The receptor potentials of hair cells (HCs) are strongly shaped by large outwardly rectifying K+ conductances that are differentially expressed according to HC type. Here we report that a specific voltage-gated K+ (KV) channel subunit participates in very different KV channels dominating the membrane conductances of type I and type II HCs in amniote vestibular organs.

Type I HCs occur only in amniote vestibular organs. Their most distinctive features are that they are enveloped by a calyceal afferent terminal (Wersäll, 1956; Lysakowski and Goldberg, 2004) and that they express gK,L (Correia and Lang, 1990; Rennie and Correia, 1994; Rüsch and Eatock, 1996a): a large noninactivating conductance with an activation range from –100 to –60 mV, far more negative than other “low-voltage-activated” KV channels. In addition to selectively attenuating and speeding up the receptor potentials of type I HCs (Correia et al., 1996; Rüsch and Eatock, 1996b), gK,L augments non-quantal transmission from type I hair cell to afferent calyx by providing open channels for K+ flow into the synaptic cleft (Contini et al., 2012, 2017, 2020; Govindaraju et al., 2023), increasing the speed and linearity of the transmitted signal (Songer and Eatock, 2013).

Type II HCs have compact afferent synaptic contacts (boutons) where the receptor potential drives quantal release of glutamate. They have fast-inactivating (A-type, gA) and delayed rectifier (gDR) conductances that are opened by depolarization above resting potential (Vrest).

The unusual properties of gK,L have long attracted curiosity about its molecular nature. gK,L has been proposed to include “M-like” KV channels in the KV7 and/or erg channel families (Kharkovets et al., 2000; Hurley et al., 2006; Holt et al., 2007). The KV7.4 subunit was of particular interest because it contributes to the low-voltage-activated conductance, gK,n, in cochlear outer hair cells, but was eventually eliminated as a gK,L subunit by experiments on KV7.4-null mice (Spitzmaul et al., 2013).

Several observations suggested the KV1.8 (KCNA10) subunit as an alternative candidate for gK,L. KV1.8 is highly expressed in vestibular sensory epithelia (Carlisle et al., 2012), particularly hair cells (Lee et al., 2013; Scheffer et al., 2015; McInturff et al., 2018), with slight expression elsewhere (skeletal muscle, Lee et al., 2013; kidney, Yao, 2002). KV1.8−/− mice show absent or delayed vestibular-evoked potentials, the synchronized activity of afferent nerve fibers sensitive to fast linear head motions (Lee et al., 2013). Unique among KV1 channels, KV1.8 has a cyclic nucleotide binding domain (Lang et al., 2000) with the potential to explain gK,L’s known cGMP dependence (Behrend et al., 1997; Chen and Eatock, 2000).

Our comparison of whole-cell currents and immunohistochemistry in type I HCs from KV1.8−/− and KV1.8+/+,+/– mouse utricles confirmed that KV1.8 expression is necessary for gK,L. More surprisingly, KV1.8 expression is also required for A-type and delayed rectifier conductances of type II HCs. In both HC types, eliminating the KV1.8-dependent major conductances revealed a smaller delayed rectifier conductance involving KV7 channels. Thus, the distinctive outward rectifiers that produce such different receptor potentials in type I and II HCs both include KV1.8 and KV7 channels.


We compared whole-cell voltage-activated K+ currents in type I and type II hair cells from homozygous knockout (KV1.8−/−) animals and their wildtype (KV1.8+/+) or heterozygote (KV1.8+/–) littermates. We immunolocalized KV1.8 subunits in the utricular epithelium and pharmacologically characterized the residual K+ currents of KV1.8−/− animals. Current clamp experiments demonstrated the impact of KV1.8-dependent currents on passive membrane properties.

We recorded from three utricular zones: lateral extrastriola (LES), striola, and medial extrastriola (MES) (Fig. 3A.1); striolar and extrastriolar zones have many structural and functional differences and give rise to afferents with different physiology (reviewed in Goldberg, 2000; Eatock and Songer, 2011). Recordings are from 412 type I and II HCs (53% LES, 30% MES, 17% striola) from mice between postnatal day (P) 5 and P370. We recorded from such a wide age range to test for developmental or senescent changes in the impact of the null mutation. Above P18, we did not see substantial changes in KV channel properties, as reported (González-Garrido et al., 2021).

As reported (Lee et al., 2013), KV1.8−/− animals appeared to be healthy and to develop and age normally.

KV1.8 is necessary for gK,L in type I hair cells

The large low-voltage activated conductance, gK,L, in KV1.8+/+,+/– type I hair cells produces distinctive whole-cell current responses to voltage steps, as highlighted by our standard type I voltage protocol (Fig. 1A). From a holding potential within the gK,L activation range (here –74 mV), voltage steps to –124 mV, which is negative to EK and the activation range, producing a large inward current through open gK,L channels that rapidly decays as the channels deactivate. The large transient inward current is a hallmark of gK,L. Steady-state activation was measured from tail currents after iterated 500-ms step voltages (Fig. 1A). We detected no difference between the Boltzmann parameters of gK,L G-V curves from KV1.8+/– and KV1.8+/+ type I HCs.

KV1.8−/− type I hair cells lacked gK,L, the dominant conductance in mature KV1.8+/+,+/– type I HCs.

Representative voltage-evoked currents in (A) a P22 KV1.8+/– type I HC and (B) a P29 KV1.8−/− type I HC. (A) Arrow, transient inward current that is a hallmark of gK,L. Note that the voltage protocol (top) in B extends to more positive voltages. Arrowheads, tail currents, magnified in insets. (C) Activation (G-V) curves from tail currents in A and B; symbols, data; curves, Boltzmann fits (Eq. 1). (D) Fit parameters from mice >P12 show big effect of KV1.8−/− and no difference between KV1.8+/– and KV1.8+/+. Asterisks (here and elsewhere): *, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001. Line, median; Box, interquartile range; Whiskers, outliers. See Table 1 for statistics.

Type I hair cell KV activation voltage dependence and kinetics. Mean ± SEM (number of cells). g is effect size, Hedge’s g. KWA is Kruskal-Wallis ANOVA.

For a similar voltage protocol, KV1.8−/− type I HCs (Fig. 1B) produced no inward transient current at the step from –74 mV to –124 mV and much smaller depolarization-activated currents during the iterated steps, even at much more positive potentials. Figure 1C compares the conductance-voltage (G-V, activation) curves fit to tail currents (Eq. 1; see insets in Fig. 1A-B): the maximal conductance (gmax) of the KV1.8−/− HC was over 10-fold smaller (Fig. 1C.1), and the curve was positively shifted by >40 mV (Fig. 1C.2). Figure 1D shows the G-V Boltzmann fit parameters for type I HCs from mice >P12, an age at which type I HCs normally express gK,L (Rüsch et al., 1998).

Suppl. Fig. 1 shows how G-V parameters of outwardly rectifying currents in type I HCs changed from P5 to P360. In KV1.8+/+,+,– mice, the parameters transitioned over the first 15-20 postnatal days from values for a conventional delayed rectifier, activating positive to resting potential, to gK,L values, as previously described (Rüsch et al., 1998; Géléoc et al., 2004; Hurley et al., 2006). Between P5 and P10, we detected no evidence of a non-gK,L KV1.8-dependent conductance in immature type I HCs (Suppl. Fig. 1B). In KV1.8− /− type I HCs, gK,L was absent and G-V parameters did not change much with age from P5 to P370.

The much smaller residual delayed rectifier activated positive to resting potential, with Vhalf ∼–40 mV and gmax density of 1.3 nS/pF. We characterize this KV1.8-independent delayed rectifier later. A much larger non-gK,L delayed rectifier conductance (“gDR,I”) was reported in our earlier publication on mouse utricular type I HCs (Rüsch et al., 1998). This current was identified as that remaining after “blocking” gK,L with 20 mM external Ba2+. Our new data suggest that there is no large non-gK,L conductance, and that instead high Ba2+ positively shifted the apparent voltage dependence of gK,L.

KV1.8 strongly affects type I passive properties and responses to current steps

While the cells of KV1.8−/− and KV1.8+/– epithelia appeared healthy, type I hair cells had smaller membrane capacitances (Cm) and presumably surface areas: 4-5 pF in KV1.8−/− type I HCs, ∼20% smaller than KV1.8+/– type I HCs (∼6 pF) and ∼30% smaller than KV1.8+/+ type I HCs (6-7 pF; Table 2). The decreased Cm may reflect deletion of a highly expressed trans-membrane protein (see discussion of gK,L channel density in Chen and Eatock, 2000, and for comparison the large decrease in outer hair cell size in the prestin null mutant (Liberman et al., 2002; Takahashi et al., 2018)).

Type I hair cell passive membrane properties. Mean ± SEM (number of cells). g is effect size, Hedge’s g. KWA is Kruskal-Wallis ANOVA.

Basolateral conductances help set the resting potential and passive membrane properties that regulate the time course and gain of voltage responses to small currents. To examine the effect of KV1.8 on these properties, we switched to current clamp mode and measured resting potential (Vrest), input resistance (Rin, equivalent to voltage gain for small current steps, △V/△I), and membrane time constant (τRC). In KV1.8−/− type I HCs, Vrest was much less negative (Fig. 2A.1), Rin was greater by ∼20-fold (Fig. 2A.2), and membrane charging times were commensurately longer (Fig. 2A.3).

KV1.8−/− type I hair cells had much longer membrane charging times and higher input resistances (voltage gains) than KV1.8+/+,+/– type I HCs.

(A) gK,L strongly affects passive membrane properties: (A.1) Vrest, (A.2) Rin, input resistance, and (A.3) membrane time constant, τRC = (Rinput * Cm). See Table 2 for statistics. (B) Current clamp responses to the same scale from (B.1) KV1.8+/– and (B.2) KV1.8−/− type I cells, both P29. Filled arrowhead (B.2), sag indicating IH activation. Open arrowhead, Depolarization rapidly decays as IDR activates. B.3, The 1st 6 ms of voltage responses to 170-pA injection is normalized to steady-state value; overlaid curves are double-exponential fits (KV1.8+/+, τ 40 μs and 2.4 ms) and single-exponential fits (KV1.8−/−, τ 1.1 ms) .

KV1.8−/− type II HCs in all zones of the sensory epithelium lacked the major rapidly inactivating conductance, gA, and had less delayed rectifier conductance. Activation and inactivation varied with epithelial zone and genotype.

(A) gA inactivation time course varied across zones. (A.1) Zones of the utricular epithelium. (A.2) Normalized currents evoked by steps from –124 mV to +30 mV with overlaid fits of Eq. 3. (A.3) τInact,Fast was faster in KV1.8+/– than KV1.8+/+ HCs, and faster in LES than other zones. Brackets show post hoc pairwise comparisons between two zones (vertical brackets) and horizontal brackets compare two genotypes; see Table 3 for statistics. (A.4) Fast inactivation was a greater fraction of total inactivation in LES than striola.

(B)Exemplars; ages, left to right, P312, P53, P287, P49, P40, P154.

(C)% inactivation at 30 mV was much lower in KV1.8−/− than KV1.8+/– and KV1.8+/+, and lower in striola than LES and MES. Interaction between zone and genotype was significant (Table 3).

(D)Exemplar currents and G-V curves from LES type II HCs show a copy number effect. (D.1) Currents for examples of the 3 genotypes evoked by steps from –124 mV to +30 mV fit with Eq. 3. (D.2) Averaged peak and steady-state conductance-voltage datapoints from LES cells (+/+, n=37; –/–, n=20) were fit with Boltzmann equations (Eq. 1) and normalized by gmax in (D.3). See Table 4 for statistics.

The differences between the voltage responses of KV1.8+/+,+/– and KV1.8−/− type I HCs are expected from the known impact of gK,L on Vrest and Rin (Correia and Lang, 1990; Ricci et al., 1996; Rüsch and Eatock, 1996b; Songer and Eatock, 2013). The large K+-selective conductance at Vrest holds Vrest close to EK (K+ equilibrium potential) and minimizes gain (△V/△I), such that voltage-gated conductances are negligibly affected by the input current and the cell produces approximately linear, static responses to iterated current steps. For KV1.8−/− type I HCs, with their less negative Vrest and larger Rin, positive current steps evoked a fast initial depolarization (Fig. 2B.2), activating residual delayed rectifiers and repolarizing the membrane toward EK. Negative current steps evoked an initial “sag” (Fig. 2B.2), a hyperpolarization followed by slow repolarization as the HCN1 channels open (Rüsch and Eatock, 1996b).

Overall, comparison of the KV1.8+/+,+/– and KV1.8−/− responses shows that with KV1.8 (gK,L), the voltage response of the type I hair cell is smaller but better reproduces the time course of the input current.

KV1.8 is necessary for both inactivating and non-inactivating KV currents in type II hair cells

Type II HCs also express KV1.8 mRNA (McInturff et al., 2018; Orvis et al., 2021). Although their outwardlyrectifying conductances (gA and gDR) differ substantially in voltage dependence and size from gK,L, both conductances were strongly affected by the null mutation: gA was eliminated and the delayed rectifier was substantially smaller. Below we describe gA and gDR in KV1.8+/+,+/– type II HCs and the residual outward rectifying current in KV1.8−/− type II HCs.

KV1.8+/+,+/– type II HCs

Most (81/84) KV1.8+/+,+/– type II HCs expressed a rapidly-activating, rapidlyinactivating A-type conductance (gA). We define A current as the outwardly rectifying current that inactivates by over 30% within 200 ms. gA was more prominent in extrastriolar zones, as reported (Holt et al. 1999, Weng and Correia 1999).

We compared the activation and inactivation time course and inactivation prominence for 200-ms steps from –124 mV to ∼30 mV. Outward currents fit with Eq. 3 yielded fast inactivation time constants (τInact, Fast) of ∼30 ms in LES (Fig. 3A.2). τInact, Fast was faster in LES than in MES or striola (Fig. 3A.3) and fast inactivation was a larger fraction of the total inactivation in LES than striola (∼0.5 vs. 0.3, Fig. 3A.4).

Type II hair cell KV currents: Activation and inactivation time course at +30 mV. Mean ± SEM. g is effect size, Hedge’s g. KWA is Kruskal-Wallis ANOVA.

To show voltage dependence of activation, we generated G-V curves for peak currents (sum of A-current and delayed rectifier) and steady-state currents measured at 200 ms, after gA has mostly inactivated (Figure 3D.2). KV1.8+/– HCs had smaller currents than KV1.8+/+ HCs, reflecting a smaller gDR (Fig. 3D) and faster fast inactivation (Fig. 3A.3). As discussed later, these effects may relate to effects of the KV1.8 gene dosage on the relative numbers of different KV1.8 heteromers.

For KV1.8+/+ and KV1.8+/– HCs, the voltage dependence as summarized by Vhalf and slope factor (S) was similar. Relative to gSS, gPeak had a more positive Vhalf (∼–21 vs. ∼–26) and greater S (∼12 vs. ∼9, Fig. 3D, Table 4). Because gPeak includes channels with and without fast inactivation, the shallower gPeak-V curve may reflect a more heterogeneous channel population. Only gPeak showed zonal variation, with more positive Vhalf in LES than striola (∼–20 mV vs. ∼–24 mV, Fig. 3D, Table 4). We later suggest that variable subunit composition may drive zonal variation in gPeak.

Type II hair cell KV currents: Activation voltage dependence. Mean ± SEM. g is effect size, Hedge’s g. KWA is Kruskal-Wallis ANOVA.

KV1.8−/− type II HCs

from all zones were missing gA and 30-50% of gDR (Fig. 3B-D). The residual delayed rectifier (1.3 nS/pF) had a more positive Vhalf than gDR in KV1.8+/+,+/– HCs (∼–20 mV vs. ∼–26 mV, Fig. 3D.2). We refer to the KV1.8-dependent delayed rectifier component as gDR(KV1.8) and to the residual, KV1.8-independent delayed rectifier component as gDR(KV7) because, as we show later, it includes KV7 channels. Supplemental Figure 3A shows the development of KV1.8-dependent and independent KV currents in type II HCs with age from P5 to over P300. In KV1.8+/+,+/– type II HCs, gA was present at all ages with a higher % inactivation after P18 than at P5-P10 (Suppl. Fig. 3A.4). gPeak did not change much above P12 except for a compression of conductance density from P13 to P370 (partial correlation coefficient = –0.4, p = 2E-5, Suppl. Fig. 3A.3).

We saw small rapidly inactivating outward currents in a minority of KV1.8−/− type II HCs (23%, 7/30), all >P12 and extrastriolar (Suppl. Fig. 4). These currents overlapped with gA in percent inactivation, inactivation kinetics, and activation voltage dependence but were very small. As discussed later, we suspect that these currents flow through homomers of inactivating KV subunits that in control hair cells join with KV1.8 subunits and confer inactivation on the heteromeric conductance.

KV1.8 affects type II passive properties and responses to current steps

In type II HCs, absence of KV1.8 did not change Vrest (Fig. 4A.1) because gA and gDR both activate positive to rest, but significantly increased Rin and τRC (Fig. 4A.2-A.3).

KV1.8−/− type II hair cells had larger, slower voltage responses and more electrical resonance.

(A) Passive membrane properties near resting membrane potential: A.1) Resting potential. Rinput(A.2) and τRC (A.3) were obtained from single exponential fits to voltage responses < 15 mV. See Table 5 for statistics.

(B)Exemplar voltage responses to iterated current steps (bottom) illustrate key changes in gain and resonance with KV1.8 knockout. (B.1) KV1.8+/– type II HC (P24, LES) and (B.2) KV1.8−/− type II HC (P53, LES). Arrowheads, depolarizing transients.

(C)Range of resonance illustrated for KV1.8−/− type II HCs (left, pink curves fit to Eq. 5) and controls (right, blue fits). (C.1) Resonant frequencies, left to right: 19.6, 18.4, 34.4, 0.3 Hz. Leftmost cell resonated spontaneously (before step). (C.2) Tuning quality (Qe; Eq. 6) was higher for KV1.8−/− type II HCs (KWA, p = 0.0064 vs. KV1.8+/+; p = 7E-8 vs. KV1.8+/–).

(D)KV1.8−/− type II HCs had higher, slower peaks and much slower rebound potentials in response to large (170-pA) current steps. (D.1) Normalized to show initial depolarizing transient (filled circles, times of peaks; horizontal arrows, peak width at half-maximum). (D.2) Longer time scale to highlight how null mutation reduced post-transient rebound.

(E)In KV1.8−/− HCs, depolarizing transients evoked by a +90-pA step were slower to peak (E.1) and (E.2) larger.

Type II hair cell passive membrane properties. Mean ± SEM (number of cells). g is effect size, Hedge’s g. KWA is Kruskal-Wallis ANOVA.

Key Resources Table

Positive current steps evoked an initial depolarizing transient in both KV1.8+/+ and KV1.8−/− type II HCs, but the detailed time course differed (Fig. 4B). Both transient and steady-state responses were larger in KV1.8−/−, consistent with their larger Rin values.

Absence of KV1.8 increased the incidence of sharp electrical resonance in type II HCs. Electrical resonance, which manifests as ringing responses to current steps, can support receptor potential tuning (Ashmore, 1983; Fettiplace, 1987; Hudspeth and Lewis, 1988; Ramanathan and Fuchs, 2002). Larger Rin values made KV1.8−/− type II HCs more prone to electrical resonance; Figure 4C.1 shows a striking example. Median resonance quality (Qe, sharpness of tuning) was greater in KV1.8−/− (1.33, n=26) than KV1.8+/+ (0.66, n=23) or KV1.8+/– (0.59, n=44) type II HCs.

KV1.8 affected the time course of the initial peak in response to much larger current injections (Fig. 4D-E). Fast activation of gA in control type II HCs rapidly repolarizes the membrane and then inactivates, allowing the constant input current to progressively depolarize the cell, producing a slow rebound (Fig 4D.2). This behavior has the potential to counter MET adaptation (Vollrath and Eatock, 2003).

KV1.8 immunolocalized to basolateral membranes of both type I and II HCs

If KV1.8 is a pore-forming subunit in the KV1.8-dependent conductances gK,L, gA, and gDR, it should localize to hair cell membranes. Figure 5 compares KV1.8 immunoreactivity in KV1.8+/+ and KV1.8−/− utricles, showing specific immunoreactivity along the basolateral membranes of both hair cell types in KV1.8+/+ utricles. To identify hair cell type and localize the hair cell membrane, we used antibodies against KV7.4 (KCNQ4), an ion channel densely expressed in the calyceal “inner-face” membrane next to the synaptic cleft (Hurley et al., 2006; Lysakowski et al., 2011), producing a cup-like stain around type I HCs (Fig. 5A). KV1.8 immunoreactivity was present in hair cell membrane apposing KV7.4-stained calyx inner face in KV1.8+/+ utricles (Fig. 5A.1, A.2) and not in KV1.8−/− utricles (Fig. 5A.3).

Type I and type II HC basolateral membranes show specific immunoreactivity to Kv1.8 antibody (magenta).

Antibodies for KV7.4 (A, green) and calretinin (B, cyan) were used as counterstains for calyx membrane (Kv7.4), type II HC cytoplasm (calretinin) and cytoplasm of striolar calyx-only afferents (calretinin). (A) Left, Cartoon showing KV7.4 on the calyx inner face membrane (CIF) and KV1.8 on the type I HC membrane. SC, supporting cell nuclei. A.1-3, Adult mouse utricle sections. KV7.4 antibody labeled calyces on two KV1.8-positive type I HCs (A.1), four KV1.8-positive type I HCs (A.2), and two KV1.8-negative type I HCs from a KV1.8−/− mouse (A.3).(B)Left, Cartoon showing cytoplasmic calretinin stain in calyx-only striolar afferents and most type II HCs, and KV1.8 on membranes of both HC types. In wildtype utricles, KV1.8 immunolocalized to basolateral membranes of type I and II HCs (B.1). KV1.8 immunolocalized to type I HCs in striola (B.2). Staining of supporting cell (SC) membranes by Kv1.8 antibody was non-specific, as it was present in KV1.8−/− tissue (B.3, B.4).

In other experiments, we used antibodies against calretinin (Calb2), a cytosolic calcium binding protein expressed by many type II HCs and also by striolar calyx-only afferents (Desai et al., 2005; Lysakowski et al., 2011) (Fig. 5B). A hair cell is type II if it is calretinin-positive (Fig. 5B.1) or if it lacks a KV7.4-positive or calretinin-positive calyceal cup (Fig. 5A.2, 5B.3, rightmost cells). Hair cell identification was confirmed with established morphological indicators: for example, type II HCs tend to have basolateral processes (feet) (Pujol et al., 2014) and, in the extrastriola, more apical nuclei than type I HC.

Previously, Carlisle et al. (2012) reported KV1.8-like immunoreactivity in many cell types in the inner ear. In contrast, Lee et al. (2013) found that gene expression reporters indicated expression only in hair cells and some supporting cells. Here, comparison of control and null tissue showed selective expression of HC membranes, and that some supporting cell staining is non-selective.

KV1.4 may also contribute to gA

Results with the KV1.8 knockout suggest that type II hair cells have an inactivating KV1 conductance that includes KV1.8 subunits. KV1.8, like most KV1 subunits, is not inactivating as a heterologously expressed homomer (Lang et al., 2000; Ranjan et al., 2019; Dierich et al., 2020), nor are the KV1.8-dependent channels in type I HCs, as we show, and cochlear inner hair cells (Dierich et al., 2020). KV1 subunits without intrinsic inactivation can produce rapidly inactivating currents by associating with KVβ1 (KCNB1) or KVβ3 subunits. KVβ1 is present in type II HCs alongside KVβ2 (McInturff et al., 2018; Jan et al., 2021; Orvis et al., 2021), which does not confer rapid inactivation (Dwenger et al., 2022).

An alternative possibility is that in type II HCs, KV1.8 subunits heteromultimerize with KV1.4 subunits – the only KV1 subunits which, when expressed as a homomer, have complete N-type (fast) inactivation (Stühmer et al., 1989). Multiple observations support this possibility. KV1.4 has been linked to gA in pigeon type II HCs (Correia et al., 2008) and is the second-most abundant KV1 transcript in mammalian vestibular HCs, after KV1.8 (Scheffer et al., 2015). KV1.4 is expressed in type II HCs but not type I HCs (McInturff et al., 2018; Orvis et al., 2021), and is not found in striolar HCs (Jan et al., 2021; Orvis et al., 2021), where even in type II HCs, inactivation is slower and less extensive (Fig. 3A).

In KV1.8+/+,+/– type II HCs, the time course (Fig. 3A, τFast,Inac of ∼30 ms +30 mV) and voltage dependence of inactivation of gA (Vhalf –41 mV, Fig. 6B.2), are consistent with heterologously expressed heteromers of KV1.4 with KV1.x and/or KVβ1 (Imbrici et al., 2006; Al-Sabi et al., 2011). In further support, we observed KV1.4-like immunoreactivity along the basolateral membranes of extrastriolar type II HCs in rat utricles (Fig. 6A).

KV1.4 subunits may contribute to gA in extrastriolar type II HCs.

(A)Immunostaining of adult rat utricular epithelium with KV1.4 antibody and TUJ-1, which labels afferent terminals, shows strong KV1.4-like immunoreactivity on the membranes of 2 type II HCs. Scale bar, 5 μm. (B)Voltage dependence of gA’s steady-state inactivation (h curve) and peak activation are consistent with KV1.4 heteromers. KV1.8+/+,+/– type II HCs, n=11, P40-P210, median P94. (B.1) The inactivation voltage protocol, bottom. Tail current is a function of the voltage dependence of accumulated steady-state inactivation. 100 μM ZD7288 in bath prevented contamination by HCN current. (B.2) Overlapping normalized activation and inactivation (“h-infinity”) G-V curves for data in B.1 at time points shown: peak currents (black squares, activation) and tail currents (red circles, inactivation). Curves, Boltzmann fits (Eq. 1). Average fit parameters for inactivation: Vhalf, –42 ±2 mV (n=11); S, 11 ± 1 mV. Activation: Vhalf, –23 ± 1 mV (n=11); S, 11.2 ± 0.4 mV.

KV7 channels contribute a small delayed rectifier in type I and type II hair cells

In KV1.8−/− HCs, absence of IK,L and IA revealed smaller delayed rectifier K+ currents that, unlike IK,L, activated positive to resting potential and, unlike IA, lacked fast inactivation. Candidate channels include members of the KV7 (KCNQ, M-current) family, which have been identified previously in rodent vestibular HCs (Kharkovets et al., 2000; Rennie et al., 2001; Hurley et al., 2006; Scheffer et al., 2015).

We test for KV7 contributions in KV1.8−/− type I HCs, KV1.8−/− type II HCs, and KV1.8+/+,+/– type II HCs of multiple ages by applying XE991 at 10 µM (Fig. 7A), a dose selective for KV7 channels (Brown et al., 2002) and close to the IC50 (Alexander et al., 2019). In KV1.8−/− HCs of both types, 10 µM XE991 blocked about half of the residual KV conductance (Fig. 7B.1), consistent with KV7 channels conducting most or all of the non-KV1.8 delayed rectifier current. In all tested HCs (P8-355, median P224), the XE991-sensitive conductance did not inactivate substantially within 200 ms at any voltage, consistent with KV7.2, 7.3, 7.4, and 7.5 currents (Wang, 1998; Kubisch et al., 1999; Schroeder et al., 2000; Jensen et al., 2007; Xu et al., 2007). We refer to this component as gDR(Kv7). The voltage dependence and Gmax density (Gmax/Cm) of gDR(Kv7) were comparable across HC types and genotypes (Figure 7B.2-4).

A KV7-selective blocker, XE991, reduced residual delayed rectifier currents in KV1.8−/− type I and II HCs.

(A)XE991 (10 μM) partly blocked similar delayed rectifier currents in type I and type II KV1.8−/− HCs and a type II KV1.8+/+ HC.

(B)Properties of XE991-sensitive conductance, gDR(KV7). (B.1) % Block of steady-state current. (B.2) tail G-V curves for KV1.8−/− type I HCs (n=8), KV1.8−/− type II HCs (9), and KV1.8+/+ type II HCs (5); mean ± SEM. (B.3) Vhalf was less negative in KV1.8+/+ type II than KV1.8−/− type I HC (p = 0.01, KWA). (B.4) Conductance density was similar in all groups (ANOVA, non-significant at 40% power (left), 20% power (right).

These results are consistent with similar KV7 channels contributing a relatively small delayed rectifier in both HC types. In addition, the similarity of XE991-sensitive currents of KV1.8+/+ and KV1.8−/− type II HCs indicates that knocking out KV1.8 did not cause general effects on ion channel expression. We did not test XE991 on KV1.8+/+,+/– type I HCs because gK,L runs down in ruptured patch recordings (Rüsch and Eatock, 1996a; Chen and Eatock, 2000; Hurley et al., 2006), which could contaminate the XE991-sensitive conductance obtained by subtraction.

In one striolar KV1.8−/− type I HC, XE991 also blocked a small conductance that activated negative to rest (Suppl. Fig. 5A-B). This conductance (Vhalf ∼= –100 mV, Suppl. Fig. 5C) was detected only in KV1.8−/− type I HCs from the striola (5/23 vs. 0/45 extrastriolar). The Vhalf and τdeactivation at –124 mV were similar to values reported for KV7.4 channels in cochlear HCs (Wong et al., 2004; Dierich et al., 2020). This very negatively activating KV7 conductance coexisted with the larger less negatively activating KV7 conductance (Suppl. Fig. 5C) and was too small (<0.5 nS/pF) to contribute significantly to gK,L (∼10-100 nS/pF, Fig. 1D).

Other channels

While our data are consistent with KV1.8- and KV7-containing channels carrying most of the outwardrectifying current in mouse utricular hair cells, there is evidence in other preparations for additional channels, including KV11 (KCNH, Erg) channels in rat utricular type I hair cells (Hurley et al., 2006) and BK (KCNMA1) channels in rat utricle and rat and turtle semicircular canal hair cells (Schweizer et al., 2009; Contini et al., 2020).

BK is expressed in mouse utricular hair cells (McInturff et al., 2018; Jan et al., 2021; Orvis et al., 2021). However, Ca2+-dependent currents have not been observed in mouse utricular HCs, and we found little to no effect of the BK-channel blocker iberiotoxin at a dose (100 nM) well beyond the IC50: percent blocked at –30 mV was 2 ± 6% (3 KV1.8−/− type I HCs); 1 ± 5% (5 KV1.8+/+,+/– type II HCs); 7% and 14% (2 KV1.8−/− type II HCs). We also did not see N-shaped I-V curves typical of many Ca2+-dependent K+ currents. In our ruptured-patch recordings, Ca2+-dependent BK currents and erg channels may have been eliminated by wash-out of the hair cells’ small CaV currents (Bao et al., 2003) or cytoplasmic second messengers (Hurley et al., 2006).

To check whether the constitutive KV1.8 knockout has strong non-specific effects on channel trafficking, we examined the summed HCN and inward rectifier currents (IH and IKir) at –124 mV, and found them similar across genotypes (Suppl. Fig. 6). In the process, we noted zonal differences in IH and IKir that have not been reported in hair cells. In type I HCs from both control and null utricles, IH and IKir were less prevalent in striola than extrastriola, and, when present, the combined inward current was smaller (Suppl. Fig. 6B).


We have shown that constitutive knockout of KV1.8 eliminated gK,L in type I HCs, and gA and much of gDR in type II HCs. KV1.8 immunolocalized specifically to the basolateral membranes of type I and II HCs. We conclude that KV1.8 is a pore-forming subunit of gK,L, gA, and part of gDR [gDR(KV1.8)]. We provide evidence that fast inactivation of gA may arise from heteromultimerization of non-inactivating KV1.8 subunits and inactivating KV1.4 subunits. Finally, we showed that a substantial component of the residual delayed rectifier current in both type I and type II HCs comprises KV7 channels.

KV1.8 is expressed in hair cells from mammalian cochlea (Dierich et al., 2020), avian utricle (Scheibinger et al., 2022), and zebrafish (Erickson and Nicolson, 2015). Our work suggests that in anamniotes, which lack type I cells and gK,L, KV1.8 contributes to gA and gDR, which are widespread in vertebrate HCs (reviewed in Meredith and Rennie, 2016). KV1.8 expression has not been detected in rodent brain but is reported in the pacemaker nucleus of weakly electric fish (Smith et al., 2018).

KV1.8 subunits may form homomultimers to produce gK,L in type I hair cells

Recent single-cell expression studies on mouse utricles (McInturff et al., 2018; Jan et al., 2021; Orvis et al., 2021) have detected just one KV1 subunit, KV1.8, in mouse type I HCs. Given that KV1.8 can only form multimers with KV1 family members, and given that gK,L channels are present at very high density (∼150 per μm2 in rat type I, Chen and Eatock, 2000), it stands to reason that most or all of the channels are KV1.8 homomers. Other evidence is consistent with this proposal. gK,L (Rüsch and Eatock, 1996a) and heterologously expressed KV1.8 homomers in oocytes (Lang et al., 2000) are non-inactivating and blocked by millimolar Ba2+ and 4-aminopyridine and >10 mM TEA. Unlike channels with KV1.1, KV1.2, and KV1.6 subunits, gK,L is not sensitive to 10 nM α-dendrotoxin (Rüsch and Eatock, 1996a). gK,L and heterologously expressed KV1.8 channels have similar single-channel conductances (∼20 pS for gK,L at positive potentials, Chen and Eatock, 2000; 11 pS in oocytes, Lang et al., 2000). gK,L is inhibited—or positively voltage-shifted— by cGMP (Behrend et al., 1997; Chen and Eatock, 2000), presumably via the C-terminal cyclic nucleotide binding domain of KV1.8.

A major novel property of gK,L is that it activates 30-60 mV negative to type II KV1.8 conductances and most other low-voltage-activated KV channels (Ranjan et al., 2019). The very negative activation range is a striking difference between gK,L and known homomeric KV1.8 channels. Heterologously expressed homomeric KV1.8 channels have an activation Vhalf of –10 to 0 mV (X. laevis oocytes, Lang et al., 2000; CHO cells, Dierich et al., 2020). In cochlear inner HCs, currents attributed to KV1.8 (by subtraction of other candidates) have a near-zero activation Vhalf (–4 mV, Dierich et al., 2020).

Possible factors in the unusually negative voltage dependence of gK,L include:

  1. elevation of extracellular K+ by the enveloping calyceal terminal, unique to type I HCs (Lim et al., 2011; Contini et al., 2012; Spaiardi et al., 2020; Govindaraju et al., 2023). High K+ increases conductance though gK,L channels (Contini et al., 2020), perhaps through K+-mediated relief of C-type inactivation (López-Barneo et al., 1993; Baukrowitz and Yellen, 1995). We note, however, that gK,L is open at rest even in neonatal mouse utricles cultured without innervation (Rüsch et al., 1998) and persists in dissociated type I HCs (Chen and Eatock, 2000; Hurley et al., 2006).

  2. The high density of gK,L (∼50 nS/pF in striolar KV1.8+/+ HCs) implies close packing of channels, possibly represented by particles (12-14 nm) seen in freeze-fracture electron microscopy of the type I HC membrane (Gulley and Bagger-Sjöbäck, 1979; Sousa et al., 2009). Such close channel packing might hyperpolarize in situ voltage dependence of gK,L, as proposed for KV7.4 channels in outer hair cells (Perez-Flores et al., 2020).

  3. Modulation by accessory subunits. Type I HCs express KVβ1 (McInturff et al., 2018; Orvis et al., 2021), an accessory subunit that can confer fast inactivation and hyperpolarize activation Vhalf by ∼10 mV. KVβ1 might interact with KV1.8 to shift voltage dependence negatively. Arguments against this possibility include that gK,L lacks fast inactivation (Rüsch and Eatock, 1996a; Hurley et al., 2006; Spaiardi et al., 2017) and that cochlear inner hair cells co-express KV1.8 and KVβ1 (Liu et al., 2018) but their KV1.8 conductance has a near-0 Vhalf (Dierich et al., 2020).

KV1.8 subunits may heteromerize with variable numbers of inactivating KV1.4 subunits to produce gA and KV1.8-dependent gDR in type II HCs

The KV1.8-dependent conductances of type II HCs vary in their fast and slow inactivation. In not showing fast inactivation (Lang et al., 2000; Ranjan et al., 2019; Dierich et al., 2020), heterologously expressed KV1.8 subunits resemble most other KV1 family subunits, with the exception of KV1.4 (for comprehensive review, see Ranjan et al., 2019). KV1.4 is a good candidate to provide fast inactivation based on immunolocalization and voltage dependence (Figs. 4, 6). We suggest that gA and gDR(KV1.8) are KV1.8-containing channels that vary in KV1.8:KV1.4 stoichiometry, with possible additional variation in KVβ2 and KVβ1 accessory subunits.

KV1.4-KV1.8 heteromeric assembly could account for several related observations. The faster τInact,Fast in KV1.8+/– relative to KV1.8+/+ type II HCs (Fig. 3A.3, Suppl. Fig. 2A.1) could reflect an increased ratio of KV1.4 to KV1.8 subunits and therefore more N-terminal inactivation domains per heteromeric channel. Zonal variation in the extent and speed of N-type inactivation (Fig. 3A) might arise from differential expression of KV1.4. The small fast-inactivating conductance in ∼20% of extrastriolar KV1.8−/− type II HCs (Suppl. Fig. 4) might flow through KV1.4 homomers.

In addition or alternatively, KVβ subunits are positioned to contribute to fast inactivation. KVβ1 is expressed in type II HCs (McInturff et al., 2018; Jan et al., 2021; Orvis et al., 2021), and, together with KV1.4, has been linked to gA in pigeon vestibular HCs (Correia et al., 2008). KVβ2, also expressed in type II HCs (McInturff et al., 2018; Orvis et al., 2021), does not confer fast inactivation but hyperpolarizes activation voltage by ∼10 mV and accelerates activation and inactivation kinetics (Heinemann et al., 1996). gA and gDR(KV1.8) are not likely to be different kinetic components of current through homogeneous incompletely inactivating KV channels. The lack of N-type inactivation of gDR(KV1.8) is readily explained by homomeric KV1.8 channels. The double-exponential decay of gPeak (comprising gA and gDR(KV1.8)) is consistent with two channel populations.

KV1.8 relevance for vestibular function

In both type I and type II utricular HCs, KV1.8-dependent channels strongly shape receptor potentials in ways that promote temporal fidelity rather than electrical tuning (Lewis, 1988), consistent with the utricle’s role in driving reflexes that compensate for head motions as they occur. This effect is especially pronounced for type I HCs, where the current-step evoked voltage response reproduces the input with great speed and linearity (Fig. 2).

gK,L dominates passive membrane properties in mature KV1.8+/+,+/– type I HCs such that KV1.8−/− type I HCs are expected to have receptor potentials with higher amplitudes but lower low-pass corner frequencies, closer to those of type II HCs and immature HCs of all types (Correia et al., 1996; Rüsch and Eatock, 1996a; Songer and Eatock, 2013). In KV1.8−/− epithelia, we expect the lack of a large basolateral conductance open at rest to reduce the speed and gain of non-quantal transmission, which depends on K+ ion efflux from the type I HC to change electrical and K+ potentials in the synaptic cleft (Govindaraju et al., 2023). In hair cells, K+ enters the mechanosensitive channels of the hair bundle from the K+-rich apical endolymph and exits through basolateral potassium conductances into the more conventional low-K+ perilymph. For the type I-calyx synapse, having in the hair cell a large, non-inactivating K+ conductance open across the physiological range of potentials avoids channel gating time and allows for instantaneous changes in current into the cleft and fast afferent signaling (Pastras et al., 2023).

In contrast, mature type II HCs face smaller synaptic contacts and have KV1.8-dependent currents that are not substantially activated at resting potential. They do affect the time course and gain of type II HC responses to input currents, speeding up depolarizing transients, producing a repolarizing rebound during the step, and reducing resonance.

Type I and II vestibular hair cells are closely related, such that adult type II HCs acquire type I-like properties upon deletion of the transcription factor Sox2 (Stone et al., 2021). In normal development of the two cell types, the Kcna10 gene generates importantly different ion channels, presenting a natural experiment in functional differentiation of sensory receptor cells.

Materials and methods


All procedures for handling animals followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of the University of Chicago and the University of Illinois Chicago. Most mice belonged to a transgenic line with a knockout allele of Kcna10 (referred to here as KV1.8−/−). Our breeding colony was established with a generous gift of such animals from Sherry M. Jones and Thomas Friedman. These animals are described in their paper (Lee et al., 2013). Briefly, the Texas A&M Institute for Genomic Medicine generated the line on a C57BL/6;129SvEv mixed background by replacing Exon 3 of the Kcna10 gene with an IRES-bGeo/Purocassette. Mice in our colony were raised on a 12:12h light-dark cycle with access to food and water ad libitum.

Semi-intact utricles were prepared from ∼150 male and ∼120 female mice, postnatal days (P) 5-375, for same-day recording. Hair cell KV channel data were pooled across sexes as most results did not appear to differ by sex; an exception was that gK,L had a more negative Vhalf in males (Suppl. Table 1), an effect not clearly related to age, copy number, or other properties of the activation curve.

Preparation, stimulation, and recording methods followed our previously described methods for the mouse utricle (Vollrath and Eatock, 2003). Mice were anesthetized through isoflurane inhalation. After decapitation, each hemisphere was bathed in ice-cold, oxygenated Liebowitz-15 (L15) media. The temporal bone was removed, the labyrinth was cut to isolate the utricle, and the nerve was cut close to the utricle. The utricle was treated with proteinase XXIV (100 μg/mL, ∼10 mins, 22°C) to facilitate removal of the otoconia and attached gel layer and mounted beneath two glass rods affixed at one end to a coverslip.


For most recordings, we used the HEKA Multiclamp EPC10 with Patchmaster acquisition software, filtered by the integrated HEKA filters: a 6-pole Bessel filter at 10 kHz and a second 4-pole Bessel filter at 5 kHz, and sampled at 10-100 kHz. Recording electrodes were pulled (PC-100, Narishige) from soda lime glass (King’s Precision Glass R-6) wrapped in paraffin to reduce pipette capacitance. Internal solution contained (in mM) 135 KCl, 0.5 MgCl2, 3 MgATP, 5 HEPES, 5 EGTA, 0.1 CaCl2, 0.1 Na-cAMP, 0.1 LiGTP, 5 Na2CreatinePO4 adjusted to pH 7.25 and ∼280 mmol/kg by adding ∼30 mM KOH. External solution was Liebowitz-15 media supplemented with 10 mM HEPES (pH 7.40, 310 ± 10 mmol/kg). Recording temperature was 22-25°C. Pipette capacitance and membrane capacitance transients were subtracted during recordings with Patchmaster software. Series resistance (8-12 MΩ) was measured after rupture and compensated 60-80% with the amplifier, to final values of ∼2 MΩ. Potentials are corrected for remaining (uncompensated) series resistance and liquid junction potential of ∼4 mV, calculated with LJPCalc software (Marino et al., 2014).

Type I HCs with gK,L were transiently hyperpolarized to –90 mV to close gK,L enough to increase Rinput above 100 MΩ, as needed to estimate series resistance and cell capacitance. The average resting potential, Vrest, was –87 mV ± 1 (41), similar to the calculated EK of –86.1 mV, which is not surprising given the large K+ conductance of these cells (Fig. 1). Vrest is likely more positive in vivo, where lower endolymphatic Ca2+ increases standing inward current through MET channels.

Voltage protocols to characterize KV currents differed slightly for type I and II HCs. In standard protocols, the cell is held at a voltage near resting potential (–74 mV in type I and –64 mV in type II), then jumped to –124 mV for 200 ms in type I HCs in order to fully deactivate gK,L and 50 ms in type II HCs in order to remove baseline inactivation of gA. The subsequent iterated step depolarizations lasted 500 ms in type I HCs because gK,L activates slowly (Wong et al., 2004) and 200 ms in type II HCs, where KV conductances activate faster. The 50-ms tail voltage was near the reversal potential of HCN channels (–44 mV in mouse utricular hair cells, Rüsch et al., 1998) to avoid HCN current contamination.

G-V (activation) parameters for control type I cells may be expected to vary across experiments on semi-intact (as here), organotypically cultured and denervated (Rüsch et al.,1998), or dissociated-cell preparations, reflecting variation in retention of the calyx (Discussion) and voltage step durations (Wong et al., 2004) which elevate K+ concentration around the hair cell. Nevertheless, the values we obtained for type I and type II HCs resemble values recorded elsewhere, including experiments in which extra care was taken to avoid extracellular K+ accumulation (Spaiardi et al., 2017, 2020). The effects of K+ accumulation on gK,L’s steady-state activation curves are small because the operating range is centered on EK and can be characterized with relatively small currents (Fig. 1A).


For most experiments, we locally perfused drug-containing solutions with BASI Bee Hive syringes at a final flow rate of 20 μL/min and a dead time of ∼30 s. Global bath perfusion was paused during drug perfusion and recording, and only one cell was used per utricle. Aliquots of test agents in solution were prepared, stored at –20°C, and thawed and added to external solution on the recording day (see Key Resources Table).


Data analysis was performed with software from OriginLab (Northampton, MA) and custom MATLAB scripts using MATLAB fitting algorithms.

Fitting voltage dependence and time course of conductances

G-V curves

Current was converted to conductance (G) by dividing by driving force (V – EK; EK calculated from solutions). For type I HCs, tail G-V curves were generated from current 1 ms after the end of the iterated voltage test step. For type II HCs, peak G-V curves were generated from peak current during the step and steady-state G-V curves were generated from current 1 ms before the end of a 200 ms step. Sigmoidal voltage dependence of G-V curves was fit with the first-order Boltzmann equation (Eq. 1).

Vhalf is the midpoint and S is the slope factor, inversely related to curve steepness near activation threshold.

Activation time course of type II HCs

For type II HCs lacking fast inactivation, outward current activation was fit with Eq. 2.

ISS is steady-state current, τw is activation time constant, n is the state factor related to the number of closed states (typically constrained to 3), and Io is baseline current.

To measure activation and inactivation time course of gA, we used Eq. 3 to fit outward K+ currents evoked by steps from –125 mV to above –50 mV (Rothman and Manis, 2003b).

Z is total steady-state inactivation (0 ≤ Z < 1 means incomplete inactivation, which allows the equation to fit non-inactivating delayed rectifier currents), f is the fraction of fast inactivation relative to total inactivation, Imax is maximal current, τzf and τzs are the fast and slow inactivation time constants. We chose to compare fit parameters at 30 ± 2 mV (91), where fast and slow inactivation were consistently separable and gA was maximized. In most KV1.8−/− and some striolar KV1.8+/+,+/– cells, where fast inactivation was absent and adjusted R2 did not improve on a single-exponential fit by >0.01, we constrained f in Eq. 3 to 0 to avoid overfitting.

For Peak G-V relations, peak conductance was taken from fitted curves (Eqs. 2 and 3). To construct ‘Steady-state’ G-V relations, we used current at 200 ms (6 ± 1 % (94) greater than steady-state estimated from fits to Eq. 3 (Fig. 3C-D)).

Percent inactivation was calculated at 30 mV with Eq. 4 :

IPeak is maximal current, and ISS is current at the end of a 200 ms voltage step.

The electrical resonance of type II HCs was quantified by fitting voltage responses to current injection steps (Songer and Eatock, 2013). We fit Eq. 5, a damped sinusoid, to the voltage trace from half-maximum of the initial depolarizing peak until the end of the current step.

VSS is steady-state voltage, Vp is the voltage of the peak response, τe is the decay time constant, fe is the fundamental frequency, and θ is the phase angle shift.

Quality factor, Qe, was calculated with Eq. 6 (Crawford and Fettiplace, 1981).


We give means ± SEM for normally-distributed data, and otherwise, median and range. Data normality was assessed with the Shapiro-Wilk test for n<50 and the Kolmogorov-Smirnov test for n>50. To assess homogeneity of variance we used Levene’s test. With homogeneous variance, we used two-way ANOVA for genotype and zone with the posthoc Tukey’s test. When variance was non-homogeneous, we used one-way Welch ANOVA with the posthoc Games-Howell test. For data that were not normally distributed, we used the non-parametric one-way Kruskal-Wallis ANOVA (KWA) with posthoc Dunn’s test. Effect size is Hedge’s g (g). For age dependence, we used partial correlation coefficients controlling for genotype and zone. Statistical groups may have different median ages, but all have overlapping age ranges. In figures, asterisks represent p-value ranges as follows: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


Mice were anesthetized with Nembutal (80 mg/kg), then perfused transcardially with 40mL of physiological saline containing heparin (400 IU), followed by 2 mL/g body weight fixative (4% paraformaldehyde, 1% picric acid, and 5% sucrose in 0.1 M phosphate buffer at pH 7.4, sometimes with 1% acrolein). Vestibular epithelia were dissected in phosphate buffer, and tissues were cryoprotected in 30% sucrose-phosphate buffer overnight at 4°C. Otoconia were dissolved with Cal-Ex (Fisher Scientific) for 10 min. Frozen sections (35 μm) were cut with a sliding microtome. Immunohistochemistry was performed on free-floating sections. Tissues were first permeabilized with 4% Triton X-100 in PBS for 1 h at room temperature, then incubated with 0.5% Triton X-100 in a blocking solution of 0.5% fish gelatin and 1% BSA for 1 h at room temperature. Sections were incubated with 2-3 primary antibodies for 72 h at 4°C and with 2-3 secondary antibodies. Sections were rinsed with PBS between and after incubations and mounted on slides in Mowiol (Calbiochem).

Data Availability

Data used in this study are available on Dryad (


  • gA,: A-type (inactivating) KV conductance in type II HCs

  • gDR,: delayed rectifier K+ conductance

  • gK,L,: low-voltage-activated K+ conductance in type I HCs

  • HC,: hair cell

  • KV,: voltage-gated K+ conductance


This study was supported by NIH grant R01 DC012347 to RAE and AL and an NSF Graduate Research Fellowship to HRM. We thank Drs. Thomas Friedman and Sherri Jones for the generous gift of the KV1.8− /− mouse line, and Drs. Zheng-Yi Chen and Deborah I. Scheffer for bringing the expression of this subunit in mouse vestibular hair cells to our attention.

We acknowledge Dr. Vicente Lumbreras for insights from his prior experiments on gA in mouse utricular hair cells, and thank him for helpful further discussions.

We thank Drs. Rebecca Lim and Ebenezer Yamoah for their critical feedback on the manuscript, and Drs. Rob Raphael and Aravind Chenrayan Govindaraju for feedback and many helpful discussions. We thank Drs. Joe Burns, Gabi Pregernig, and Lars Becker (Decibel Therapeutics, Inc.) for helpful discussions.

Author Contributions

HRM designed and performed experiments, analyzed data, and wrote the paper; RAE helped design experiments, analyze data, and write the paper; AL performed immunohistochemistry experiments.

Supplemental Figures

Developmental changes in type I HC KV conductances.

(A) Parameters from Boltzmann fits of tail G-V relations for type I HCs plotted against age.

(B) Conductance density is similar in young (P5-P10) type I HCs that lack gK,L. gK,L is defined here as having a Vhalf negative to –55 mV. KV1.8+/+,+/– with gK,L, 17 ± 5 nS/pF (19); KV1.8+/+,+/– without gK,L, 3.7 ± 0.4 nS/pF (22); KV1.8−/−, 1.8 ± 0.4 nS/pF (13). KV1.8+/+,+/– with gK,L vs. KV1.8−/−: p = 0.007, KWA, g 1.0.

For type II HCs older than P12, KV conductance activation and inactivation differed across zones and genotypes.

(A) τinact,Fast at 30 mV was fastest in LES in KV1.8+/+ and KV1.8+/– HCs, and faster in KV1.8+/– than KV1.8+/+ HCs (see Table 3 for p-values).

(B) Fast inactivation was a larger fraction of the total in LES than striola.

(C) τAct at 30 mV was slower in KV1.8−/− than KV1.8+/+ and KV1.8+/–, and slower in striola than LES and MES.***(D) Percent inactivation at 30 mV was lowest in striola (zone effect), and lowest in KV1.8−/− HCs (genotype effect).

For type II HCs older than P12, KV conductances were stable.

(A-C) Parameters from Boltzmann fits of peak G-V relations and (D) % inactivation at +30 mV plotted against age from all zones. Overlaid curves are smoothing cubic β-splines. Note the seven extrastriolar KV1.8−/− type II HCs with % inactivation >30%.

A minority of extrastriolar KV1.8−/− type II HCs had a very small fast-inactivating outward rectifier current.

(A) All extrastriolar KV1.8+/+,+/– type II HCs inactivated by >30%. Most mature (>P12) extrastriolar KV1.8−/− type II HCs inactivated by <30% but some inactivated by >30% (7/30, 23%) because they had fast inactivation (B). (B) Exemplar residual fast inactivation (τFastInact = 10 ms at +30 mV). For the 7 cells in this group, τFastInact = 30 ± 6 ms, amplitude of fast inactivation = 310 ± 70 pA; activation peak Vhalf = –15 ± 2 mV and slope factor = 12.4 ± 0.9 mV. These parameters are similar to gA but for the much smaller conductance (one-way ANOVAs).

A minority of striolar KV1.8−/− type I HCs had a small low-voltage-activated outward rectifier current.

(A) Low-voltage-activated current from one cell was isolated by 10 μM XE991 (P39), suggesting it was a KV7 current. Deactivation of XE991-sensitive current after step from –64 mV to –124 mV (arrow) was fit with exponential decay (τ = 21 ms). (B) Tail G-V curve fit with a sum of two Boltzmann equations: Vhalf,1 = –102 ± 4 mV (n=5) and Vhalf,2 = –41 ± 1 mV. Ages: P11, 39, 202, 202, 202. (C) Bimodal Vhalf distribution was specific to striolar type I HCs. 5/23 (22%; P6-P370) of striolar type I HCs had this low-voltage-activated component, but no extrastriolar type I HCs (0/45; P6-277).

No difference was detected in H (HCN) and KIR (fast inward rectifier) currents between KV1.8+/+ and KV1.8−/− hair cells, consistent with a specific involvement of KV1.8 in Kcna10 expression.

(A) Hyperpolarizing voltage steps evoked IKIR and IHCN in KV1.8+/+,+/–,–/– type I and II HCs. Note the prominent fast activation of IKIR in type II but not type I HCs. Arrows in top panel show deactivation of gK,L. IH and IKir were measured as inward current after 250 ms at –124 mV. (B) Summed IKIR and IH density was the same across genotypes but smaller in striola than extrastriola (see Supplemental Table 2 for statistics).

Test of sex differences in hair cell KV channel data.

Detected zonal but not genotype differences in hair cell IKIR and IH.