The potassium channel subunit K_V1.8 (Kcna10) is essential for the distinctive outwardly rectifying conductances of type I and II vestibular hair cells

Reviewer #1 (Public Review):

Summary:
In this paper, the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage-activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:
The experiments are comprehensive and clearly described, both in the text and in the figures. Statistical analyses are provided throughout.

Weaknesses:
None.

Reviewer #2 (Public Review):

The focus of this manuscript was to investigate whether Kv1.8 channels, which have previously been suggested to be expressed in type I hair cells of the mammalian vestibular system, are responsible for the potassium conductance gK,L. This is an important study because gK,L is known to be crucial for the function of type I hair cells, but the channel identity has been a matter of debate for the past 20 years. The authors have addressed this research topic by primarily investigating the electrophysiological properties of the vestibular hair cells from Kv1.8 knockout mice. Interestingly, gK,L was completely abolished in Kv1.8-deficient mice, in agreement with the hypothesis put forward by the authors based on the literature. The surprising observation was that in the absence of Kv1.8 potassium channels, the outward potassium current in type II hair cells was also largely reduced. Type II hair cells express the largely inactivating potassium conductance g,K,A, but not gK,L. The authors concluded that heteromultimerization of non-inactivating Kv1.8 and the inactivating Kv1.4 subunits could be responsible for the inactivating gK,A. Overall, the manuscript is very well written and most of the conclusions are supported by the experimental work. The figures are well described, and the statistical analysis is robust.

My only comment relates to the statement regarding the results providing "evidence" that Kv1.4 form heteromultimers with Kv1.8 channels (see Discussion). The only data I can see from the results is that Kv1.4 channels are expressed in the membrane of type II hair cells, which is not sufficient evidence for the above claim. Is the distribution of Kv1.8 and Kv1.4 overlapping in type II hair cells? Have the authors attempted to perform some pharmacological studies on Kv1.4? For example, would gK,A be completely blocked by a Kv1.4 antagonist? Addressing at least some of these questions would strengthen your argument.

Reviewer #3 (Public Review):

Summary:
This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts, and heterozygotes) are examined here and whole-cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in the current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition, immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that the removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

1. From previous work it is known that GK,L in type I hair cells have unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+, and modulated by cyclic GMP. The question arises, if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells and immunostaining for Kv1.4 channels in rat utricle presented here in Fig. 6. However, hair cell-specific partner interactions with Kv1.8 that result in GK,L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

2. Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

3. Strong immunosignal appears in the cuticle plates of hair cells in addition to signal in basal regions of hair cells and supporting cells. Please provide a possible explanation for this.

4. A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be very interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Author Response

eLife assessment

This study provides direct evidence showing that Kv1.8 channels underly several potassium currents in the two types of sensory hair cells found in the mouse vestibular system. This is an important finding because the nature of the channels underpinning the unusual potassium conductance gK,L in type I hair cells has been under scrutiny for many years. Although most of the experimental evidence is compelling and the analysis is rigorous, the evidence supporting some of the claims related to Kv1.4 channels is incomplete. The study will be of interest to cell and molecular biologists and auditory neuroscientists.

We are thankful to the editor and reviewers for their thorough assessment of our work and insightful feedback. Our responses to the comments and suggestions are below.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage-activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:

The experiments are comprehensive and clearly described, both in the text and in the figures. Statistical analyses are provided throughout.

Weaknesses:

None.

Reviewer #2 (Public Review):

The focus of this manuscript was to investigate whether Kv1.8 channels, which have previously been suggested to be expressed in type I hair cells of the mammalian vestibular system, are responsible for the potassium conductance gK,L. This is an important study because gK,L is known to be crucial for the function of type I hair cells, but the channel identity has been a matter of debate for the past 20 years. The authors have addressed this research topic by primarily investigating the electrophysiological properties of the vestibular hair cells from Kv1.8 knockout mice. Interestingly, gK,L was completely abolished in Kv1.8-deficient mice, in agreement with the hypothesis put forward by the authors based on the literature. The surprising observation was that in the absence of Kv1.8 potassium channels, the outward potassium current in type II hair cells was also largely reduced. Type II hair cells express the largely inactivating potassium conductance gK,A, but not gK,L. The authors concluded that heteromultimerization of non-inactivating Kv1.8 and the inactivating Kv1.4 subunits could be responsible for the inactivating gK,A. Overall, the manuscript is very well written and most of the conclusions are supported by the experimental work. The figures are well described, and the statistical analysis is robust.

My only comment relates to the statement regarding the results providing "evidence" that Kv1.4 form heteromultimers with Kv1.8 channels (see Discussion). The only data I can see from the results is that Kv1.4 channels are expressed in the membrane of type II hair cells, which is not sufficient evidence for the above claim. Is the distribution of Kv1.8 and Kv1.4 overlapping in type II hair cells? Have the authors attempted to perform some pharmacological studies on Kv1.4? For example, would gK,A be completely blocked by a Kv1.4 antagonist? Addressing at least some of these questions would strengthen your argument.

Author response: With respect to the “evidence” for heteromultimerization of Kv1.4 and Kv1.8: We agree that there is not conclusive evidence but have pulled together reasons to suggest that the fast inactivation of Kv1.8-dependent gA in type II hair cells reflects a contribution from Kv1.4 subunits. The reasons we note are mostly from other sources: 1) Kv1.4 subunits are the only Kv1 alpha subunits known to make channels with intrinsic rapid inactivation (Bertoli et al., 1994); 2) Kv1.4 is highly expressed in type II hair cells, but not type I hair cells, in mouse utricle (McInturff et al., Biol. Open., 2018; Jan et al., Cell Reports, 2021; Orvis et al., Nat. Methods, 2021); 3) previous work from M. Correia and colleagues suggested Kv1.4 as the likely source of A-current in pigeon vestibular hair cells; 4) some rat type II hair cells show comparatively strong Kv1.4-like immunoreactivity (our Fig. 5). While we consider heteromultimerization of Kv1.4 and Kv1.8 alpha subunits a plausible explanation consistent with available data from different sources, we agree that the question is not at all settled, and indeed raise the possibility that KV beta subunits, which are also differentially expressed by type I and II hair cells, play a role. Experiments to definitively advance or refute this hypothesis are beyond the scope of this paper.

Reviewer #3 (Public Review):

Summary:

This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular, amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts, and heterozygotes) are examined here and whole-cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in the current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition, immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that the removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

1. From previous work it is known that GK,L in type I hair cells has unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+, and modulated by cyclic GMP. The question arises, if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells and immunostaining for Kv1.4 channels in rat utricle presented here in Fig. 6. However, hair cell-specific partner interactions with Kv1.8 that result in GK,L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

Author response: Our results raise the question of how Kv1.8/Kcna10 is regulated to produce gK,L in type I hair cells, which has different properties from the Kv1.8 conductance expressed heterologously (Lang et al., Am. J. Physiol. Renal Physiol., 2000; Ranjan et al., Front. Cell. Neurosci., 2019; Dierich et al., Cell Reports, 2020) and the Kv1.8 conductance inferred in inner hair cells (Dierich et al., 2020). We lay out several possibilities in the Discussion, but testing these suggestions is beyond the scope of the present paper.

The relatively high Cs+ permeability of gK,L (0.31 pCs/pK, Rüsch & Eatock, J. Neurophysiol., 1996; Rennie & Correia, J. Membr. Biol., 2000) suggests there is something different about the selectivity filter and pore region of gK,L relative to most Kv1 family members. Although the intrinsic Cs+ permeability of heterologously expressed Kv1.8 is not reported. While we note that the pore region in Kv1.8 differs from other Kv1 subunits by a single amino acid (a glycine instead of alanine at position 411 – placed by AlphaFold in the pore helix of hKCNA10, Jumper et al., Nature, 2021), the effect of this difference is not known. A separate study is needed to determine why gK,L has a high Cs+ permeability relative to other Kv channels.

For type II hair cells, the Cs+ permeability of Kv currents has not been fully characterized. Internal Cs+ does appear to reduce outward current more effectively in type II hair cells (Lang & Correia, J. Neurophysiol., 1989; Sokolowski et al., Dev. Biol., 1993) than in type I hair cells (Rüsch & Eatock, J. Neurophysiol., 1996; Rennie & Correia, J. Membr. Biol., 2000).

With respect to cochlear inner hair cells, note that the assignment of Kv1.8 by Dierich et al. (2021) to a delayed rectifier in cochlear inner hair cells (IHCs) was based on inference – that is, existing inner ear expression databases show that Kv1.8 is expressed in IHCs, and heterologous Kv1.8 channels have a current resembling that observed in IHCs after block of multiple other K channels. We agree with Dierich et al. that Kv1.8 is an attractive candidate for the residual conductance in cochlear IHCs based on comparison with its properties in heterologous expression data. Together their study and our study suggest that Kv1.8 takes on quite different voltage dependence depending on the hair cell environment, and it will be an interesting challenge to sort out the reasons.

1. Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

Author response: Our pharmacology with XE991 suggests a small but significant population of Kv7 channels in type I and II hair cells (Fig 7). With the immunogold technique, Kharkovets et al. (PNAS, 2000) and Hurley et al. (J. Neurosci., 2006) counted significant Kv7.4 particles in type I hair cells, although the particles occurred at much greater density in the postsynaptic calyx membrane facing the hair cell. These results lead us to propose that the Kv7 channel we identified pharmacologically includes the Kv7.4 subunit, possibly in combination with other Kv7 subunits (Lysakowski et al., J. Neurosci., 2011). By this argument, the absence of clear hair cell staining in the confocal images of Fig. 5A is likely to reflect differences in methods, which include the use of different mouse strains, different sensitivities of immunogold vs. confocal imaging, and different antibodies.

Holt et al. (J. Neurosci., 2007) indeed saw inhibition of gK,L in hair cells grown in organotypic cultures of the neonatal mouse utricle after viral expression of a dominant negative Kv7.4 construct. However, other studies show that Kv7 antagonists do not block gK,L (Hurley et al., J. Neurosci., 2006), and the Jentsch group, which first proposed Kv7.4 as a likely candidate for gK,L (Kharkovets et al., PNAS, 2000), ultimately showed that knocking out Kv7.4 and Kv7.5 expression failed to eliminate gK,L (Spitzmaul et al., J. Biol. Chem., 2013). Together, these results suggest that in Holt et al. (2007), the inhibition of gK,L by transfection with the dominant negative KCNQ4 construct may have occurred through unintended interactions with native gK,L channels. The young age of the neonatal cultured and transfected utricles raises the possibility of a developmental effect – that functional Kv7 channels are needed for the developmental transition to a Kv1.8 conductance. Consistent with this idea is the observation that Kv7 current is present in neonatal hair cells, where it is a relatively large proportion of Kv current in type I HCs before they acquire gK,L (Hurley et al., J. Neurosci., 2006). Alternatively, the overexpression of nonfunctional Kv7.4 channels in virally-transfected hair cells may have inhibited or delayed gK,L acquisition through a more general effect on membrane proteins.

1. Strong immunosignal appears in the cuticle plates of hair cells in addition to signal in basal regions of hair cells and supporting cells. Please provide a possible explanation for this.

Author response: There is significant non-specific staining of apical cell surfaces and supporting cell membranes in addition to specific staining of hair cell basolateral membranes. We infer non-specific staining when immunolabeling is present in the knockout tissue, as it is for the apical surfaces and supporting cell membranes—compare Fig. 5B.3 (control tissue) with Fig. 5B.4 (Kv1.8 null mutant). Non-specific immunostaining can occur with polyclonal antibodies (specific to several epitopes) if the antibodies are not affinity-purified, but we used an affinity-purified antibody. The apical surfaces are reputed to be “sticky” (susceptible to non-specific staining) but the non-specific labeling in the basal parts of supporting cells is more puzzling. One possibility is that the Kv1.8 antibody weakly recognized closely related Kv1.1 channels, which are more strongly expressed in supporting cells than hair cells (Scheffer et al., J. Neurosci., 2015).

1. A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be very interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Author response: We agree; some of these questions are the subject of another paper in preparation.