Research Article

Structural Biology and Molecular Biophysics

ER-luminal [Ca²⁺] regulation of InsP₃ receptor gating mediated by an ER-luminal peripheral Ca²⁺-binding protein

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, United States
Department of Statistics, University of Pittsburgh, United States
Department of Neurobiology and Behavior, University of California, United States
Proteomics Core Facility, The Children’s Hospital of Philadelphia, United States
Department of Physiology and Biophysics, University of California, United States
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, United States

May 18, 2020

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Abstract

Modulating cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP₃R) Ca²⁺-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP₃R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca²⁺ concentration ([Ca²⁺]_ER) is poorly understood and controversial. We discovered that the InsP₃R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca²⁺]_ER. The widely-expressed Ca²⁺-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca²⁺]_ER inhibition of InsP₃R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP₃-mediated Ca²⁺ signaling events. Thus, [Ca²⁺]_ER is a major regulator of InsP₃R channel activity and InsP₃R-mediated [Ca²⁺]_i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca²⁺-binding protein, likely ANXA1.

Introduction

Modulating cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) is a universal signaling pathway that regulates numerous cell-physiological processes (Berridge, 2016). Ubiquitous endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) Ca²⁺-release channels play a central role in this pathway (Foskett et al., 2007). InsP₃ generated in response to extracellular stimuli binds to and activates InsP₃R channels to release Ca²⁺ stored in the ER lumen, generating diverse local and global [Ca²⁺]_i signals (Berridge, 2016). Whereas much is known regarding regulation of InsP₃R channel gating by multiple processes, including binding of cytoplasmic ligands (Ca²⁺, InsP₃ and ATP^4–), post-translational modifications, interactions with proteins, clustering and differential localization (Berridge, 2016), the regulation of InsP₃R channel activity by Ca²⁺ concentration in the ER lumen ([Ca²⁺]_ER) is poorly understood and controversial (see (Caroppo et al., 2003; Yamasaki-Mann and Parker, 2011) and references therein). InsP₃R activity influences [Ca²⁺]_ER, which is critical for many processes, including regulation of bioenergetics, protein biogenesis and folding, and Ca²⁺ signaling (Corbett and Michalak, 2000). [Ca²⁺]_ER dysregulation is associated with pathological conditions, including ER stress responses, diabetes, cardiac dysfunction, neurodegeneration, defective cell proliferation and cell death (Berridge, 2016). Mechanisms that link cellular processes with [Ca²⁺]_ER, and the connections between [Ca²⁺]_ER dysregulation and disease pathogenesis are ill-defined and poorly understood (Mekahli et al., 2011).

For a long time, regulation of InsP₃R channel activity by [Ca²⁺]_ER has primarily been examined by approaches that relied on changes in [Ca²⁺]_i or [Ca²⁺]_ER to infer channel activity (Caroppo et al., 2003; Yamasaki-Mann and Parker, 2011) and references therein), with no rigorous control of both [Ca²⁺]_ER and [Ca²⁺]_i simultaneously. It has therefore been difficult to differentiate feed-through effects in which Ca²⁺ flux through channel raises [Ca²⁺]_i at the cytoplasmic Ca²⁺-binding sites to modulate channel activity from direct effects of [Ca²⁺]_ER on the luminal aspect of the InsP₃R. Patch-clamp electrophysiology allows rigorous, simultaneous control of [Ca²⁺] on both sides of the membrane, but intracellular ER membranes are not accessible to patch pipettes. To overcome this limitation, we made use of the fact that the ER membrane is continuous with the outer nuclear membrane (Dingwall and Laskey, 1992) and pioneered the use of nuclear patch-clamp electrophysiology on isolated nuclei (Mak et al., 2005). This enables the activities of single InsP₃R channels to be recorded in their native ER membrane and luminal milieus. Different patch-clamp configurations have been used to study single InsP₃R channels under rigorously controlled [Ca²⁺]_ER and [Ca²⁺]_i: including on-nucleus (on-nuc, cytoplasmic aspect of the channel faces into the pipette solution with the luminal milieu intact, Figure 1A), excised luminal-side-out (lum-out, cytoplasmic aspect of the channel faces into the pipette solution with the luminal aspect exposed to the bath solution, Figure 1B) and excised cytoplasmic-side-out (cyto-out, cytoplasmic aspect of the channel perfused by the bath solution with the luminal aspect facing the pipette solution, Figure 1C; Mak et al., 2013a; Mak et al., 2013b). Because of the relatively low selectivity of InsP₃R channels for Ca²⁺ vs K⁺ (15.2 : 1 [Vais et al., 2010a]) and orders of magnitude higher [K⁺] (140 mM) than that of other ions in the physiological solutions used in our experiments (70 nM to ≤600 μM [Ca²⁺]_free; 0 or 200 μM [Mg²⁺]_free), the electrical currents through open InsP₃R channels are overwhelmingly carried by K⁺ in all our patch-clamp electrophysiology experiments, enabling the kinetics of channel gating to be studied with both luminal and cytoplasmic [Ca²⁺] well-defined and controlled.

Figure 1

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Schematic diagram illustrating the orientation of InsP₃R channels in isolated nuclear membrane patches and InsP₃-containing solution relative to the micropipette in various configurations of nuclear patch-clamping.

(A) On-nucleus configuration with outer nuclear membrane intact, (B) excised luminal-side-out configuration, (C) excised cytoplasmic-side-out configuration.

Using the nuclear patch-clamp approach in a previous study (Vais et al., 2012), we demonstrated that InsP₃R channel activity can be modulated by [Ca²⁺]_ER indirectly via feed-through effects of Ca²⁺ flux driven through an open channel by high [Ca²⁺]_ER that raises the local [Ca²⁺]_i in the channel vicinity to regulate its activity through its cytoplasmic activating and inhibitory Ca²⁺-binding sites (Figure 2A). That study demonstrated that these feed-through effects can be completely abrogated by sufficient Ca²⁺ chelation on the cytoplasmic side to buffer local [Ca²⁺]_i at cytoplasmic Ca²⁺ binding sites of the InsP₃R. In the presence of 5 mM 5,5’-diBromo BAPTA (a fast acting Ca²⁺ chelator) on the cytoplasmic side in the lum-out excised patch configuration (Figure 1B), the open probability P_o of the InsP₃R channel did not change discernably when the [Ca²⁺]_ER was switched between 70 nM and 300 μM, no matter whether saturating 10 μM [InsP₃] (Figure 2C in Vais et al., 2012) or sub-saturating 3 μM [InsP₃] (Figure 7B in Vais et al., 2012) was present in the micropipette (2 μM [Ca²⁺]_i was in the micropipette in all experiments described here). Thus, as long as [Ca²⁺]_free on the cytoplasmic side was well buffered, switching [Ca²⁺]_ER between 70 nM and 300 μM in lum-out excised membrane patches had no effect on channel activity. This definitively ruled out the possibility that InsP₃R activity is modulated directly by an intrinsic Ca²⁺-regulatory site on the luminal side that is either part of the InsP₃R or of a protein that constitutively interacts with the channel (Figure 2B).

Figure 2

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Possible mechanisms of [Ca²⁺]_ER regulation of InsP₃R channel activity.

(A) Ca²⁺ flux through an open InsP₃R channel driven by high [Ca²⁺]_ER raises local [Ca²⁺]_i in the pore vicinity to regulate the channel through its cytoplasmic activating and inhibitory Ca²⁺-binding sites. (B) Ca²⁺ binds directly to an intrinsic site on the luminal side of the channel to regulate its activity. (C) Ca²⁺ binding to a peripheral protein in the ER lumen regulates channel activity indirectly, by promoting interaction of the peripheral protein with the InsP₃R.

Notably, however, in all previous studies of InsP₃R channel gating, nuclei were isolated into a bath solution with low [Ca²⁺]_free to simulate physiological resting [Ca²⁺]_i(Mak and Foskett, 2015). Although sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is present in the nuclear envelope (Lanini et al., 1992), the bath solutions contained no MgATP to energize it. Thus, passive Ca²⁺ leaks equilibrated [Ca²⁺]_free in the perinuclear space of the isolated nuclei (effectively [Ca²⁺]_ER) with bath [Ca²⁺] at unphysiologically low levels. Accordingly, possible [Ca²⁺]_ER regulation of channel activity by peripheral proteins in the lumen that interact with the InsP₃R only in the presence of high, physiological [Ca²⁺]_ER (Figure 2C) would have been undetected in previous studies.

Here we specifically assessed whether such a peripheral luminal protein (PLP)-mediated [Ca²⁺]_ER regulation of InsP₃R channel activity exists. We discovered a strong Ca²⁺ flux-independent inhibition of InsP₃R channel activity mediated by accessory ER-luminal protein(s) only in the presence of high, physiological [Ca²⁺]_ER. We identified the region of the InsP₃R that is involved in this [Ca²⁺]_ER-dependent inhibition, and discovered that the widely expressed Ca²⁺-binding protein annexin A1 (ANXA1) localizes not only to the cytoplasm but also to the perinuclear space inside the nuclear envelope, and possibly in the ER lumen as well, where it plays a critical role in channel inhibition through high [Ca²⁺]_ER-mediated interaction with the InsP₃R. Reducing this interaction enhanced agonist-induced InsP₃R-mediated Ca²⁺ release, and increased local elementary Ca²⁺-release events in intact cells. Thus, [Ca²⁺]_ER is a major regulator of InsP₃R-channel activity and InsP₃R-mediated [Ca²⁺]_i signaling in cells by controlling an interaction of the channel with a luminal peripheral membrane-associated Ca²⁺-binding protein, likely ANXA1.

Results

Novel regulation by [Ca²⁺]_ER of InsP₃R single-channel activity

We performed single-channel patch-clamp electrophysiology on outer membranes of nuclei isolated from mutant chicken B cells in which all three endogenous InsP₃R genes were ablated and stably replaced with wild-type rat type-3 InsP₃R (DT40-r3 cells) (Mak et al., 2005), so that homotetrameric rat type-3 InsP₃R channels (Vais et al., 2012) were recorded in our experiments. In on-nuc patch-clamp experiments with the bath containing no MgATP, the [Ca²⁺]_ER in the perinuclear space (Figure 3A) equilibrated with [Ca²⁺]_bath (70 nM). Linear, ohmic InsP₃R-channel current vs. applied potential (i_ch-V_app) curves were observed with high single-channel conductance, as expected (Figure 3B). In contrast, with 1 mM MgATP in the bath solution (Figure 3C), SERCA activity raised [Ca²⁺]_ER to physiological levels, if the outer and inner membranes of the isolated nuclei remained intact. The i_ch-V_app curves (Figure 3D) observed were non-ohmic and asymmetric with respect to the origin, and single-channel conductance was reduced, both caused by permeant-ion (Ca²⁺) block of the measured K⁺ current due to high [Ca²⁺]_ER. Thus, absence or presence of MgATP in the bath provides a mechanism to alter [Ca²⁺]_ER in intact nuclei. In a previous study examining the Ca²⁺ permeant-ion effect on K⁺ conductance through InsP₃R channels (Vais et al., 2010a), we derived an empirical equation that describes, with high accuracy, the InsP₃R channel slope conductance over a broad range of [Ca²⁺]_ER(0 to 1.1 mM). This enabled us to use the single-channel conductance, rather than Ca²⁺ indicator dye fluorescence, to estimate that [Ca²⁺]_ER is ~300 μM in the perinuclear space of intact isolated DT40-r3 nuclei in the presence of bath MgATP. Subsequent excision of the membrane patch into the lum-out patch configuration (Mak et al., 2013c) exposed its luminal side to 70 nM [Ca²⁺]_bath (Figure 3E), which restored linear I_ch-V_app curves and high single-channel conductance (Figure 3F).

Figure 3 with 1 supplement see all

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Raising [Ca²⁺]_ER in intact isolated nuclei affects activities of InsP₃R channels.

(**A and B**) On-nucleus (on-nuc) patch-clamp configuration and single-channel current-voltage (i_ch-V_app) plot of InsP₃R channels in bath solution with 0-MgATP so SERCA was not active and [Ca²⁺]_ER equilibrated with [Ca²⁺]_bath. (**C and D**) On-nuc configuration and i_ch-V_app plot of InsP₃R channels in bath solution with 1 mM MgATP to activate SERCA to move Ca²⁺ from the bath into the perinuclear space, raising [Ca²⁺]_ER to ~300 μM. ([Ca²⁺]_ER was estimated from the magnitude of the InsP₃R channel current size [Vais et al., 2010a]). (**E and F**) Excised luminal-side-out (lum-out) configuration and i_ch-V_app plot of InsP₃R channels in isolated membrane patch perfused with 70 nM [Ca²⁺]_ER and 0-MgATP bath solution. The baseline closed-channel currents were subtracted from the i_ch-V_app plots. Cytoplasmic [InsP₃] in the pipette solution = 3 μM for sub-saturating level, and 10 μM for saturating level. V_app ramped from –50 to 50 mV (w.r.t. ground electrode in the bath) in 2 s. 10–20 ramps were averaged for each graph. Colored lines: linear (**B, F**) and 4^th order polynomial (D) fits to open-channel data points in graphs. In this and all subsequent patch-clamp experiments, optimal cytoplasmic [Ca²⁺]_free (2 μM) was used. In experiments shown here, [Ca²⁺]_free in bath solution = 70 nM. (G) Overlay of three fitted curves in (**B, D and F**). (H) Typical single-channel current traces recorded under constant V_app (–30 mV) in on-nuc configuration, with pipette solution containing sub-saturating 3 µM InsP₃, and bath solutions containing 0- (red) or 1 mM-MgATP (blue). In this and all subsequent current traces, arrow on left of trace indicates closed-channel current level. (I) Corresponding current traces recorded with pipette solution containing saturating 10 µM InsP₃. (J) Typical current trace recorded in lum-out configuration with constant V_app = –30 mV. (K) Typical current trace recorded as lum-out patch was perfused by solution with 70 nM Ca²⁺_free (grey bar) and then switched to one with 300 μM Ca²⁺_free (black bar). (L) P_o from individual current traces (filled circles), and their averages (thick horizontal bars) and s.e.m. (error bars) observed in on-nuc patch-clamp configuration with 3 µM InsP₃ in pipette solution, and with 0- (red) or 1 mM (blue) MgATP in bath solutions. [Ca²⁺]_free on luminal side tabulated at x-axis. Numbers of current traces tabulated next to corresponding averages. In this and all subsequent data plots, symbols °, °° and °°° indicate t-test p value < 0.05, 0.005 and 0.001, respectively. (M) P_o from individual current traces (circles), averages (thick bars) and s.e.m. (error bars) observed with 10 µM InsP₃ in pipette solution. Red symbols: P_o in on-nuc configuration with 0-MgATP in bath so [Ca²⁺]_ER = [Ca²⁺]_bath = 70 nM. Blue symbols: P_o in on-nuc configuration with 1 mM MgATP in bath so [Ca²⁺]_ER ~300 μM. Filled circles: P_o from experiments in which only the on-nuc configuration was achieved. Open circles: P_o from experiments in which lum-out configuration was achieved after on-nuc channel activity had been recorded. Green symbols: P_o observed in lum-out patches whose luminal side was perfused with solution containing 70 nM Ca²⁺_free. Open circles connected with grey lines: P_o observed in same patch before and after membrane excision. Brown symbols: P_o observed in lum-out membrane patches after switching to perfusing solution containing 300 μM Ca²⁺_free, as in (K). Open circles connected with dashed grey lines: P_o observed in same lum-out membrane patch before and after perfusion-solution switching. Purple symbols: P_o in on-nuc configuration with 1 mM bath MgATP and 2.5 μM thapsigargin.

In on-nuc patches of DT40-r3 nuclei, InsP₃R channel open probability (P_o) was high with 70 nM Ca²⁺_ER (Figure 3H and L, red) whereas it was profoundly reduced with [Ca²⁺]_ER raised to 300 μM by addition of MgATP to the bath (Figure 3H and L, blue). Even in saturating (10 μM) [InsP₃], 300 μM Ca²⁺_ER strongly reduced P_o (Figure 3I and M, blue), to a level indistinguishable from that observed in 3 μM InsP₃ (Figure 3L, blue). In contrast, when [Ca²⁺]_ER = 70 nM, P_o was higher in 10 μM InsP₃ (Figure 3M, red) than in 3 μM InsP₃ (Figure 3L, red). With the bath solution containing 1 mM MgATP and the SERCA inhibitor thapsigargin (2.5 μM) (Figure 3M, purple and Figure 3—figure supplement 1), P_o was indistinguishable from that in 0-MgATP_bath, confirming that bath MgATP raised [Ca²⁺]_ER in intact nuclei by supporting SERCA activity. Because high concentrations of Ca²⁺ chelator (5 mM diBrBAPTA) buffered [Ca²⁺]_i at cytoplasmic Ca²⁺ regulatory sites, possible feed-through effects were eliminated (Vais et al., 2012). Thus, with an intact ER-luminal milieu and [Ca²⁺]_ER at a level characteristic of replete ER stores, InsP₃R activity is profoundly suppressed by a mechanism unrelated to but as powerful as the well-known high-[Ca²⁺]_i inhibition of InsP₃R activity. Because luminal [Ca²⁺] does not directly affect InsP₃R channel activity (Vais et al., 2012), these results suggest a novel regulatory mechanism, possibly mediated by a resident luminal Ca²⁺-binding protein.

After recording channel activity in the on-nuc patch-clamp configuration, patches were excised into the lum-out configuration to observe gating of channels with their luminal sides exposed to bath [Ca²⁺]_free = 70 nM. Since [Ca²⁺]_i and [Ca²⁺]_ER experienced by channels in this configuration were effectively the same as those in the on-nuc configuration with no bath MgATP, it was expected that the inhibitory luminal-protein-mediated effect would be abrogated. In agreement, similar P_o were observed for channels in this lum-out configuration (Figure 3J and M, green) and channels in the on-nuc configuration in 0-MgATP (Figure 3I and M, red). Importantly, channel P_o remained high during subsequent perfusion with 300 μM Ca²⁺_free (Figure 3K and M, brown). Lack of inhibition when [Ca²⁺] was raised to 300 μM suggests that the luminal effector mediating high-[Ca²⁺]_ER inhibition is only loosely associated with the InsP₃R, becoming irretrievably lost when the luminal side of the isolated patch was perfused by low-[Ca²⁺]_free solution.

Since the levels of suppression of InsP₃R channel activities in the presence of high [Ca²⁺]_ER were independent of [InsP₃] used (Figure 3L and M), all subsequent experiments were performed with saturating 10 μM [InsP₃] to avoid possible ambiguous results due to insufficient stimulation of InsP₃R channels.

To determine whether peripheral luminal-protein (PLP) inhibition affects endogenous InsP₃R in different cell types, similar on-nuc patch-clamp experiments were conducted with intact nuclei (verified by asymmetric i_ch-V_app curves [Figure 4A–C] and reduced single-channel conductance [Figure 4D–F]) isolated from mouse N2a cells, which predominantly express type 1 InsP₃R (InsP₃R–1) (Wojcikiewicz, 1995), rat PC-12 cells expressing mainly InsP₃R-1 and InsP₃R-3 (Newton et al., 1994), and wild-type (WT) chicken DT40 cells, which express all three isoforms (Sugawara et al., 1997). In a bath containing 0-MgATP, channels from the different cell types exhibited different maximum P_o (Figure 4D–F red traces, and Figure 4G). With high [Ca²⁺]_ER generated by 1 mM bath MgATP, P_o of the various endogenous InsP₃R channels were strongly suppressed (Figure 4D–F blue traces, and Figure 4H). Interestingly, P_o of the different InsP₃Rs were strongly suppressed to very similar extents (P_o reduction: WT DT40 92%, PC-12 86%, N2a 90% (Figure 4I), comparable to that of InsP₃R-3 in DT40-r3 cells (85%) (Figure 3M, red and blue). This suggests that the inhibitory protein is present in the ER lumen of many cell types and interacts with all three InsP₃R isoforms under high [Ca²⁺]_ER.

Figure 4

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Activities of InsP₃R channels from a number of cell lines expressing various InsP₃R isoforms in different proportions are similarly affected by rise in [Ca²⁺]_ER in intact isolated nuclei.

(**A–C**) Single-channel *I-V*_app plots of on-nuc patch-clamp experiments of endogenous InsP₃R channels from WT DT40 (A), PC12 (B), and N2a (C) cells. Channels activated by 10 μM InsP₃ with (blue) or without (red) 1 mM MgATP. Data obtained using protocol as in Figure 3 with V_app ramped from –40 to 40 mV in 2 s. Lines: linear (red) and 4^th order polynomial (blue) fits to open-channel data points. (D–F) Typical current traces for endogenous InsP₃R channels from WT DT40 (D), PC12 (E), and N2a (F) cells, in presence (blue) and absence (red) of 1 mM MgATP in bath with V_app = –40 mV. (G) Optimal P_o activated by 10 μM InsP₃ and 2 μM Ca²⁺_i in individual experiments (circles) and averages and s.e.m. (bars) for endogenous WT DT40 (magenta), PC12 (orange) and N2a (purple) InsP₃R channels in bath without MgATP. (H) Optimal P_o in bath with 1 mM bath MgATP activated by the same ligand conditions as (G) for the same endogenous InsP₃R channels. (I) Normalized P_o, their averages and s.e.m. for endogenous InsP₃R channels in bath with 1 mM MgATP, relative to respective average optimal P_o in (G).

A luminal loop of the InsP₃R is critical for luminal protein-mediated [Ca²⁺]_ERregulation

We hypothesized that the PLP interacts directly with the InsP₃R. Only a small fraction of the InsP₃R sequence is exposed to the ER lumen, consisting of three loops (L1, L2, and L3) connecting transmembrane helices in the pore-forming domain. We selected human sequences (Figure 5A–C) based on structural information about the InsP₃R (Fan et al., 2015). Since the inhibitory effects were observed in cells expressing different channel isoforms, we selected sequences conserved in all three InsP₃R isoforms. We excluded the P-region (orange stripe) and selectivity filter (green stripe) involved in other crucial channel functions (Figure 5C). Only four short sequences (magenta stripes in Figure 5B–C) fit the criteria to be luminal region(s) of the InsP₃R that could interact with the PLP.

Figure 5

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Identification of the InsP₃R region involved in [Ca²⁺]_ER regulation of channel activity.

(A–C) Sequences of L1 (A), L2 (B) and L3 (C) loops of human InsP₃R isoforms exposed to ER lumen according to h-InsP₃R-1 cryo-EM structure in Fan et al., 2015. Residues conserved over all three InsP₃R isoforms are in red; similar residues in black; and different residues in grey. Highly-conserved sequences: magenta stripes. Sequence of pL2 (partial L2) peptide used in experiments is from h-InsP₃R-3: purple stripe. L2 loop sequence of chicken InsP₃R-1 also shown (B, top line) revealing highly-conserved L2 loop sequence. (D–F) Cartoons showing [Ca²⁺]_ER-dependent interaction between luminal part of InsP₃R and a peripheral protein in ER lumen in various pipette and bath solutions, during on-nuc or cyto-out nuclear patch-clamp experiments, as labeled. (G–I) Typical InsP₃R-3 current traces in cyto-out experiments with 0- or 1 mM MgATP in bath, and 0- or 10 μM pL2 peptide in pipette solution, as tabulated. Traces in (G), (H) and (I) recorded in experiments depicted in (D), (E) and (F), respectively. In this and all subsequent cyto-out experiments, V_app = +30 mV and channels were activated by perfusion solutions containing optimal 2 μM Ca²⁺_i, saturating 10 μM InsP₃ and 0.5 mM ATP^4–. (J) P_o of individual current traces (circles) and averages and s.e.m. (horizontal bars) observed under conditions as tabulated. For comparison, averages and s.e.m. of P_o in on-nuc experiments (red and blue horizontal bars) from Figure 3J are shown.

We first investigated whether the conserved region of the L2 loop (pL2, Figure 5B) is involved, by recording InsP₃R-3 channels in the cytoplasmic-side-out (cyto-out) configuration (Mak et al., 2013b) with pipette solutions that faced the luminal aspect of the channel containing synthetic pL2 peptides. Nuclei were first exposed to [Ca²⁺]_bath = 70 nM and 0-MgATP, so [Ca²⁺]_ER equilibrated with [Ca²⁺]_bathat 70 nM (Figure 5D, left) promoting dissociation of PLP from the InsP₃R. When the cyto-out patch configuration was achieved (Figure 5D, right), the luminal aspect of the channel faced the pipette solution containing [Ca²⁺]_pip = 300 μM. Because the PLP had already dissociated from the channel before patch excision (Figure 5D, left), we expected no inhibition to be observed. Indeed, when the patch was perfused with saturating 10 μM InsP₃ and optimal 2 μM Ca²⁺_i (buffered with 5 mM dibromoBAPTA) (Vais et al., 2010b), channels were activated with reduced conductance due to permeant Ca²⁺ block caused by high [Ca²⁺]_ER in the pipette solution, but they exhibited high P_o (~0.6) (Figure 5G and J, magenta) indistinguishable from that observed in on-nuc experiments under similar conditions (red bars in Figure 5J). Alternately, nuclei were first exposed to 1 mM MgATP in the bath to raise [Ca²⁺]_ER to ~300 μM to promote an interaction between the PLP and the InsP₃R (Figure 5E, left). Because the pipette solution contained 300 μM Ca²⁺, the interaction remained intact when the cyto-out patch configuration was achieved (Figure 5E, right). Indeed, low channel P_o was observed with maximal stimulation (Figure 5H and J, purple), similar to the observations made using the on-nuc configuration (Figure 5J, blue bars). Having therefore established that the inhibitory effect of the PLP could be observed in the cyto-out patch configuration, we included 10 μM pL2 peptide in the pipette solution (Figure 5F, left). With pL2 peptides present on the cytoplasmic side in the on-nuc configuration before establishing the cyto-out configuration, InsP₃R remained bound to the PLP before membrane excision. However, after achieving the cyto-out configuration, the luminal side of the InsP₃R became exposed to the peptides. We reasoned that if the pL2 region is involved in the inhibitory effects through interactions with the PLP, then pL2 peptide in high concentrations should compete the PLP away from the InsP₃R, and thereby raise P_o despite presence of 300 μM Ca²⁺ (Figure 5F, right). Indeed, inhibition was completely abolished such that the P_o of channels with pL2 peptides on their luminal side (Figure 5I–J, brown) was indistinguishable from P_o recorded in on-nuc (red) or cyto-out patch-clamp experiments with 0-MgATP in the bath (magenta). This indicates that the pL2 region interacts with the PLP to suppress InsP₃R channel activity, and that the inhibitory effect of the PLP is mostly or completely mediated by the pL2-PLP interaction.

Identification of the inhibitory peripheral protein(s) in the ER lumen

To identify the PLP, peptides with a modified pL2 sequence (mpL2) or scrambled mpL2 (smpL2) were used for in-vitro pull-downs from soluble fractions of bovine hepatocyte ER microsomes (Figure 6—figure supplement 1). Eluates from the pull-downs were analyzed by mass spectrometry. Among >460 proteins detected, we looked for those enriched in the mpL2, that are Ca²⁺-binding proteins (UniProt protein database) in the ER lumen (or in topologically-equivalent locations), and that are broadly expressed. The annexin (ANX) protein family met these criteria. Of 12 members of the ANX family, five were identified to have statistically-significant preference to bind to the mpL2 peptide over the smpL2 peptide (Figure 6—source data 1, also see ‘Ca²⁺-dependent affinity enrichment mass spectrometry’ in Materials and methods).

ANX proteins are ubiquitously expressed, Ca²⁺-dependent phospholipid-binding proteins (Raynal and Pollard, 1994). Ca²⁺ binding to ANX (dissociation constant K_d ~25 μM to 1 mM) promotes its association with negatively-charged phospholipid-containing membranes (Raynal and Pollard, 1994; Gerke and Moss, 2002). Although ANX proteins are known to be cytoplasmic, many studies have reported their presence in locations topologically equivalent to the ER lumen, including the extracellular side of the plasma membrane (PM) (Solito et al., 1994; Brownstein et al., 2001; McArthur et al., 2009; Yáñez et al., 2012; Mirkowska et al., 2013), within intracellular and secretory granules (McArthur et al., 2009; Leoni et al., 2015; Chi et al., 2006; Boudhraa et al., 2016; Perretti et al., 2000), and in the extracellular space (McArthur et al., 2009; Boudhraa et al., 2016; Arur et al., 2003; Fan et al., 2004; Scannell et al., 2007; D'Acquisto et al., 2007; Iwasa et al., 2012; Belvedere et al., 2014), suggesting that ANX can also be present in the ER lumen.

Among annexins, ANXA1 is most widely reported to be present in locations topologically equivalent to the ER lumen. To confirm that ANXA1 was present in the ER lumen, we localized it by immunofluorescence microscopy. A549 cells were briefly exposed to a low concentration of digitonin to permeabilize the PM to release cytoplasmic ANXA1. Remaining ANXA1 co-localized with the ER marker calnexin in the nuclear envelope and in cytoplasmic puncta (Figure 6A). ANXA1 localization to the nuclear envelope was also observed in HeLa and Panc-1 cells (Figure 6B–D). The inability to observe ANXA1 throughout the ER could be the result of its low expression in the bulk ER lumen. Therefore, to further establish localization to the ER lumen, we performed capture ELISA of secreted proteins. The non-secreted β-actin was detected only in the lysate (Figure 6E, blue), indicating that the HEKtsA201 cells remained healthy and intact during the 3 day incubation period. In contrast, the signal for extracellular ANXA1 was robust (Figure 6E, red), suggesting that ANXA1 was present in the ER lumen en route to be secreted.

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Localization of AnxA1 in the ER lumen.

(A–B) Confocal images of permeablized A549 (A) and HeLa (B) cells. Native ANXA1 (red), calnexin (green), nuclei (blue). ANXA1 co-localized with calnexin in nuclear envelope (white arrow). ANXA1 also found in puncta without (white arrowhead) or with (cyan arrowhead) calnexin. (C–D) Confocal images of intact (C) and digitonin permeabilized (D) Panc-1 cells. Native ANXA1 (red), nuclei (blue). White arrow in (D) indicates nuclear envelope labeled with ANXA1. (E) ANXA1-(red) and β-actin-(blue) capture ELISA of supernatant (left) and whole-cell lysate (right). Dots: signals detected in individual assays (n = 6); horizontal bars: mean, s.e.m.

Figure 6—source data 1 Abundance of annexin proteins (quantified as total spectral counts by mass spectrometry analysis) detected in experiment eluates collected from magnetic beads covalently linked to peptides with modified pL2 sequence, and in control eluates collected from beads linked to peptide with scrambled modified pL2 sequence. The spectral count entry is black when the count ratio (experiment count/control count) is ≥1.2; grey when 1.2 > count ratio≥1.0; and grey italics when count ratio <1.0. lfsr is the local false sign rate (see Statistical analysis of mass spectrometry output in the Materials and methods section). The lfsr ≤0.2 (corresponds to a global false discovery rate ≤5%) are bolded.: https://cdn.elifesciences.org/articles/53531/elife-53531-fig6-data1-v2.docx
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Luminal ANXA1 can mediate [Ca²⁺]_ER-dependent InsP₃R channel inhibition

We examined direct effects of ANXA1 on channel gating in cyto-out patch-clamp experiments with pipette solutions (bathing the luminal side) containing 1 μM recombinant full-length human ANXA1 under conditions that prevented permeant Ca²⁺ feed-through effects (Figure 7—figure supplement 1). ANXA1 was without effect on maximally-stimulated channel P_o (≈ 0.65) in [Ca²⁺]_ER between 70 nM and 55 μM (Figure 7A–C and F). In contrast, with [Ca²⁺]_ER >55 μM, channel P_o dropped abruptly with increasing [Ca²⁺]_ER (Figure 7D–F). Fitting the P_o data with a simple inhibitory Hill equation (Figure 7F):

P_{o} = P_{m a x} {1 + {(\frac{[C a^{2 +}]_{E R}}{K_{i n h}})}^{H_{i n h}}}^{- 1}

Figure 7 with 1 supplement see all

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[Ca²⁺]_ER inhibition of InsP₃R-3 channel activity is mediated by a specific interaction between the channel and ANXA1 in the ER lumen.

(A–E) Typical current traces of InsP₃R-3 channels in cyto-out configuration with 1 μM ANXA1 and various [Ca²⁺]_ER in pipette solution. In *C–E* with [Ca²⁺]_ER >40 μM, channel conductance reduced due to permeant-ion block. (F) P_o of individual current traces (blue symbols) and averages and s.e.m. (red horizontal bars) observed under conditions in (*A–E*). Red curve: least-squares fit to average P_o at various [Ca²⁺]_ER using simple inhibitory Hill equation with parameters tabulated. (G) Averages and s.e.m. of normalized probability of detection of InsP₃R channels (P_d) in cyto-out experiments with 0 (black) or 1 μM (red) ANXA1 in pipette solutions with various [Ca²⁺]_ER. (H–I) Averages and s.e.m. of InsP₃R-3 channel open (H) and closed (I) durations in cyto-out patch-clamp experiments in (F). (J–K) Typical current traces in on-nuc patches with pipette (cytoplasmic) solutions containing 2 μM Ca²⁺_i and 10 μM InsP₃, with 0 (J) or 1 μM (K) ANXA1. Bath solution: 70 nM Ca²⁺_ER with 0-MgATP. (L) Po of individual current traces (brown: no ANXA1; light blue: 1 μM ANXA1 on cytoplasmic side) and averages and s.e.m. (respective horizontal bars) observed under conditions in (**J–K**).

showed that the half-maximal inhibitory [Ca²⁺]_ER (K_inh) is 260 ± 14 μM with Hill coefficient (H_inh) of 2.3 ± 0.3 (Figure 7F). The observed K_inh is within observed physiological levels of [Ca²⁺]_ER (~100 μM to 1 mM) (Zampese and Pizzo, 2012) and K_d of ANX binding to Ca²⁺. Without affecting the number of channels observed (Figure 7G), ANXA1 inhibited P_o mainly by increasing channel-closed durations (Figure 7H–I).

ANXA1 is primarily localized to the cytoplasm. However, ANXA1 on the cytoplasmic side of the InsP₃R was without affect (Figure 7J–L). Thus, ANXA1 inhibits InsP₃R activity exclusively from the luminal side.

ANXA1 inhibits InsP₃R channel gating by interacting with the L2 loop region

To determine if ANXA1 inhibition is mediated by interaction with the L2 loop, we performed cyto-out patch-clamp experiments with the pipette solution facing the luminal aspect containing 1 μM ANXA1 and 40 μM of peptides with either the L2 sequence or a scrambled L2 (sL2) sequence (Figure 8A–B, respectively) to compete with the InsP₃R for binding to ANXA1 (Figures 5J and 7F). The L2 peptide, but not the sL2 peptide, completely abrogated inhibition of InsP₃R activity by ANXA1 (Figure 8C). This suggests that suppression of channel activity by ANXA1 is specifically mediated by the L2 region of the InsP₃R.

Figure 8

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L2 peptide, but not scrambled L2 peptide, was able to compete ANXA1 from the InsP₃R channel and restore high channel activity.

(A–B) Typical current traces in cyto-out experiments with pipette solutions containing 300 Ca²⁺_ER, 1 μM ANXA1, and 40 μM of L2 (A) or scrambled L2 (sL2) (B) peptides. Peptide sequences shown below corresponding traces. (C) P_o of individual current traces (orange: L2 peptide; yellow: sL2 peptide) and averages and s.e.m. (horizontal bars) observed under conditions in (**A–B**). For comparison, averages and s.e.m. of P_o in similar cyto-out experiments without peptide, and without (magenta bars) and with (light green bars) ANXA1, from Figures 4J and 6F, respectively.

Native PLP-mediated and recombinant ANXA1-mediated [Ca²⁺]_ER-dependent InsP₃R channel inhibition have similar features

In sub-physiological 70 nM Ca²⁺_ER, neither the native PLP (Figure 9, Lane 2) nor recombinant ANXA1 (Figure 9, Lane 5) suppressed channel activities, since P_o observed were indistinguishable from those with no PLP on the luminal side (Figure 9, Lane 1). In 300 μM Ca²⁺_ER, both native PLP (Figure 9, Lane 3) and luminal ANXA1 (Figure 9, Lane 6) inhibited the P_o. Inhibition by PLP or ANXA1 was completely abrogated by L2 loop peptides (Figure 9, Lanes 4 and 7, respectively). Such similarities suggest that ANXA1 is the major PLP mediating [Ca²⁺]_ER-dependent inhibition of InsP₃R channels.

Figure 9

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Modulation of homotetrameric InsP₃R channel activity by native inhibitory peripheral luminal protein or ANXA1 under various experimental conditions: 70 nM, 300 or 600 μM Ca²⁺_ER; in the absence or presence of peptides with L2 sequences (pL2, L2 or sL2); and in on-nuc or cyto-out patch-clamp configurations.

Averages and s.e.m. of InsP₃R-3 P_o are shown as tabulated.

Although native PLP and luminal ANXA1 both inhibit InsP₃R activity, P_o of PLP-inhibited InsP₃R channels (Figure 9, Lane 3) was lower than that of ANXA1-inhibited channels (Figure 9, Lanes 6 and 8). However, in higher [Ca²⁺]_ER (600 μM), ANXA1-inhibited channel P_o (Figure 9, Lane 9) was comparable to the native PLP-inhibited P_o (Figure 9, Lane 3). This suggests that the luminal concentration of endogenous ANXA1 is effectively higher than 1 μM, and/or that other factor(s) enhance the [Ca²⁺]_ER sensitivity of the channel to inhibition by ANXA1.

Annexin A1 specifically mediates [Ca²⁺]_ER-dependent InsP₃R channel inhibition

Multiple annexins were identified in our mass spectroscopy analysis, including ANXA6, ANXA2 and ANXA11 (Figure 6—source data 1). However, recombinant ANXA6 or ANXA2 had no effect on channel P_o (Figure 10A–B). Similar experiments with ANXA11 were precluded by its low solubility. The ANXA6 used had an N-terminal His tag. However, suppression of InsP₃R activities by ANXA1 with and without an N–terminal His tag was equivalent (Figure 10—figure supplement 1), indicating that the observed effects were not impacted by presence or absence of the tag.

Figure 10 with 1 supplement see all

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[Ca²⁺]_ER inhibition of InsP₃R channel activity is specifically mediated by ANXA1 through interaction with the L2 loop of InsP₃R.

(A) Typical current traces of InsP₃R-3 in cyto-out experiments with pipette containing 300 μM Ca²⁺_ER and 1 μM ANXA6 or A2. Isolated patches perfused with 2 μM Ca²⁺_free, 0.5 mM ATP^4– with 0 or 10 μM InsP₃ as indicated. (B) P_o of individual current traces (circles) and average and s.e.m. (horizontal bars) for cyto-out experiments with ANXA6 (blue) or ANXA2 (cyan). For comparison are averages and s.e.m. of P_o in similar experiments with no ANXA1 (magenta bars) and ANXA1 (green bars), from Figures 5J and 6F, respectively. (C) Synthetic peptide with 41 residues of N-terminal domain of ANXA1. (D) Typical current traces of InsP₃R-3 in cyto-out configuration with pipette containing 300 μM Ca²⁺_ER, with or without 32 μM ANXA1 N-terminal peptide. Isolated patches perfused with solution containing 2 μM Ca²⁺_i, 10 μM InsP₃ and 0.5 mM ATP^4–. (E) P_o of individual current traces (circles) and average and s.e.m. (horizontal bars) for cyto-out experiments with (black) or without (grey) 32 μM ANXA1 N-terminal peptides.

Annexins have a conserved C-terminal ‘annexin core’ domain with multiple ‘annexin-type’ Ca²⁺-binding sites (Gerke and Moss, 2002). In contrast, the N-terminal regions are diverse (Raynal and Pollard, 1994). That ANXA1, but neither ANXA6 nor ANXA2, inhibited gating of InsP₃R channels suggests that inhibition is mediated by interaction of its N-terminal domain with the InsP₃R. To determine if the N-terminal domain itself can affect channel activity, we included 32 μM N-terminal peptide of human ANXA1 (Figure 10C) in the pipette solution with 300 μM Ca²⁺_ER, and found that channel P_o was unaffected (Figure 10D–E). This suggests that whereas the N-terminal domain may mediate interaction between ANXA1 and the InsP₃R L2 region, the ANXA1 C-terminal domain is likely needed to localize ANXA1 to the two-dimensional surface of the ER membrane to increase its local concentration to facilitate N-terminus binding to the InsP₃R to suppress channel activity.

Endogenous ANXA1 inhibits InsP₃R-mediated Ca²⁺release in intact cells

To determine effects of ANXA1 on cytoplasmic Ca²⁺ signaling, we used siRNA to reduce ANXA1 protein expression after 48 hr to ~35% of WT levels in HEKtsA201 cells (Figure 11A–B), and measured magnitude (ΔR_max) and rate of change (1/τ) of Fura-2 fluorescence ratio in populations of cells responding to the muscarinic receptor agonist carbachol (Figure 11C) in a bath solution containing 1.5 mM CaCl₂. These parameters capture the initial InsP₃R-mediated Ca²⁺-release phase of the response to agonist. Both parameters were enhanced specifically in the ANXA1 siRNA-treated cells (Figure 11D–E, respectively), suggesting that endogenous ANXA1 inhibits InsP₃R-mediated Ca²⁺ release.

Figure 11

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Endogenous ANXA1 inhibits InsP₃R-mediated Ca²⁺ release.

(A) Western blot of ANXA1 and β-actin in lysates from HEKtsA201 cells treated with transfection medium (left) or *ANXA1* siRNA (right) (one of four similar blots shown). (B) Summary of ANXA1 protein knockdown. HEKtsA201 cells treated with *ANXA1* siRNA (four samples), transfection medium only (two samples) or with non-targeting (N-T) siRNA (two samples). (C) Typical trace of fura-2 fluorescence ratio (ΔR) observed in population of intact HEKtsA201 cells stimulated by 10 μM carbachol (CCH). Maximal rise in ΔR (ΔR_max): blue double arrow; time constant (τ) of single exponential fit to rising phase of fluorescence ratio (red curve) is time for ΔR to rise to [1–1/e] of ΔR_max: red double arrow. (D) Normalized ΔR_max of individual ΔR traces (circles) and averages and s.e.m. (horizontal bars) for *ANXA1* siRNA-treated (right) and control cells (left). Number of traces tabulated next to horizontal bars. (E) Normalized rate of change of ΔR (1/τ) for ΔR traces using convention as (D). (F) ANXA1 immunofluorescence intensity of HeLa cells treated with non-targeting (N-T) or *ANXA1* siRNA. Number of cells tabulated below corresponding circles. (G) Fractions of N-T or *ANXA1* siRNA-treated HeLa cells that responded by ER Ca²⁺ release through InsP₃R when stimulated by sub-saturating 10 (red) or saturating 100 (blue) μM histamine. (**H–I**) Traces of mean normalized ER Ca²⁺ release from N-T (red) or *ANXA1* (black) siRNA-treated HeLa cells responding to 100 μM (H) or 10 μM (I) histamine. (J) Fractions of N-T or *ANXA1* siRNA-treated HeLa cells that oscillated in response to 10 (red) or 100 (blue) μM histamine. (K) Selected traces showing different kinds of Ca²⁺ signals in *AnxA1* siRNA-treated HeLa cells responding to sub-saturating 10 μM histamine. (L) Typical fluorescence amplitude (ΔF/F₀) traces showing local Ca²⁺ release events (puffs) in HEK293 cells treated with N-T (black) or *ANXA1* (red) siRNA. Cells stimulated by photolysis of caged i-InsP₃ using sub-maximal 50 ms UV flash. (**M–P**) Puffs generated by 50 ms UV flash were subsequently observed for 30 s in eight imaging fields (M); puffs generated by maximal 150 ms UV flash and subsequently observed for 10 s in six imaging fields (N). Dots indicate numbers of puffs observed for N-T (black) and *ANXA1* (red) siRNA-treated cells. Means and s.e.m. indicated by bars. (**O–P**) Dot plots of ΔF/F₀ of individual Ca²⁺ puffs observed in N-T (black) and *ANXA1* (red) siRNA-treated cells, generated by 50 ms (O) and 150 ms (P) UV flashes, respectively. Means and s.e.m. indicated by diamonds and bars, respectively.

We also measured histamine-induced changes in Fura-2 fluorescence ratio (R/R₀) in individual ANXA1 and non-targeting (N-T) siRNA-treated HeLa cells (Figure 11F). Saturating (100 µM) histamine elicited similar responses in both kinds of siRNA-treated cells (Figure 11H). In contrast, in sub-saturating (10 µM) histamine, an increase in both the fraction of responding cells (Figure 11G) and the maximal increase in R/R₀ was observed in the ANXA1 siRNA-treated cells (Figure 11I). We classified responding cells as ‘oscillatory’ or ‘non-oscillatory’ (Figure 11K). In 100 µM histamine, the fraction of ocillatory cells was indistinguishble between AnxA1 or N-T siRNA-treated cells; whereas in 10 µM histamine, a significantly higher fraction of ANXA1 siRNA-treated cells showing either ‘random spiking’ or ‘periodic oscillations’ (Figure 11J).

We explored this further by visualizing elementary Ca²⁺-release events (Ca²⁺ puffs) generated by clusters of InsP₃R stimulated by uncaging of a non-metabolizable InsP₃ analogue (Figure 11L; Lock et al., 2018). More Ca²⁺ puffs were observed in the cells treated with ANXA1-siRNA than in cells treated with non-targeting siRNAs (Figure 11M–N), with larger amplitudes (Figure 11O–P), regardless of whether they were generated by a sub-saturating UV flash (50 ms, Figure 11M and O) or a saturating one (150 ms, Figure 11N and P). Taken together, these results suggest that endogenous luminal ANXA1 inhibits InsP₃R-mediated Ca²⁺ release in vivo, in strong agreement with the strong inhibition by ANXA1 of InsP₃R channel activity observed in vitro.

Discussion

A novel mechanism of strong regulation of InsP₃R channel activity by ER [Ca²⁺]

Our study has established that physiological [Ca²⁺]_ER in the normally-replete ER lumen strongly inhibits the activity of the InsP₃R Ca²⁺-release channel, with consequent effects on InsP₃-mediated Ca²⁺ signals in cells. This inhibitory effect is not mediated by direct Ca²⁺ binding to luminal sites on the InsP₃R nor by flux-through effects of Ca²⁺ on cytoplasmic regulatory sites. Rather, it is mediated by a direct interaction of the InsP₃R with a lumen-localized membrane-associated protein, which is likely to be ANXA1.

We first established that high [Ca²⁺]_ER strongly inhibits InsP₃R channel activity by a mechanism involving a putative ER-luminal peripheral protein. To identify the protein, we focused on a segment that is highly conserved in all three InsP₃R isoforms and which, according to a 2015 cryo-EM structure (Fan et al., 2015), is located as a luminal loop (L2) between transmembrane helices TM3 and TM4 of InsP₃R-1. Interestingly, the L2 sequence was assigned to a different location in a recent cryo-EM structure of InsP₃R-3 (Paknejad and Hite, 2018). Nevertheless, we discovered ANXA1 by pull-down with the pL2 peptide, and inhibition of channel activity by high [Ca²⁺]_ER was relieved by inclusion of L2 peptides in the ER lumen. These experiments establish that the inhibition of InsP₃R activity is mediated through a protein interaction with the InsP₃R L2 region. The structures in Paknejad and Hite, 2018 have closed channel pores despite binding of InsP₃ and Ca²⁺. Of note, ANXA1 reduces, but does not completely abrogate activity of the InsP₃R. It is possible that the topology of the L2 region is different in functionally distinct channel-activation states, with some states enabling it to interact in the ER lumen with ANXA1.

Whereas the predominance of our data suggests that the luminal protein responsible for InsP₃R inhibition in high [Ca²⁺]_ER is ANXA1, some results give us pause. First, we have been unable to co-immunoprecipitate (co-IP) the two proteins from cell lysates. There are many reasons why co-IP can fail, including low expression of ANXA1 in the ER lumen, particularly in comparison with its strong expression in the cytoplasm; and that the interacting regions in InsP₃R and ANXA1 are small, particularly in the case of the InsP₃R. Second, whereas we have been able to apply immunofluorescence microscopy to localize ANXA1 to the nuclear envelope in different permeabilized cells with cytoplasmic ANXA1 washed out, localization to the ER has been unconvincing (Figure 3A–E). This may reflect a very low abundance of luminal ANXA1 in the peripheral ER that escapes detection. Third, effects of ANX1A knockdown on intracellular Ca²⁺ signaling were modest (Figures 11D–E,G–J,M–P). This could be due to the fact that knockdown reduced the ANXA1 expression level by only about 65% (Figure 11B and F). Taken together, these results may suggest that ANXA1 is not the bona fide PLP. Other ER-luminal proteins have also been reported to regulate InsP₃R activity, but their effects are all very different from those of the PLP defined here. Direct interaction with chromogranins enhances activity (Yoo and Lewis, 2000), and BiP/Grp78 promotes tetramerization of InsP₃R-1, thereby enhancing Ca²⁺ release (Higo et al., 2010). In contrast, the PLP here inhibits InsP₃R channel activity. Thioredoxin ERp44 interacts directly with InsP₃R to suppress channel activity (Higo et al., 2005). However, ERp44-mediated inhibition is reduced as [Ca²⁺]_ER increases beyond 100 μM, a [Ca²⁺]_ER dependence opposite to that of the PLP defined here. Furthermore, ERp44 interacts specifically with InsP₃R-1, whereas the PLP here inhibits all InsP₃R isoforms.

Annexins are defined by a conserved C-terminal Ca²⁺ binding domain (Gerke and Moss, 2002). Although the intrinsic affinity of ‘annexin-type’ Ca²⁺ binding sites is low (K_d up to mM range) (Raynal and Pollard, 1994), the presence of phospholipids, especially those with negatively-charged headgroups, strongly enhances Ca²⁺ affinity (Gerke and Moss, 2002). Conversely, the affinity of annexins for acidic phospholipids increases dramatically in the presence of high [Ca²⁺]_free(Raynal and Pollard, 1994; Gerke and Moss, 2002). This feature is likely the mechanism underlying the [Ca²⁺]_ER dependence of ANXA1 inhibition of InsP₃R activity. In low [Ca²⁺]_ER, ANXA1, having low affinity for phospholipids, remains in the bulk ER lumen, rendering an interaction between ANXA1 and InsP₃R channels that are confined to the ER membrane highly unfavorable entropically. With no ANXA1 interaction, the channel can gate robustly. In contrast, in high [Ca²⁺]_ER (>100 μM, Figure 7F), the ANXA1 C-terminus binds to phospholipids with high affinity and become restricted to the surface of the ER membrane. This substantially increases the chances for ANXA1 to interact with the InsP₃R, which promotes channel inhibition.

Physiological implications of ANXA1 regulation of the InsP₃R

Inhibition of InsP₃R by the PLP is very potent. With [Ca²⁺]_ER sufficiently high (≥300 μM), the PLP and ANXA1 strongly suppressed channel activation even in optimal concentrations of cytoplasmic Ca²⁺ and InsP₃, such that channel P_o is limited to ~0.1 (Figure 7F), merely 15% of the maximal P_o elicited by saturating [InsP₃] in absence of PLP (ANXA1) (Vais et al., 2012). This powerful effect is comparable in magnitude to the maximal inhibition of InsP₃R activity by high (50 μM) [Ca²⁺]_i(Vais et al., 2012). Furthermore, the range of [Ca²⁺]_ER over which this regulation occurs is within the physiological range. [Ca²⁺]_ER in fully-replete stores has been measured to be 250 μM – 1 mM (Zampese and Pizzo, 2012). We observed strong channel inhibition at 300 μM, with more profound inhibition at 600 μM. During agonist stimulation, bulk [Ca²⁺]_ER falls to concentrations that activate STIM1, which has an apparent luminal-Ca²⁺ affinity of 200–400 μM (Suzuki et al., 2014; Stathopulos et al., 2008). We observed apparent Ca²⁺ affinity of ANXA1-mediated inhibition of ~250 μM, suggesting that relief of inhibition will occur under physiological conditions that activate STIM1 and store-operated Ca²⁺ entry.

It has been suggested that most InsP₃R channels (~95%) in cells are unresponsive to experimentally-induced elevations of [InsP₃], with responsive channels limited to those in immobile clusters from which Ca²⁺ blips and puffs originate (Lock et al., 2019; Keebler and Taylor, 2017; Thillaiappan et al., 2017). The mechanisms that silence the majority of InsP₃R are unknown, but our results indicate that the PLP defined here (ANXA1) could play a role. In this scenario, most InsP₃R are normally associated with the PLP, rendering them unresponsive to InsP₃. However, channels in some clusters may lose their association with the PLP, possibly because of a unique phospholipid composition of the ER membrane there, or because the L2 sequence is not exposed in InsP₃R channels in clusters, or for other reasons. Channels in such clusters would be much more responsive to InsP₃ and ‘licensed’ to respond even with the ER fully replete with Ca²⁺. In addition, the strong inhibition of InsP₃R channel activity by ANXA1 could possibly help shape intracellular InsP₃-induced Ca²⁺ signals. PLP (ANXA1) inhibition will reduce the amount of Ca²⁺ released by shortening the open durations of activated channels (Figure 7H) and reducing their frequency by lengthening their closed durations (Figure 7I). With fewer Ca²⁺ release events with reduced magnitude, coordination by released Ca²⁺ from channels in a cluster to generate Ca²⁺ puffs can be hampered, preventing puffs from being organized into global Ca²⁺ signals. Such suppression of Ca²⁺ release by the PLP could ensure spatial fidelity of local Ca²⁺ signals and be a fail-safe mechanism to prevent excessive, detrimental release of Ca²⁺ from the ER. At reduced [Ca²⁺]_ER, the PLP regulation of InsP₃R activity could also be a mechanism to preserve fidelity of Ca²⁺ signals. As [Ca²⁺]_ER drops, the [Ca²⁺] gradient across the InsP₃R is reduced, and the Ca²⁺ flux it drives through the pore decreases. Between 100 and 600 μM [Ca²⁺]_ER, the six-fold reduction in Ca²⁺ flux could be compensated by the ~6.6 fold increase in the P_o of the InsP₃R channels (from ~0.09 to 0.6) as PLP inhibition of channel activity is alleviated (Figure 7F). This could allow the amount of Ca²⁺ released to be maintained despite the drop in [Ca²⁺]_ER to preserve Ca²⁺ signals required to regulate downstream cellular processes.

Limitations of the current study

As noted, co-immunoprecipitation of ANXA1 with the InsP₃R was not successful. Further studies, using different methods and antibodies should be performed to determine whether the proteins physically interact.
As noted, ANXA1 was secreted into the bath (Figure 6E), but its localization in permeabilized cells was evident only in the nuclear envelope (Figure 6A–D), and not in the peripheral ER. On the other hand, AnxA1 was initially identified as a potential protein that interacts with the InsP₃R through in-vitro pull-down assays and mass spectrometry, using ultra-centrifuged ruptured ER microsome fraction devoid of nuclei. Additional studies employing different antibodies and imaging techniques, including super-resolution light microscopy and electron microscopy should be performed to establish the presence of ANXA1 in the lumen of the entire ER.
We demonstrated increased channel P_o in the presence of the pL2 peptide and concluded that it was caused by the peptide reversal of the inhibition by luminal Ca²⁺. A control experiment to demonstrate that the pL2 peptide does not increase channel P_o under conditions with reduced luminal [Ca²⁺] should be performed. We note however that the channel P_o is already very high under conditions of reduced luminal [Ca²⁺]. In a control experiment, we could change the conditions, for example use lower [InsP₃] to observe channels with a low channel P_o in conditions of reduced luminal [Ca²⁺].

Materials and methods

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Peptide, recombinant protein	Anx A1	Abcam	ab86446
Peptide, recombinant protein	Anx A1 with N-terminal His tag	Abcam	ab184588
Peptide, recombinant protein	Anx A2	Abcam	ab93005
Peptide, recombinant protein	Anx A6	Abcam	ab92934
Peptide, recombinant protein	L2 peptide (GNRGTFIRGYKAMVMDMEFLYHVGYILTSVLGLFAHEL)	Peptide 2.0	custom
Peptide, recombinant protein	sL2 peptide (DVVIALHGNAMMYLLVFEHTSTGIGKFLRFYGERLMYG)	Peptide 2.0	custom
Peptide, recombinant protein	pL2 peptide (GNRGTFIRGYKAMVMDME)	Peptide 2.0	custom
peptide, recombinant protein	mpL2 peptide (K-Ahx-GNRGTFIRGYRAMVMDME, Ahx stands for 6-aminohexanoate residue)	Peptide 2.0	custom
Peptide, recombinant protein	smpL2 peptide (K-Ahx-RDYRGMRMIMGETFNVGA, Ahx stands for 6-aminohexanoate residue)	Peptide 2.0	custom
Peptide, recombinant protein	ANXA1 N-terminal peptide (MAMVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPYPT)	Peptide 2.0	custom
Chemical compound, drug	siRNA buffer	Dharmacon	B-002000-UB-100
Chemical compound, drug	siRNA transfection reagent	Dharmacon	T-2001–02
Chemical compound, drug	diBrBAPTA (5,5′-dibromo1,2-bis(o-amino phenoxy)ehane -N,N,N′,N′-tetraacetic acid)	Invitrogen	D-1211
Chemical compound, drug	diBrBAPTA (5,5′-dibromo1,2-bis(o-amino phenoxy)ehane -N,N,N′,N′-tetraacetic acid)	Santa Cruz Biotechnology	sc-2273516
Chemical compound, drug	2Hydroxyethyl)ethylenediaminetriacetic acid (HEDTA)	Sigma	H7154
Chemical compound, drug	inositol 1,4,5-trisphosphate	Invitrogen	I-3716
Chemical compound, drug	inositol 1,4,5-trisphosphate	Santa Cruz Biotechnology	sc-201521
Chemical compound, drug	NHS-activated magnetic beads	Pierce	88826
Chemical compound, drug	Protein A Dynabeads	ThermoFisher	10006D
Chemical compound, drug	Anti-Flag M2 agarose beads	Sigma	A2220
Chemical compound, drug	Fura-2 AM	Molecular Probes	I-1225
Chemical compound, drug	siGLO Red transfection indicator	Dharmacon	D-001630-02-05
Chemical compound, drug	Cal-520/AM	AAT Bioquest	21130
Chemical compound, drug	Caged Ins(1,4,5)P3/PM (caged InsP3)	Sirius Fine Chemical SiChem GmbH	cag-iso-2–145-
Chemical compound, drug	EGTA/AM	ThemoFisher	E1219
Cell line (Gallus gallus)	DT40 cells (wild-type)	Riken Bioresource Center	RCB1464; RRID:CVCL_0249
Cell line (Gallus gallus)	DT40-KO cells (with all three InsP₃R genes disrupted)	Riken Bioresource Center	RCB1467; RRID:CVCL_4634
Cell line (Gallus gallus)	DT40-r3 cells	ref. (Mak et al., 2013b) in this study	NA
Cell line (Homo-sapiens)	HEK293 cells	ATCC	CRL-1573; RRID:CVCL_0045
Cell line (Homo-sapiens)	HEK-3KO cells	Kerafast	EUR030; RRID:CVCL_HB82
Cell line (Homo-sapiens)	HEK293-3KO-r InsP3R-3 cells	this study	NA
Cell line Mus musculus	N2a cells	ATCC	CCL-131; RRID:CVCL_0470
Cell line (Rattus rattus)	PC12 cells	ATCC	CRL-1721; RRID:CVCL_0481
Cell line (Homo-sapiens)	tsA201 cells	Sigma-Aldrich	96121229; RRID:CVCL_2737
Cell line (Homo-sapiens)	A549 cells	ATCC	CCL-185; RRID:CVCL_0023
Genetic reagent (Homo sapiens)	Anx A1 siRNA	Dharmacon	M-011161-01-0005
Genetic reagent (Homo sapiens)	Non-targeting siRNA	Dharmacon	D-001206-13-05
Antibody	rabbit polyclonal anti-AnxA1 antibody	Proteintech	21990–1-AP; RRID:AB_11182596	WB: 1:1000-1:4000 IP: 1:1000-1:10000 IHC: 1:50-1:500 IF: 1:20-1:200
Antibody	mouse monoclonal anti-AnxA1 antibody	ECM Biosciences	AM0211	ELISA 1:1000 ICC 1:100 IP 1:100 WB 1:1000
Antibody	rabbit polyclonal anti-FLAG antibody	Cell Signaling	14793S; RRID:AB_2572291	WB: 1:1000 IP: 1:50 IHC: 1:800 IF: 1:800 FC: 1:1600 Chromatin IP: 1:50
Antibody	goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568	Invitrogen	A-11011; RRID:AB_143157	FC: 1–10 µg/mL ICC: 2 µg/mL IF: 2 µg/mL
Antibody	mouse monoclonal anti-calnexin antibody	Chemicon	MAB3126 RRID:AB_143157	ICC: 1:100-1:250 WB: 1:200-1:2000 IP: 1:200-1:1000
Antibody	rabbit polyclonal anti-β actin antibody	Cell Signaling	7881S; RRID:AB_1549731	capture Elisa: 1:100
Antibody	goat polyclonal anti-mouse IgG-HRP antibody	Cell Signaling	7074S; RRID:AB_2099233	capture Elisa: 1:1000-1:3000
Antibody	horse polyclonal anti-mouse IgG-HRP antibldy	Cell Signaling	7076S; RRID:AB_330924	capture Elisa: 1:1000-1:3000
Antibody	mouse monoclonal anti-βactin antibody	Cell Signaling	8H10D10; RRID:AB_2242334	WB: 1:1000 IHC: 1:8000-1:32000 IF: 1:2500-1:10000 FC: 1:200-1:800
Antibody	mouse monoclonal anti-type 3 InsP3R antibody	BD Transduction Laboratories	610312; RRID:AB_397704	WB: 1:2000-1:4000
Software, algorithm	QuB	refs. (Qin et al., 2000) and (Bruno et al., 2013) in this study		Quantitative single-channel analysis
Software, algorithm	IGOR Pro	Wavemetrics		Figure production and data fitting
Software, algorithm	Metamorph v7.7	Universal Imaging/Molecular Devices		Image analysis
Software, algorithm	Flika	Ellefsen et al., 2014		Image processing
Software, algorithm	Microcal Origin v6.0	OriginLab		Data analysis and graphing
Software, algorithm	Max Chelator	online freeware		Calculation of ion concentrations
Software, algorithm	MaxQuant, version 1.6.1.0	online freeware		Database search

Proteins

Recombinant full-length human annexin proteins from Abcam were used in our experiments: ANXA1 (cat. # ab86446 with no tag; ab184588 with N-terminal His tag), Anx A2 (cat. # ab93005 with N-terminal His tag), and Anx A6 (cat. # ab92934 with N-terminal His tag).

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Schematic diagram illustrating the orientation of InsP3R channels in isolated nuclear membrane patches and InsP3-containing solution relative to the micropipette in various configurations of nuclear patch-clamping.

Possible mechanisms of [Ca2+]ER regulation of InsP3R channel activity.

Raising [Ca2+]ER in intact isolated nuclei affects activities of InsP3R channels.

Activities of InsP3R channels from a number of cell lines expressing various InsP3R isoforms in different proportions are similarly affected by rise in [Ca2+]ER in intact isolated nuclei.

Identification of the InsP3R region involved in [Ca2+]ER regulation of channel activity.

Localization of AnxA1 in the ER lumen.

Figure 6—source data 1

[Ca2+]ER inhibition of InsP3R-3 channel activity is mediated by a specific interaction between the channel and ANXA1 in the ER lumen.

L2 peptide, but not scrambled L2 peptide, was able to compete ANXA1 from the InsP3R channel and restore high channel activity.

[Ca2+]ER inhibition of InsP3R channel activity is specifically mediated by ANXA1 through interaction with the L2 loop of InsP3R.

Endogenous ANXA1 inhibits InsP3R-mediated Ca2+ release.

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Horia Vais

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Min Wang

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Karthik Mallilankaraman

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Riley Payne

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Chris McKennan

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Jeffrey T Lock

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Lynn A Spruce

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Carly Fiest

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Matthew Yan-lok Chan

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Ian Parker

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Steven H Seeholzer

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J Kevin Foskett

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For correspondence

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Don-On Daniel Mak

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Schematic diagram illustrating the orientation of InsP₃R channels in isolated nuclear membrane patches and InsP₃-containing solution relative to the micropipette in various configurations of nuclear patch-clamping.

Possible mechanisms of [Ca²⁺]_ER regulation of InsP₃R channel activity.

Raising [Ca²⁺]_ER in intact isolated nuclei affects activities of InsP₃R channels.

Activities of InsP₃R channels from a number of cell lines expressing various InsP₃R isoforms in different proportions are similarly affected by rise in [Ca²⁺]_ER in intact isolated nuclei.

Identification of the InsP₃R region involved in [Ca²⁺]_ER regulation of channel activity.

[Ca²⁺]_ER inhibition of InsP₃R-3 channel activity is mediated by a specific interaction between the channel and ANXA1 in the ER lumen.

L2 peptide, but not scrambled L2 peptide, was able to compete ANXA1 from the InsP₃R channel and restore high channel activity.

[Ca²⁺]_ER inhibition of InsP₃R channel activity is specifically mediated by ANXA1 through interaction with the L2 loop of InsP₃R.

Endogenous ANXA1 inhibits InsP₃R-mediated Ca²⁺ release.