N-terminus. A more detailed electrophysiological characterization of the CBDless mutant channel is warranted.

• Since many studies are done on HEK293, B16F10 melanoma or Jurkat cells transfected with WT Kv1.3 or the CBDless Kv1.3 mutant, it is essential to show electrophysiology data for both channels in these cells. Mutations of 1 or 2 of the three aromatic residues in the CBD may be sufficient to reduce caveolin-1 binding and be less disruptive to the channel. Point mutations in the CBD of HCN4 are sufficient to alter caveolin-1 binding (J Mol Cell Cardiol. 2012;53:187-95).

• If Kv1.3's association with caveolin-1 is essential to localize Kv1.3 in lipid raft membrane microdomains in the plasma membrane, how do Kv1.3 channels in caveolin-1 deficient Jurkat cells translocate to the plasma membrane and generate normal Kv1.3 currents (J Biol Chem. 2001;276:12249-56)? In Jurkat cells transfected with WT Kv1.3 versus CBDless Kv1.3 channels, is the Kv1.3 membrane-to-mitochondrial ratio, mitochondrial morphology and mitochondrial function different?

MitoKv1.3, mitochondrial dysfunction, apoptosis

• In Figure 3, please show membrane Kv1.3 expression normalized to Na-K-ATPase in cells transfected with WT Kv1.3 or CBDless Kv1.3. This could be correlated with Kv1.3 channel density determined electrophysiologically.

• In Figure 6C, one Kv1.3-spot is shown in HEK293 cells transfected with WT Kv1.3, and in Figure 6E, one Kv1.3-spot is shown in HEK293 cells transfected with CBDless Kv1.3. Wouldn't you expect more Kv1.3 channels spots in the mitochondria? In Figure 7C, Kv1.3 is shown inside the organelle and not at the inner mitochondrial membrane. Do you have any images showing Kv1.3 and caveolin-1 in the inner mitochondrial membrane?

• If caveolin-1's interaction with mitoKv1.3 alleviates the pro-apoptotic activity of mitochondrial Kv1.3, caveolin-1 deficient Jurkat cells (that contain mitoKv1.3 unbound to caveolin-1) should be more prone to apoptosis than caveolin-1 and Kv1.3 containing cells, and over-expression of caveolin-1 in Jurkat cells should suppress apoptosis. This might be worth testing.

• Is increased apoptosis in 3T3-L1 cells following caveolin-1 knockdown due to the pro-apoptotic effects of mitoKv1.3 (as you suggest), or is it due to p53-p21-dependent induction of mitochondrial dysfunction and cellular senescence caused by caveolin-1 deficiency (Aging Cell 2017;16:773-784)? One could distinguish between these possibilities by over-expressing Kv1.3 in 3T3-L1 cells such that there is an excess of mitoKv1.3 over caveolin-1. If apoptosis is enhanced, it would suggest that mitoKv1.3 is a driver of apoptosis.

• Human T cells express caveolin-1, and caveolin-1 deficiency in T cells reduces effector function (J Immunol. 2017; 199: 874-884). Based on your results, caveolin-1 deficient primary human T cells would be predicted to express less surface Kv1.3 and more mitoKv1.3, exhibit altered mitochondrial morphology and physiology, and be more susceptible to apoptosis. This might be worth testing.

• Accumulation of Kv1.3 in the ER and Golgi when CBD is mutated (Figure 2D) may affect the physiology of these organelles. The authors may wish to address/discuss this.

Reviewer #3:

Capera et al. cover an interesting topic in their manuscript, which reports a novel role of the interaction between caveolin 1 and Kv1.3 channels. The Authors present a detailed analysis on the possible function of caveolin and Kv1.3 in cellular processes such as apoptosis. They revealed that mitochondrion associated Kv1.3 without caveolin 1 binding domain is pro-apoptotic, WT Kv1.3 show colocalization with caveolin in the mitochondrial inner membrane, contributes to the mitochondrial dysfunction via destructing the structure of cisterna. Authors concluded that Kv1.3-caveolin 1 "crosstalk" can sensitize the cancer cells to apoptosis. Based on these my questions and comments are the following:

1. Authors in a series of experiments use Cav1 knock-down HEK cell line. However, I did not find in the manuscript if they applied it in the mitochondrial function and apoptosis experiments. As all the experiments related to Cav1-driven Kv1.3 targeting I suppose it would be practical to use a cell line lacking endogenous Cav1 and then "re-add" Cav1 with transfection of Cav1 plasmid e.g. for organelle-localization experiments, mitochondrial location, apoptosis.

2. For electrophysiology Authors chose oocytes instead of HEK cells. Why did not the authors show data which were measured in HEK cells? In their previous paper the authors demonstrated that in Cav1- HEK cells the Kv1.3 channel had slower inactivation kinetics than in those with Cav1 expression, and not a drastic reduction in the current happened upon Cav1 knock-down. Here the data show that prevention of Cav1 binding to Kv1.3 channels results in a drastic change. I think these outcomes, at least in part, are contradictory. Do the Authors have any explanation for that? Is it possible that mutation eliminated the interaction between the channel and another (not Cav1) protein necessary for PM targeting?

3. For the TEM images the authors mentioned that Kv1.3 channels are localized to mitochondria detected using the immunogold labeling technique. I could observe only one-two gold beads/mitochondria (or organelles), which I suppose is unexpectedly low as Kv1.3 protein was overexpressed (and also large fraction of channels target to mitochondria). On the other hand, I wonder if any specific markers for organelles were applied to identify them.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "A novel mitochondrial Kv1.3-caveolin axis controls cell survival and apoptosis" for further consideration by eLife. Your revised article has been evaluated by Kenton Swartz (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. One of the claims made in the paper: " interactions between caveolin and Kv1.3 are essential for apoptosis" seems slightly overstated. We recommend the authors to soften this claim and discuss alternative possibilities. Their work clearly provides evidence for improper trafficking of Kv1.3 CBDless to the plasma membrane and ectopic expression in mitochondria, resulting in apoptosis. They have also shown that caveolin is involved in modulating apoptosis. Previous work from their lab has shown caveolin interacts with Kv1.3 and increases C-type inactivation and fails to interact with CBDlessKV1.3. It remains unclear whether there is any direct interaction/cross-modulation between Kv1.3 and caveolin in the mitochondria, causing apoptosis, and that requires more direct evidence to support the claim.

There is no doubt that both caveolin and Kv1.3 are involved in apoptosis and apoptosis IS dependent on Kv1.3-caveolin axis, but the direct interaction between these two proteins in the mitochondria warrants further investigations. The experimental evidence provided in the manuscript are suggestive of such an interaction in mitochondria and resulting apoptosis, but it is not definitive. Addressing it in the discussion will benefit the readers.

2. Regarding the electrophysiological properties of the mutant channel, against the view of the authors and even though the construct appears toxic to mammalian cells and the experiments need to be done in oocytes, if the mutant activates „earlier", it is in our view more active at any potential. Also, in oocytes, if the transport to the membrane is altered, then it would be expected that the currents are smaller. A decrease in the input resistance of the oocyte would also rather indicate a "higher" basal activity. If we understand correctly, the role of Kv1.3 in apoptosis relies on the fact that reduction of its activity through bax blockade triggers ∆ψm hyperpolarization and then apoptosis, so it is counterintuitive how a "more active" channel is proapoptotic. Importantly, how activation of Kv1.3 happens with the values of ∆ψm in the context of mitochondria (140 mV negative to the activation potential of Kv1.3) remains an unanswered question. We think that this issue merits a few words in the discussion. Moreover, Cav1 maintains mitochondrial depolarization, as its knockdown induces hyperpolarization. This is against what one would expect if it acts partly through Kv1.3. The concept "Kv1.3-mediated cell death" is somewhat misleading if Kv1.3 blockade is what triggers apoptosis. Why does then mitoKv1.3 overexpression promote apoptosis? We might well have got the point wrong, but we think other readers would have the same problem.

3. Disregarding the fact that Kv1.3 overexpression on its own reduces respiration by almost one-half (Figure 5), the mitochondrial fragmentation induced by Kv1.3CBDless deserves some comments in this context.

Reviewer #1:

This manuscript addresses the importance of Cav1 interaction for Kv1.3 targeting to the plasma membrane and the relevance of the interaction for the impact of Kv1.3 in apoptosis. In this revised version, the authors have provided further evidence of the specificity of the effect. Nevertheless, I have still some substantial questions that the authors probably can address by changing the wording of their statements.

Regarding the electrophysiological properties of the mutant channel, against the view of the authors and even though the construct appears toxic to mammalian cells and the experiments need to be done in oocytes, if the mutant activates „earlier", it is in my view more active at any potential. Also, in oocytes, if the transport to the membrane is altered, then it would be expected that the currents are smaller. A decrease in the input resistance of the oocyte would also rather indicate a "higher" basal activity. If I understand correctly, the role of Kv1.3 in apoptosis relies on the fact that reduction of its activity through bax blockade triggers ∆ψm hyperpolarization and then apoptosis, so it is counterintuitive how a "more active" channel is proapoptotic. Importantly, how activation of Kv1.3 happens with the values of ∆ψm in the context of mitochondria (140 mV negative to the activation potential of Kv1.3) remains an unanswered question. I think that this issue merits a few words in the discussion. Moreover, Cav1 maintains mitochondrial depolarization, as its knockdown induces hyperpolarization. This is against what one would expect if it acts partly through Kv1.3. The concept "Kv1.3-mediated cell death" is somewhat misleading if Kv1.3 blockade is what triggers apoptosis. Why does then mitoKv1.3 overexpression promote apoptosis? I might well have got the point wrong, but I think other readers would have the same problem.

Disregarding the fact that Kv1.3 overexpression on its own reduces respiration by almost one-half (Figure 5), the mitochondrial fragmentation induced by Kv1.3CBDless deserves some comments in this context.

Figure S6. This is indeed a very nice control. However, it is very hard to assess how efficiently YMVIii is targeted to the mitochondria. Loss of membrane targeting would, in any case, increase Pearsons's correlation against any intracellular marker. It would be much more convincing if the copurification experiments in Figure 3 or 7 would be reproduced here.

Reviewer #2:

The authors have presented an elegant work on the role of Kv1.3 in cellular homeostasis. Authors clearly demonstrated that CBDless Kv1.3 failed to localize on the plasma membrane. The work highlights the significance of interactions between Kv1.3 and caveolin in cellular apoptosis. The data presented supports the notion that improper trafficking of Kv1.3 to plasma membrane can result in ectopic expression in the mitochondrial membrane, which can dysregulate protein interactions, causing apoptosis.

In my opinion, the authors have addressed all the reviewers comments and the manuscript is ready to be accepted for publication. No further edits are required. Congratulations to all the authors for an intensive work presented in the manuscript.

This is one minor suggestion to the authors for future work (and is not required for this manuscript): To test the currents in oocytes where caveolin and Kv1.3 are co-expressed and compare it with CBDless clones (with and without the co-expression of caveolin). This might provide more direct evidence into Kv1.3 modulation by caveolin. This, of course, might not address their role pertaining to mitochondrial functions, as oocyte expression will be in the plasma membrane, but will certainly provide insights into functional interactions. Lack of interaction between CBDless Kv1.3 and caveolin in oocytes will provide validation that many reviewers raised. Again, such intensive study is outside the scope of current manuscript and is merely suggested here for future work.

https://doi.org/10.7554/eLife.69099.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

This manuscript described the effects of the ablation of the Caveolin 1 (Cav1) interaction site in Kv1.3. on subcellular distribution, functional properties and effects on apoptosis. The main conclusion of the paper is that the interaction between Kv1.3 and Cav1 plays a crucial role not only in the proper plasma membrane localization of Kv1.3, resulting in an enrichment of mitochondrial localization, but that the interaction with Cav1 at the mitochondria also modulates the induction of apoptosis. Such an effect would justify the proposal of a Cav1-Kv1.3 axis, and would be a very relevant finding adding to the emerging importance of both Cav1 and Kv1.3 in physiology and pathology, especially in cancer.

The main concern is the possibility that an "overload" of the mitochondria with either wild type or CBDless Kv1.3 is responsible for the observed effects.

First of all, we apologize for the delay answering the reviewers concerns. The initial and total lockdown in both Spain and Italy plus further partial lockdowns during the last months have made very difficult to address all requests in due time.

Thank you very much for your kind review considering our findings very relevant to physiology and pathophysiology of, especially but not limited to, cancer cells. Indeed one of the most relevant findings has been the identification of the essential role of the mitochondrial Cav/Kv1.3 association controlling cell survival and apoptosis.

Although the overload of either Kv1.3 could exert some observed effects, this is unlikely because Kv1.3 WT sensitizes to apoptotic stimuli rather than generating apoptosis by itself, with no changes in mitochondrial morphology in the presence of caveolin (Figure 4-7). Only the CBDless mutant triggers massive apoptosis (Figure 4 and 6); thereby arguing against the observation of the Reviewer. To further examine this point, we performed new experiments using primary human T-cells and 3T3 fibroblasts, where we modulated only expression of caveolin (but not of Kv1.3). These new data further support our findings (Figure 7-10). Importantly, additional, new data exploiting the YMVIii mutant that, being intracellular retained (Martinez-Marmol et al., 2013; J Cell Sci 126, 5681-5691), partially shares CBDless intracellular localization and also targets to mitochondria, but preserves cav 1 interaction, did not generate apoptosis (Supplemental Figure 6).

1. Whereas the enrichment in mitochondrial Kv1.3 in CBDless channels leaves little doubt, to what extent that interaction at the mitochondria is important for apoptosis is less clear. Kv1.3 wild type overexpression induces an increase of apoptosis on its own.

Kv1.3, as mentioned above, sensitives to apoptosis rather generates apoptosis (see Figure 4-7). The Reviewer is right that in HEK293 cells (see Figure 6A), the % of apoptotic cells is relatively high after expression of WT Kv1.3, but it is significantly less than in the case of cells expressing Kv1.3 CBDless. Please note also that in Suppl. Figure 5, where the B16F10 melanoma cells were first sorted following transfection and then treated with pro-apoptotic stimuli (or left untreated), the % of apoptotic cells is again much higher in the case of the cells expressing the mutant channels. Importantly, even in the HEK293 transfected with Kv1.3 WT, the cells look healthy and mitochondria maintained correct cristae organization (Figures 6 and 7) and the mitochondrial membrane potential as well the maximal respiration are similar to that observed in control cells (Figure 5). In addition, results obtained with the Kv1.3 YMVIii mutant also supports our claims (Supplemental Figure 6).

If Cav1 binding-defective mutants are more abundant at the mitochondria, this alone could explain the increase in apoptosis described.

The channel mitochondrial accumulation is not exclusively responsible for the apoptotic effects. Indeed, YMVIii mutant reroutes to mitochondria but generates no apoptosis (Supplemental Figure 6). In addition, the expression of WT Kv1.3 in T-cells (Jurkat and primary T-lymphocytes, low cav) promotes similar apoptosis than that caused by Kv1.3 CBDless, but the introduction and/or the endogenous expression of Cav partially protects cells from apoptosis in Kv1.3WT but not in CBDless (Figure 7 and 10. The ablation of cav 1 in 3T3 fibroblasts with no alteration of Kv1.3 further supports our data (Figure 7). Therefore, all these data strongly suggest that the apoptosis is mostly related to the absence of cav/Kv1.3 association rather than to the unique expression of Kv1.3 by itself. In fact, caveolin by itself is claimed to be anti-apoptotic (just few examples: Yang et al., Cancer Invest. 2012, 30:453-462; Codenotti et al., Cancer Lett. 2021, 505:1-12; Aberg et al., Apoptosis. 2020, 25:519-534).

The effects reported in both Jurkat and 3T3-L1 could in principle be also explained if the excess of Kv1.3 is equally targeted to mitochondria in Jurkat cells regardless of CBD due to the low Cav1 level, and if knockdown of Cav1 reduces PM Kv1.3 in preadipocytes and therefore increases mitoKv1.3 (at least it seems to alter the ratio between mature and non-glycosylated channel, Figure 7F).

Yes, the reviewer is correct. When Kv1.3 does not associate with cav, the channel impairs membrane expression (lower non-glycosylated band). However, the level of Kv1.3 rerouting to mitochondria is not proportional to the level of apoptosis. In addition, the new YMVIii mutant data, which targets to mitochondria but associates with caveolin, clearly supports that the level of Kv1.3 channel in mitochondria is not the unique cause (supplemental Figure 6). Thus, the mitoKv1.3 association with caveolin rather than the level of mitoKv1.3 by itself should be the responsible of such effect. Furthermore, we have incorporated data with fresh human primary T-cells, which naturally lack expression of Cav. In such cells, both Kv1.3 channels (WT and CBDless) generate similar level of basal apoptosis, but the introduction of external caveolin partially protects the cells when the channel can interact with Cav, i.e. only in Kv1.3 WT (Figure 10). These data therefore further support Jurkat and 3T3-L1 results (Figure 7).

2. As the authors state, it appears that the reduced PM activity of Kv1.3CBDless is due to trafficking defects (less targeting to the membrane and shorter half-life). Actually, the mutant is activated at less depolarized potentials, suggesting a more favorable transition to an open state. Inhibition of mitoKv1.3 by Bax or pharmacological agents triggers apoptosis in several cell types. It is therefore counterintuitive, or at least it is not directly explained in the manuscript, that overexpression of a variant of the channel, which is in fact "more active" than the wild type induces more apoptosis, unless the effect is different from the physiological role of mitoKv1.3.

To our knowledge, the fact that CBDless activates earlier does not mean that is more active. In fact, the current generated by Kv1.3 CBDless is much lower. The electrophysiology was performed in oocytes because the plasma membrane of mammalian cells was severely altered by the Kv1.3 CBDless-related massive apoptosis. Therefore, trying to establish a close relationship between channel kinetics and physiology is misleading. Because we could not perform patch clamp in CBDless-transfected mammalian cells, we analyzed the lipid composition of the PM under apoptosis. CBDless dependent apoptotic cells exhibited a reduction in membrane cholesterol (12%), phosphatidylserine + phosphatidylinositol (10%) and sphingolipids (5%), which severely affects the membrane integrity to be patch-clamped. Seals broke and cells died. Voltage clamp studies in oocytes solely demonstrate a functional channel which generates tetramers despite altering an important motif lying close the Shaker tetramerization domain. In fact, we also saw important effects on the membrane resistance of oocytes (input resistance, Supplemental Figure 3), which further supports the weakness of the membrane in severely apoptotic mammalian cells (see Figure 6D). Our results demonstrate that CBDless-injected oocytes displayed about half membrane resistance than WT (Supplemental Figure 3). These changes in membrane (HEK and oocytes) are concomitant to important losses of membrane integrity during apoptosis.

On our opinion the data presented here do not contradict the well-delineated mechanism by which mitoKv1.3 block triggers apoptosis. We here further decipher a new mechanism involving Kv1.3, which does not argue against the previous one and further reinforces the role of mitoKv1.3 in this important process. The mitoCav, acting as antiapoptotic (as widely demonstrated) regulates the mitoKv1.3-dependent apoptotic effects. The possibility of a competition between the Bax effect and the Cav effect is feasible and would fine-tune the mitoKv1.3-related apoptotic control under different insults, but addressing this point would be far beyond the scope of the present manuscript.

3. It would be extremely interesting to know what the effect of mitochondria-targeted Kv1.3 inhibitors like the ones described previously by some of the authors have on cells overexpressing Kv1.3 (wild type and CBDless), and if such agents can counteract the effect of Cav1 knockdown. One would expect that such treatment would be protective rather than apoptosis-inducing.

We thank the reviewer for this inquire. However, we do not fully understand her/his claims in the context of our work. We humbly think that the reviewer misunderstands the CBDless activity (see above). Anyway, The PAPTP mitoKv1.3 inhibitor has been used as requested. However, the data generates misunderstandings as expected. As previously published, PAPTP (Leanza et al., 2017, Cancer Cell 31, 516–531) triggered apoptosis in the Kv1.3 expressing cells similarly to the intrinsic apoptosis inducer staurosporine. As previously published, cells with low activity of Kv1.3, such as primary human fibroblasts, MSCs and, in our case, with CBDless channel, PAPTP has little effects. As abovementioned, we claim that the CBDless channel is not more active. Basal apoptosis in the CBDless-expressing cells was extremely high; thereby, the slightly increase of apoptosis triggered by PAPTP, although logical, is difficult to interpret. We do not understand why PAPTP should counteract the apoptosis in CBDless. Furthermore, the absence of caveolin-Kv1.3 association would further increase apoptosis because caveolin should be contemplated as anti-apoptotic in many cell models.

The effectivity of mitoKv1.3 inhibitors, such as PAPTP, might be influenced by different factors. Thus, (i) the activity and expression of Kv1.3 channels (Arcangeli et al., 2009; Leanza et al., 2013); (ii) cells with elevated Kv1.3 exhibit a hyperpolarized IMM (Hockenbery, 2010); (iii) redox state (e.g., Sabharwal and Schumacker, 2014) and (iv) membrane permeability to molecules (such as PAPTP) increase oxidative stress above a critical threshold triggering apoptosis. In fact, loss of mitochondrial membrane potential (ΔΨm) has been shown to be an early event during apoptosis in some systems. Thus, we show that both channels (WT and CBDless) triggered hyperpolarization in both HEK 293 and oocytes (Figure 5 and Suppl Figure 3).

The main issue of our manuscript deals with the association of Kv1.3 with caveolin rather than the Kv1.3CBDless activity by itself. Therefore, many uncertain inputs, such as changes in membrane lipids, massive apoptosis, minor membrane resistance and more permeable plasma membrane in CBDless channel, indicate that raising conclusions about blocking channel activity should be discarded. However, our data is in accordance with what it has been described before – PAPTP triggers apoptosis – and the massive apoptosis generated by the CBDless channel slightly increased nor decreased by PAPTP. In fact as abovementioned, the CBDless channel is not more active; thereby, the data is much debatable. In this scenario, although we include this data in Author response image 1 for the internal report evaluation, the inclusion of this uncertain data in the main body of the manuscript would be unclear. We humble demand not to be included in the manuscript because would raise confusion.

Author response image 1

Reviewer #2:

The comparison of WT-Kv1.3 and the CBDless Kv1.3 mutant (166-FQRQVWLLF-174 to 166-AQRQVGLLA-174) is the central focus of this paper. In an earlier paper (Sci Rep 2016;6:22453), the authors reported that the CBDless Kv1.3 mutant did not interact with caveolin-1 and exhibited reduced lipid raft portioning. In the present paper, the authors report that the CBDless Kv1.3 mutant preferentially accumulates in mitochondria, resulting in altered mitochondrial physiology and cell survival.

First of all, we apologize for the delay answering the reviewers concerns. The initial and total lockdown in both Spain and Italy plus further partial lockdowns during the last months have made very difficult to address all requests in due time.

Thank you very much for your kind report. We agree that this is indeed one message in the paper, which, however additionally highlight for the first time that mitoKv1.3 association to caveolin is able to fine-tune cell survival and apoptosis. Caveolin has been claimed as antiapoptotic protector (just few examples: Yang et al., Cancer Invest. 2012, 30:453-462; Codenotti et al., Cancer Lett. 2021, 505:1-12; Aberg et al., Apoptosis. 2020, 25:519-534) but we here show that it functions by specific associations with a novel target, mitoKv1.3.

CBDless Kv1.3 mutant

• In Xenopus oocytes, the CBDless Kv1.3 mutant produced roughly 1/4th the current amplitude of WT Kv1.3. The authors conclude that the reduced current density is the consequence of reduced surface abundance. However, reduced current density could also be due to decreased production of the CBDless Kv1.3 mutant. A Western blot comparing Kv1.3 protein expression in Xenopus oocytes injected with WT Kv1.3 versus the CBDless mutant could exclude this possibility.

The reviewer is correct. Although our data indicated that the main reason triggering lower activity is the intracellular retention, following reviewer’s suggestion, we now include a Western blot comparing Kv1.3 protein expression in Xenopus oocytes injected with WT Kv1.3 versus the CBDless mutant (Supplemental Figure 3A). Protein expression demonstrated that the level of protein in CBDless-injected oocytes is similar to that of the WT channel.

• In Xenopus oocytes, the CBDless triple mutation in the N-terminus alters voltage-dependence of activation and accelerates C-type inactivation (which involves changes in the outermost selectivity filter; J Gen Physiol 141: 151-160; Nat Struct Mol Biol. 2017; 24: 857-865). Clearly, the CBDless mutation impacts regions of Kv1.3 distant from the N-terminus. A more detailed electrophysiological characterization of the CBDless mutant channel is warranted.

While we acknowledge the useful suggestion of the Reviewer, we feel that being the main aim of the present work that of deciphering whether the association of Kv1.3 and caveolin influences cell survival and apoptosis, the electrophysiological characterization of the CBDless mutant is not strictly related to this story. Nonetheless, as requested by the reviewer, we introduced into this version new biophysical data from the CBDless mutant channel (Supplemental Figure 3). This include membrane potential, membrane resistance, conductance and cumulative inactivation analysis. The rationale for including a partial electrophysiological characterization of the CBDless mutant was the fact that we were concerned about the possibility that modifying the CBD, next to the tetramerization domain, could impair the oligomerization of the channel. In our previous version, we analyzed few parameters to demonstrate that the CBDless was functional in terms of tetramerization and conductivity. The effects for a limited caveolin interaction were addressed previously as you mention above (Sci Rep 2016;6:22453). In fact, Kv1.3CBDless-exressing cells undergo severe apoptosis and changes in membrane lipid composition and membrane integrity are evident (Figure 6). Because of that, patch-clamp was impossible to achieve in mammalian culture cells. We analyzed the lipid composition of Kv1.3CBD-less HEK cell membrane and they exhibited about 12% cholesterol, 10% phosphatidylserine + phosphatidylinositol and 5% sphingolipids less in their membranes than Kv1.3 WT cells. This clearly will affect the integrity of the membrane as well as the kinetic of channels (Cholesterol regulation of Ion channels and receptors, I. Levitan and F.J. Barrantes eds. 2012. DOI: 10.1002/9781118342312. John Wiley and Sons, Inc). The data is concomitant with a 50% decrease in membrane input resistance of Kv1.3 CBDless-injected oocytes. In this scenario, with such amount of inputs affecting electrophysiological parameters, we honestly think that raising conclusions from those results would be extremely uncertain. Indeed, we mention when describing the newly introduced biophysical parameters that any biophysical conclusion from this CBDless mutant should be taken with caution.

• Since many studies are done on HEK293, B16F10 melanoma or Jurkat cells transfected with WT Kv1.3 or the CBDless Kv1.3 mutant, it is essential to show electrophysiology data for both channels in these cells.

We initially desired to perform the electrophysiology in our mammalian cell models rather than in oocytes. However, as abovementioned, Kv1.3CBDless triggers important changes at the cell physiology and membrane integrity. First, membrane cholesterol and sphingolipids decreased, triggering membrane weakness impeding patch clamp experiments. Therefore, seals were extremely difficult to achieve and cells died just applying the whole cell configuration. Second, severe apoptotic cells exhibit an extremely sensitive cell membrane integrity (see input resistance oocytes, Supplemental Figure 3). This is clearly observed in electron micrographs throughout the manuscript (i.e. Figure 6). We do not know whether the former or the last goes first but, this clearly impeded the study. Finally, we chose oocytes because, under these circumstances, they exhibit an extremely resistant membrane for the validation of channel tetramerization and functionality. To sum up, mammalian cell models were not suitable for electrophysiology with the CBDless mutant.

Mutations of 1 or 2 of the three aromatic residues in the CBD may be sufficient to reduce caveolin-1 binding and be less disruptive to the channel. Point mutations in the CBD of HCN4 are sufficient to alter caveolin-1 binding (J Mol Cell Cardiol. 2012;53:187-95).

Although the reviewer is correct regarding HCN4, in our case what matters is the disruption of caveolin association with Kv1.3 rather than a disruption of the channel activity (or protein). In fact, the CBDless channel tetramerizes (Supplemental Figure 2), is functional (Supplemental Figure 3) and associates to Kvβ subunits (see Author response image 2). That interacts with Kv1 channels at a site that lies next to the CBD.

Author response image 2

Consensus sequences for the caveolin binding domains are: ΦXΦXXXXΦ,ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ = Trp, Phe, or Tyr (Couet et al., J Biol Chem. 1997;272(10):6525-33). As suggested by the reviewer, we have performed the experiments, which include mutations of 1 or 2 residues (new Supplemental Figure 1). Only when the second cluster of aromatic residues was substituted, the association to caveolin is impaired. However, both clusters seem to cooperatively participate in the Kv1.3/cav colocalization. We believe that although the second cluster mutant triggers minor caveolin/Kv1.3 association, the data do not alter the significance of our claims.

• If Kv1.3's association with caveolin-1 is essential to localize Kv1.3 in lipid raft membrane microdomains in the plasma membrane, how do Kv1.3 channels in caveolin-1 deficient Jurkat cells translocate to the plasma membrane and generate normal Kv1.3 currents (J Biol Chem. 2001;276:12249-56)?

The reviewer is correct and this issue opens an exciting debate. This is one of our current laboratory goals. Our working hypothesis is that in cells, with a low expression of caveolin, other post-translational mechanisms or protein interactions could take the lead. We are currently deciphering the role of these mechanisms on the Kv1.3-dependent physiology in lymphocytes but addressing this issue in the present manuscript is beyond the scope in our opinion.

In Jurkat cells transfected with WT Kv1.3 versus CBDless Kv1.3 channels, is the Kv1.3 membrane-to-mitochondrial ratio, mitochondrial morphology and mitochondrial function different?

This is a very relevant question. In addition, some additional inquiries from the editorial report demanded experiments in primary human T lymphocytes. Therefore, to concentrate our efforts, in order to answer this query, we preferred to use freshly isolated human T lymphocytes rather than a cell line. These, more physiological data, shows that in fresh blood human T-lymphocytes, which do not express caveolin, Kv1.3 WT and Kv1.3CBDless channels triggered similar effects. Thus, the membrane and mitochondrial colocalization (confocal studies, Figure 8), the mitochondrial morphology (confocal studies, Figure 9) and the mitochondrial function by tetramethylrhodamine, methyl ester (TMRM) dye (mitochondrial membrane potential, Figure 10) were similar with both channels. In addition, annexin V (apoptosis) showed that both channels triggered similar cell apoptosis but, when caveolin was introduced in T-lymphocytes, apoptosis was partially counteracted in cells expressing Kv1.3 WT (Figure 10 B). Furthermore, new data with a selected Jurkat cell line that endogenously expresses caveolin (Figure 7) further confirms that the presence of caveolin triggers certain protection against apoptosis with Kv1.3WT but not with CBDless.

MitoKv1.3, mitochondrial dysfunction, apoptosis

• In Figure 3, please show membrane Kv1.3 expression normalized to Na-K-ATPase in cells transfected with WT Kv1.3 or CBDless Kv1.3.

We thank the reviewer for this suggestion. A new panel in Figure 3B has been incorporated as required.

This could be correlated with Kv1.3 channel density determined electrophysiologically.

We humbly apologize, but this correlation cannot be obtained because it was impossible to perform electrophysiology on mammalian cells expressing Kv1.3 CBDless (see above).

• In Figure 6C, one Kv1.3-spot is shown in HEK293 cells transfected with WT Kv1.3, and in Figure 6E, one Kv1.3-spot is shown in HEK293 cells transfected with CBDless Kv1.3. Wouldn't you expect more Kv1.3 channels spots in the mitochondria?

The Reviewer is correct, however in the same figure (H), there are two spots in mitochondria of HEK293 cells transfected with CBD-less Kv1.3. Figure 7C also shows two spots for Kv1.3 and only one spot for Cav, while both proteins are well-detectable in purified mitochondria by Western blot. We have to take into consideration that in the TEM images, using a very limited dilution of antibody, as we did use, we assure that the staining is specific. More staining often represents increase in background, false labeling and poor structural preservation.

In Figure 7C, Kv1.3 is shown inside the organelle and not at the inner mitochondrial membrane. Do you have any images showing Kv1.3 and caveolin-1 in the inner mitochondrial membrane?

New images have been added as requested.

• If caveolin-1's interaction with mitoKv1.3 alleviates the pro-apoptotic activity of mitochondrial Kv1.3, caveolin-1 deficient Jurkat cells (that contain mitoKv1.3 unbound to caveolin-1) should be more prone to apoptosis than caveolin-1 and Kv1.3 containing cells, and over-expression of caveolin-1 in Jurkat cells should suppress apoptosis. This might be worth testing,

We want to thank the reviewer for this inquiry. Evidence in the literature suggest that some leukocytes, that do not initially express caveolin, may express the protein under certain states of activation (Hatanaka et al., Biochem Biophys Res Commun 1998, 253: 382387). Indeed, we managed to isolate some Jurkat cells that started to express endogenously caveolin (most probably due to several freezing/thawing cycles they underwent due to the lock down). New data (Figure 7 D, E) with these Jurkat cav+ cells have been incorporated. As suggested by the reviewer, in Jurkat cells, which do express caveolin endogenously, the apoptosis was partially counteracted. However, it must be said that regular, primary Tlymphocytes are clearly defective in caveolin (see Figure 8). Indeed, new data from primary fresh human T-cells (Figure 8-10), which do not express caveolin, further support our claims. Similar to Jurkat, Kv1.3 WT and CBDless channels trigger similar results, but when caveolin 1 was introduced, as requested, only in WT-transfected cells, apoptosis was partially counteracted (Figure 10).

• Is increased apoptosis in 3T3-L1 cells following caveolin-1 knockdown due to the pro-apoptotic effects of mitoKv1.3 (as you suggest), or is it due to p53-p21-dependent induction of mitochondrial dysfunction and cellular senescence caused by caveolin-1 deficiency (Aging Cell 2017;16:773-784)? One could distinguish between these possibilities by over-expressing Kv1.3 in 3T3-L1 cells such that there is an excess of mitoKv1.3 over caveolin-1. If apoptosis is enhanced, it would suggest that mitoKv1.3 is a driver of apoptosis.

We thank the reviewer for this suggestion. The reviewer is right that the paper published in Aging cell confers such a role to Cav. On the other hand, deficiency of caveolin itself may trigger mitodysfunction and cellular senescence. In fact, caveolin has been postulated as protective against apoptosis and cell death (just few examples: Yang et al., Cancer Invest. 2012, 30:453462; Codenotti et al., Cancer Lett. 2021, 505:1-12; Aberg et al., Apoptosis. 2020, 25:519534). In this context, we claim that the function of Kv1.3 should be considered within the association to caveolin. All our data support this claim and is concomitant with a partial role for caveolin as mentioned by the reviewer.

Unfortunately, 3T3-L1 cells are extremely resistant to transfection and the experiment suggested is hard to achieve. We tried several times to perform this experiment, but over-expression of Kv1.3 (chemical transfection or electroporation), getting healthy cells to work with, was impossible to achieve in our hands. However, merging the rest of experiments, we may suggest that the axis of interaction between Kv1.3 and caveolin, not just Kv1.3, is at the onset of the apoptotic response. Thus:

1. The overexpression of Kv1.3 CBDless, but not Kv1.3 WT, in HEK-293 and B16F10 melanoma cells, which accumulates in mitochondria, without caveolin association, triggers apoptosis.

2. The overexpression of Kv1.3 WT and CBDless in regular Jurkat (cav -) cells yielded similar apoptosis. However, the use of Jurkat (cav+) cells – see above – indicated that apoptosis was partially prevented solely in Kv1.3 WT, which has the capacity of caveolin association.

3. Similar results were obtained with fresh peripheral human T-lymphocytes. These native cells, as many leukocytes, do not express caveolin (Hatanaka et al., Biochem Biophys Res Commun 1998, 253: 382-387; Vallejo and Hardin, FASEB J. 2005, 19:586-587; Sawada et al., Blood. 2010, 115:2220-2230). The introduction of caveolin protected partially against apoptosis in Kv1.3 WT, but not in Kv1.3 CBDless.

4. In 3T3-L1 cells, caveolin may be ablated by shRNA, thereby an increase in the Kv1.3/caveolin ratio, which surely impairs the Kv1.3-caveolin association, results in more apoptosis. These results parallel T-cell data.

• Human T cells express caveolin-1, and caveolin-1 deficiency in T cells reduces effector function (J Immunol. 2017; 199: 874-884).

Although Borger et al. claim the expression of caveolin 1, caveolin 1 is only observed upon concentration by immunoprecipitation. In contrast, the defective expression of caveolin in lymphocytes has been reported in several papers by different groups (Hatanaka et al., Biochem Biophys Res Commun 1998, 253: 382387; Vallejo and Hardin, FASEB J. 2005, 19:586-587; Sawada et al., Blood. 2010, 115:22202230). It is true that Hatanaka et al., 1998 suggest that certain cell lines may express caveolin under certain states of activation. Our data agrees with that observation (see our new panel in Figure 7D). In fact, we detected, in a regular WB with no immunoprecipitation, a faint band of cav 1 (Figure 7D). Therefore, we mention that cav1 expression is limited. Anyway, Kv1.3 is much more abundant than caveolin. However, as abovementioned, we managed to select some Jurkat cav+ (Figure 7D) and results in this cell clone, as well as primary T-lymphocytes support our claim.

Based on your results, caveolin-1 deficient primary human T cells would be predicted to express less surface Kv1.3 and more mitoKv1.3, exhibit altered mitochondrial morphology and physiology, and be more susceptible to apoptosis. This might be worth testing.

As abovementioned, in deficient Jurkat T-cells (regular cav-) both channels (WT and CBDless) triggered similar level of apoptosis and this is partially counteracted by the endogenous expression of caveolin (Jurkat cav+) (Figure 7E). However, as suggested by the reviewer, we have performed several studies in freshly isolated primary human Tlymphocytes (Figure 8-10). In these T-cells, with no endogenous caveolin, apoptosis was partially counteracted in Kv1.3 WT-expressing cells, but not in CBDless cells, once cav was introduced. In addition, both Kv1.3 channels share similar location in primary T-cells with no caveolin (Figure 8). As mentioned by the reviewer, Pearson’s coefficient of colocalization is higher in mitochondria than membrane. However, this should be taken with caution because membrane and mitochondrial marker staining are quite different.

In addition, new data on primary human T-lymphocytes (Figure 9-11) demonstrate that the reviewer is correct and the expression of Kv1.3 triggers “altered mitochondrial morphology and physiology, and cells are more susceptible to apoptosis”. Overall, our data supports that the association Kv1.3/cav is important for fine-tuning the apoptosis susceptibility.

• Accumulation of Kv1.3 in the ER and Golgi when CBD is mutated (Figure 2D) may affect the physiology of these organelles. The authors may wish to address/discuss this.

Thank the reviewer for this suggestion. The increment of colocalization of Kv1.3CBDless with these organelles is a result of a massive increase of intracellular retention. However, Supplemental Figure 7 we show no ER stress. Anyway, in order to address this specific concern, in this new version, we have used a Kv1.3 mutant (YMVIii) that shows massive ER intracellular retention and does not reach the membrane surface (Martinez-Marmol et al., J Cell Sci. 2013, 126:5681-5691). The Kv1.3 YMVIii mutant, associates with caveolin, colocalizes within the ER (Martinez-Marmol et al., 2013), routes to mitochondria, targets no plasma membrane (Martinez-Marmol et al., 2013) and causes no apoptosis (Supplemental Figure 6D). Therefore, data obtained with this channel further support that the Kv1.3/caveolin association rather than the intracellular retention by itself is the main responsible for our claims. These results are introduced now as a Supplemental Figure 6.

Reviewer #3:

Capera et al. cover an interesting topic in their manuscript, which reports a novel role of the interaction between caveolin 1 and Kv1.3 channels. The Authors present a detailed analysis on the possible function of caveolin and Kv1.3 in cellular processes such as apoptosis. They revealed that mitochondrion associated Kv1.3 without caveolin 1 binding domain is pro-apoptotic, WT Kv1.3 show colocalization with caveolin in the mitochondrial inner membrane, contributes to the mitochondrial dysfunction via destructing the structure of cisterna. Authors concluded that Kv1.3-caveolin 1 "crosstalk" can sensitize the cancer cells to apoptosis. Based on these my questions and comments are the following:

Thank you very much for your kind report considering that our novel work covers an interesting topic undertaking a detailed study. We apologize for the delay answering the reviewers concerns. The initial and total lockdown in both Spain and Italy plus further partial lockdowns during the last months have made very difficult to address all requests in due time.

1. Authors in a series of experiments use Cav1 knock-down HEK cell line. However, I did not find in the manuscript if they applied it in the mitochondrial function and apoptosis experiments. As all the experiments related to Cav1-driven Kv1.3 targeting I suppose it would be practical to use a cell line lacking endogenous Cav1 and then "re-add" Cav1 with transfection of Cav1 plasmid e.g. for organelle-localization experiments, mitochondrial location, apoptosis.

We only used the HEK cav1- cell line when FRET between cav and Kv1.3 was analyzed (Figure 1D). The rest of the paper deals mostly with HEK-293 wt cells and lymphocytes. In fact, the HEK cav1- was not of interest in this work. This cell line was widely characterized in a previous work (Perez-Verdaguer et al., Sci Rep. 2016, 6:22453). As requested, we have introduced a set of new experiments with primary human T-cells, which have limited expression of cav, and introduced cav in order to evaluate what the Reviewer proposes. Importantly, our new data in primary human T-cells (Figure 8-10) and Jurkat (Figure 7) demonstrate that the presence of cav partially protects from apoptosis. In summary, taking into account all cellular models (HEK, B16F10 melanoma, Jurkat, primary human T-cells and NIH 3T3 L1 cells) allows us achieving relevant conclusions.

2. For electrophysiology Authors chose oocytes instead of HEK cells. Why did not the authors show data which were measured in HEK cells?

The aim of the present work was to decipher whether the association of Kv1.3 and caveolin influences cell survival and apoptosis. We initially wanted to perform the electrophysiology in our mammalian cell models rather than in oocytes. However, as abovementioned, Kv1.3CBDless generates important changes at the membrane physiology and integrity triggering membrane weakness impeding patch clamp experiments.

As mentioned above, CBDless cells undergo severe apoptosis and changes in membrane lipid composition and membrane integrity are evident. Because of that, and patch-clamp being impossible to achieve in mammalian culture cells, we analyzed the cholesterol and sphingolipid concentration of Kv1.3CBDless HEK cells. CBDless cells exhibited a reduction of 12% cholesterol, 10% phosphatidylserine + phosphatidylinositol and 5% sphingolipids in their membranes. This clearly will affect the integrity of the membrane as well as the kinetic of channels (Cholesterol regulation of Ion channels and receptors, I. Levitan and F.J. Barrantes eds. 2012. DOI: 10.1002/9781118342312. John Wiley and Sons, Inc).

Probably because of this reason, seals were extremely instable and cells died just applying the whole cell configuration. To sum up, mammalian cell models were not suitable for electrophysiology.

Our choice of using oocytes was driven by the observation that these cells exhibit an extremely resistant membrane for the validation of channel tetramerization and functionality. In any case, the rationale for the electrophysiological characterization of the CBDless mutant was the fact that altering the CBD, lying next to the tetramerization domain, could impair the oligomerization and functionality of the channel. Therefore, we wanted to exclude the possibility that Kv1.3 CBDless gives rise to a non-functional channel, but the detailed electrophysiological characterization of this mutant was not the primary scope of the current study. In our previous version, we analyzed few parameters to demonstrate that the CBDless was functional in terms of tetramerization and conductivity. The effects of a limited caveolin interaction of the channel on the electrophysiological parameters of the CBDless mutant were addressed previously (Perez-Verdaguer et al., Sci Rep 2016;6:22453).

In their previous paper the authors demonstrated that in Cav1- HEK cells the Kv1.3 channel had slower inactivation kinetics than in those with Cav1 expression, and not a drastic reduction in the current happened upon Cav1 knock-down. Here the data show that prevention of Cav1 binding to Kv1.3 channels results in a drastic change. I think these outcomes, at least in part, are contradictory. Do the Authors have any explanation for that?

The Reviewer raises and interesting point that is currently under study in our laboratory. In our previous study, knock-down of Cav1 still allowed Kv1.3 to reach the plasma membrane, at least partially. Here we show that the same is true for the Kv1.3 CBDless, where there is no interaction at all with Cav1, and thus the intracellular retention of the mutant channel is further enhanced. As mentioned above, to establish a direct correlation to answer the question of the reviewer, the electrophysiological parameters should be studied in HEK293 cells, which is however impossible in the case of the CBD-less Kv1.3.

Is it possible that mutation eliminated the interaction between the channel and another (not Cav1) protein necessary for PM targeting?

The reviewer is correct and because the CBD lies close the tetramerization domain as well as to the Kvβ binding domain, we have checked both possibilities. Throughout the manuscript we show that Kv1.3CBD less mutant tetramerizes (Supplemental Figure 2) being functional (Supplemental Figure 3) and we now include for the internal reviewer’s evaluation some preliminary raw data showing that the CBDless mutant is also able to associate to Kvβ subunits. Although we have addressed the possible elimination of the channel interaction with the auxiliary subunit due to mutation, lying next to the CBD cluster, the possibility of any alternative protein remains open; therefore further studies need to be performed, that are out of the scope of the paper on our opinion. For this reason, we decided to show these data here to the Reviewer, but not to include it into the manuscript. Another important issue could be the interaction of Kv1.3 with lipids. The apoptosis generated by the CBDless mutant trigger an important decrease of membrane cholesterol. The effects of cholesterol on ion channels have been widely documented (Cholesterol on Ion channels, book, Levitan and Barrantes editors).

3. For the TEM images the authors mentioned that Kv1.3 channels are localized to mitochondria detected using the immunogold labeling technique. I could observe only one-two gold beads/mitochondria (or organelles), which I suppose is unexpectedly low as Kv1.3 protein was overexpressed (and also large fraction of channels target to mitochondria). On the other hand, I wonder if any specific markers for organelles were applied to identify them.

The reviewer is right, but please note that in the immunogold TEM, images of a single, tiny section of mitochondria are visible. Figure 7C shows two spots for Kv1.3 and only one spot for Cav, while both proteins are well-detectable in purified mitochondria by Western blot. We have to take into consideration that in the TEM images, using a very limited dilution of antibody, as we did use, we assure that the staining is specific. More staining often represents increase in background, false labeling and poor structural preservation.

[Editors’ note: what follows is the authors’ response to the second round of review.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. One of the claims made in the paper: " interactions between caveolin and Kv1.3 are essential for apoptosis" seems slightly overstated. We recommend the authors to soften this claim and discuss alternative possibilities. Their work clearly provides evidence for improper trafficking of Kv1.3 CBDless to the plasma membrane and ectopic expression in mitochondria, resulting in apoptosis. They have also shown that caveolin is involved in modulating apoptosis. Previous work from their lab has shown caveolin interacts with Kv1.3 and increases C-type inactivation and fails to interact with CBDlessKV1.3. It remains unclear whether there is any direct interaction/cross-modulation between Kv1.3 and caveolin in the mitochondria, causing apoptosis, and that requires more direct evidence to support the claim.

There is no doubt that both caveolin and Kv1.3 are involved in apoptosis and apoptosis IS dependent on Kv1.3-caveolin axis, but the direct interaction between these two proteins in the mitochondria warrants further investigations. The experimental evidence provided in the manuscript are suggestive of such an interaction in mitochondria and resulting apoptosis, but it is not definitive. Addressing it in the discussion will benefit the readers.

We thank the Reviewer for this important comment. Indeed, we agree that direct interaction of mitoKv1.3 with mitochondria-located caveolin is not proven in our experiments. Thus, as kindly suggested, our claims have been soften thoroughly the manuscript.

We have modified the Discussion accordingly and a new sentence has been included (p 11, l 16-19): “Although a direct interaction of these proteins in mitochondria has not been confirmed and warrants further investigation, evidence suggests that apoptosis is dependent on mitoKv1.3-caveolin functional axis”.

In addition, we have deleted the following sentence from the Discussion: “The severity of the Kv1.3-mediated events highly depends on its intramitochondrial association with Cav1.”

We have also modified the subheading (p 7) and a sentence in the Results section: “Our data indicated that altering the functional crosstalk between Cav1 and Kv1.3 plays a crucial role in determining the sensitivity of cells to apoptosis” (p 8, l 21-22). This statement emphasizes that interaction between mitoKv1.3 and mitochondria-located Cav1 is functional (not necessarily physical). To note that, functional crosstalk is supported by several observations reported in our work, including that, unlike Kv1.3 CBDless, Kv1.3 (YMVIii) partially locates to mitochondria, associates to Cav 1 (Suppl Figure 6C), but does not trigger relevant apoptosis (Suppl Figure 6D).

2. Regarding the electrophysiological properties of the mutant channel, against the view of the authors and even though the construct appears toxic to mammalian cells and the experiments need to be done in oocytes, if the mutant activates „earlier", it is in our view more active at any potential. Also, in oocytes, if the transport to the membrane is altered, then it would be expected that the currents are smaller. A decrease in the input resistance of the oocyte would also rather indicate a "higher" basal activity. If we understand correctly, the role of Kv1.3 in apoptosis relies on the fact that reduction of its activity through bax blockade triggers ∆ψm hyperpolarization and then apoptosis, so it is counterintuitive how a "more active" channel is proapoptotic.

We would like to emphasize that currents generated by the CBDless mutant should be taken with caution and mostly considered artefacts because the massive fail of cellular integrity. The electrophysiological characterization of Kv1.3 CBDless was undertaken exclusively to demonstrate that the mutant channel was functional because the CBD lies near the tetramerization domain. CBDless mutant currents are deeply affected by the massive cellular apoptosis, which affects the integrity of their plasma membrane and lipid composition (see Cholesterol Regulation of Ion Channels and Receptors, Levitan and Barrantes eds. John Wiley and Sons Inc; 2012).

Anyway, the reviewer is correct and the conductance/voltage curve of the Kv1.3 CBDless shifts to left indicating that the channel activates a more negative potentials (earlier activation). However, because the membrane expression is very different the K+ conductance at -40 mV (close to the resting membrane potential of oocytes) is similar for CBDless and WT (5.89 ± 1.20 μS, n=28, vs 5.93 ± 0.98 μS, n=23, for CBD-less and WT, respectively) what explains why the membrane potential of both groups was similar. These values cannot be correlated with input resistance because the input resistance is measured at -100/-80 mV and Kv1.3 is not active at the plasma membrane. Therefore, this value only indicates passive properties of oocyte membrane. The fact that the CBDless mutant exhibited a less input resistance is because the membrane is more leaky that WT because cells are sick (apoptosis).

Importantly, how activation of Kv1.3 happens with the values of ∆ψm in the context of mitochondria (140 mV negative to the activation potential of Kv1.3) remains an unanswered question. We think that this issue merits a few words in the discussion. Moreover, Cav1 maintains mitochondrial depolarization, as its knockdown induces hyperpolarization. This is against what one would expect if it acts partly through Kv1.3. The concept "Kv1.3-mediated cell death" is somewhat misleading if Kv1.3 blockade is what triggers apoptosis. Why does then mitoKv1.3 overexpression promote apoptosis? We might well have got the point wrong, but we think other readers would have the same problem.

Many thanks for calling our attention to the need of explaining better the somewhat complex physiology of mitochondrial channels. The Reviewer is correct that unfortunately it is still unknown how mitoKv1.3 can be active at the highly negative membrane potential found across the inner mitochondrial membrane (IMM) (around -180mV). As mentioned in the Introduction, what was shown is that acute inhibition of mitoKv1.3 causes IMM hyperpolarization, and this triggers ROS production with subsequent opening of the permeability transition pore (PTP). When PTP opens, this leads to loss of mitochondrial integrity, IMM depolarization, swelling and cytochrome c release (now we added this sentence to accentuate the consequences of PTP opening). PTP opening can be triggered, among other factors, either by ROS (triggered by hyperpolarization due to the chemical reduction of respiratory chain complexes) or by IMM depolarization above a certain threshold. Overexpression of K+ transporting pathways in mitochondria causes depolarization, as they allow the influx of depolarizing K+ into the matrix following the electrochemical gradient for this ion and cause mitochondrial depolarization as well as changes in ultrastructure (see e.g. Figure 3d, Paggio et al., Nature. 2019;572(7771):609-613.). Thus, acute changes in the membrane potential, upon block of the channel, induce ROS and PTP opening, while overexpression of mito-located Kv1.3, that is more prominent in the case of CBDless mutant or in the absence of Cav1 (for both WT and CBD-less Kv1.3), causes sustained depolarization that sensitizes the cells to apoptotic stimuli (Suppl Figure 5). PTP opening then further depolarizes IMM and reduces respiration due to swelling and loss of cytochrome c from the respiratory chain. Overexpression of WT or CBDless equally depolarizes mitochondria and causes apoptosis in primary T cells that lack endogenous Cav1. It has to be mentioned that while overexpression promotes apoptosis, the lack of the channel (WT Kv1.3) renders the cells resistant to apoptosis (Szabo et al., PNAS, 2008). This situation is similar to other ones, where the pharmacological block of a channel (e.g. KATP; Miki et al., Proc Natl Acad Sci U S A 1998;95(18):10402-6) does not yield the same effect of KO, as the former one triggers a series of signaling events that cannot be triggered in the cells lacking the channel. We now introduced the above considerations in the Introduction (page 4, lines 18-19) and Discussion (p 9, l 18-34) as suggested.

In addition, the role of Cav1 in mitochondrial function is under debate. While the reviewer mentions: “Cav1 maintains mitochondrial depolarization, as its knockdown induces hyperpolarization”, Yu et al., (Aging Cell. 2017; 16(4):773-784) claim that Cav-1 knockdown prevents mitochondrial respiration and ATP production. In fact, the enzymatic activity of OXPHOS complex I after Cav-1 knockdown was reduced to approximately 40%. This fact would trigger depolarization rather hyperpolarization. Furthermore, we have recently shown that mitoKv1.3 interacts with complex I and inhibition of mitoKv1.3 reduces complex I activity (Peruzzo et al., 2020 Redox Biology; 37:101705). Therefore, it is tempting to speculate that if Cav1 knockdown reduces complex I activity, and mitoKv1.3 blockage reduces complex I activity, a functional link between Cav1 and mitoKv1.3 would exist at the level of complex I further supporting our claim. However, this is far beyond the scope of the present manuscript.

3. Disregarding the fact that Kv1.3 overexpression on its own reduces respiration by almost one-half (Figure 5), the mitochondrial fragmentation induced by Kv1.3CBDless deserves some comments in this context.

Basal (without addition of oligomycin) (Figure 5B) and maximal respiration (Figure 5C) were slightly reduced (by 15%) in the case of WT Kv1.3, while reduction was significantly higher for Kv1.3 CBD-less. The cells were transfected directly in the Seahorse plate and efficiency (more than 60% of the cells were transfected and transfection efficiency was controlled by YFP fluorescence, as mentioned in the Materials and methods section). In any case, in Figure 5C data were normalized with respect to the basal respiration and thus show a significant decrease in maximal respiration independently of the cell number. In general, mitochondrial fission is regulated in a way to couple mitochondrial morphology to the energetic status of the cell (see e.g. Giacomello et al., 2020, Nature reviews Molecular cell biology 21, 204-224). Fission is induced by phosphorylation mediated by AMPK, able to sense the changes in the energy status of a cell and consequently adapt mitochondrial function and dynamics. AMPK facilitates mitochondrial fission downstream of mitochondrial dysfunction caused by mitochondrial respiration inhibition. This mechanism was proposed to facilitate apoptosis in cells with severely dysfunctional mitochondria (Toyama et al., 2016, Science (New York, NY) 351, 275-281). The above explanation has now been added to the Discussion section (p 10, l 30-36).

https://doi.org/10.7554/eLife.69099.sa2

Article and author information

Author details

  1. Jesusa Capera

    Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain
    Present address
    Kennedy Institute of Rheumatology, University of Oxford, Oxford, United Kingdom
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Mireia Pérez-Verdaguer
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8123-7725
  2. Mireia Pérez-Verdaguer

    Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain
    Present address
    Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, United States
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Jesusa Capera
    Competing interests
    No competing interests declared
  3. Roberta Peruzzo

    Department of Biology, University of Padova, Padova, Italy
    Contribution
    Formal analysis, Investigation, Methodology
    Contributed equally with
    María Navarro-Pérez
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9209-9068
  4. María Navarro-Pérez

    Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing
    Contributed equally with
    Roberta Peruzzo
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8106-9787
  5. Juan Martínez-Pinna

    Dept de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Armando Alberola-Die

    Dept de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5391-5739
  7. Andrés Morales

    Dept de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
    Contribution
    Formal analysis, Funding acquisition, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  8. Luigi Leanza

    Department of Biology, University of Padova, Padova, Italy
    Contribution
    Formal analysis, Supervision, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  9. Ildiko Szabó

    Department of Biology, University of Padova, Padova, Italy
    Contribution
    Conceptualization, Supervision, Funding acquisition, Validation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    ildi@mail.bio.unipd.it
    Competing interests
    No competing interests declared
  10. Antonio Felipe

    Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain
    Contribution
    Conceptualization, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    afelipe@ub.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7294-6431

Funding

Ministerio de Ciencia, Innovación y Universidades (BFU2017-87104-R)

  • Antonio Felipe

Ministerio de Ciencia, Innovación y Universidades (PID2020-112647RB-I00)

  • Antonio Felipe

Ministerio de Ciencia, Innovación y Universidades (CSD2008-00005)

  • Andrés Morales

Italian Association for Cancer Research (20286)

  • Ildiko Szabó

Ministero dell'Istruzione, dell'Università e della Ricerca (PRIN 20174TB8KW_004)

  • Ildiko Szabó

Associazione Italiana Sclerosi Multipla

  • Ildiko Szabó

European Regional Development Fund

  • Antonio Felipe

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

Supported by the Ministerio de Ciencia e Innovación (MICINN), Spain (BFU2017-87104-R and PID2020-112647RB-I00 to AF; CSD2008-00005 to AM), the Italian Association for Cancer Research (AIRC IG grant 20286 to IS), the Italian Ministry of University and Education (PRIN 20174TB8KW_004 to IS), the Italian Association for Multiple Sclerosis (to IS), and the European Regional Development Fund. Italian and Spanish laboratories share equal co-responsibility of the work. JC and MPV contributed equally. RP and MNP contributed equally. JC, MPV, and MNP hold fellowships from the Fundación Tatiana Pérez de Guzmán el Bueno and MICINN, respectively. Authors thank to Prof. G Fernández-Ballester (University Miguel Hernández) for his help with the molecular model and Prof. C Deutsch (University of Pennsylvania) for useful discussion. The English editorial assistance of the American Journal Experts is also acknowledged.

Ethics

Human subjects: The protocol was reviewed and approved by the Ethics Committee of the Universitat de Barcelona and the Banc de Sang i Teixits de Catalunya (BST). Institutional Review Board (IRB00003099). All procedures followed the rules of the Declaration of Helsinki Guidelines. All donors signed a written informed consent and samples were totally anonymous and untraceable.

Animal experimentation: Animal handling was carried out in accordance with the guidelines for the care and use of experimental animals adopted by the E.U (RD214/1997).

Senior Editor

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Reviewing Editor

  1. Baron Chanda, Washington University in St. Louis, United States

Reviewer

  1. Shrinivasan Raghuraman, University of Utah, United States

Publication history

  1. Received: April 5, 2021
  2. Accepted: June 22, 2021
  3. Version of Record published: July 1, 2021 (version 1)
  4. Version of Record updated: July 6, 2021 (version 2)

Copyright

© 2021, Capera et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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