The large low-voltage-activated conductance, g_K,L, in Kcna10^+/+,+/– type I HCs produces distinctive whole-cell current responses to voltage steps, as highlighted by our standard type I voltage protocol (Figure 1A). From a holding potential within the g_K,L activation range (here –74 mV), the cell is hyperpolarized to –124 mV, negative to E_K and the activation range, producing a large inward current through open g_K,L channels that rapidly decays as the channels deactivate. We use the large transient inward current as a hallmark of g_K,L. The hyperpolarization closes all g_K,L channels, and then the activation function is probed with a series of depolarizing steps, obtaining the maximum conductance from the peak tail current at –44 mV (Figure 1A). We detected no difference between the Boltzmann parameters of g_K,L G–V curves from Kcna10^+/– and Kcna10^+/+ type I HCs.

For a similar voltage protocol, Kcna10^–/– type I HCs (Figure 1B) produced no inward transient current at the step from holding potential 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 (Equation 1; see insets in Figure 1A, B): the maximal conductance (g_max) of the Kcna10^–/– HC was over 10-fold smaller (Figure 1C.1), and the curve was positively shifted by >40 mV (Figure 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 g_K,L (Rüsch et al., 1998).

In type I HCs from Kcna10^+/+,+,– mice, the G–V parameters of outwardly rectifying currents transitioned over the first 15–20 postnatal days from values for a conventional delayed rectifier, activating positive to resting potential, to g_K,L values (Figure 1—figure supplement 1A), as previously described (Rüsch et al., 1998; Géléoc et al., 2004; Hurley et al., 2006). Between P5 and P10, some type I HCs have not yet acquired the physiologically defined conductance, g_K,L. No effects of K_V1.8 deletion were seen in the delayed rectifier currents of immature type I HCs (Figure 1—figure supplement 1B), showing that they were not immature forms of the K_V1.8-dependent g_K,L channels.

Kcna10^–/– type I HCs had a much smaller residual delayed rectifier that activated positive to resting potential, with V_half ~–40 mV and g_max density of 1.3 nS/pF. No additional K⁺ conductance activated up to +40 mV, and G–V parameters did not change much with age from P5 to P370. We characterize this K_V1.8-independent delayed rectifier later. A much larger non-g_K,L delayed rectifier conductance (g_DR,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’ g_K,L with 20 mM external Ba²⁺. Our new data suggest that there is no large non-g_K,L conductance, and that instead high Ba²⁺ positively shifted the apparent voltage dependence of g_K,L.

While the cells of Kcna10^–/– and Kcna10^+/– epithelia appeared healthy, type I HCs had smaller membrane capacitances (C_m): 4–5 pF in Kcna10^–/– type I HCs, ~20% smaller than Kcna10^+/– type I HCs (~6 pF) and ~30% smaller than Kcna10^+/+ type I HCs (6–7 pF; Table 2). While C_m scales with surface area, the lack of change in soma sizes by deletion of K_V1.8 (Supplementary file 1b) suggests that surface area was not different. Instead, C may be higher in Kcna10^+/+ cells because of g_K,L for two reasons. First, highly expressed trans-membrane proteins (see discussion of g_K,L channel density in Chen and Eatock, 2000) can affect membrane thickness (Mitra et al., 2004), which is inversely proportional to specific C_m. Second, resistive current through g_K,L could contaminate estimations of capacitive current, which is calculated from the decay time constant of transient current evoked by a small voltage step negative to –90 mV, where we measured C_m (see Methods).

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 K_V1.8 on these properties, we switched to current clamp mode and measured resting potential (V_rest), input resistance (R_in, equivalent to voltage gain for small current steps, ΔV/ΔI), and membrane time constant (τRC). In Kcna10^–/– type I HCs, V_rest was much less negative (Figure 2A.1), R_in was greater by ~20-fold (Figure 2A.2), and membrane charging times were commensurately longer (Figure 2A.3).

The differences between the voltage responses of Kcna10^+/+,+/– and Kcna10^–/– type I HCs are expected from the known impact of g_K,L on V_rest and R_in (Correia and Lang, 1990; Ricci et al., 1996; Rüsch and Eatock, 1996b; Songer and Eatock, 2013). The large K⁺-selective conductance at V_rest holds V_rest close to E_K (K⁺ equilibrium potential) and minimizes gain (ΔV/ΔI), 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 Kcna10^–/– type I HCs, with their less negative V_rest and larger R_in, positive current steps evoked a fast initial depolarization (Figure 2B.2), activating residual delayed rectifiers and repolarizing the membrane toward E_K. Negative current steps evoked an initial hyperpolarization followed by a slowly repolarizing ‘sag’ (Figure 2B.2) as the HCN1 channels open (Rüsch and Eatock, 1996b; Horwitz et al., 2011).

Overall, comparison of the Kcna10^+/+,+/– and Kcna10^–/– responses shows that with K_V1.8 (g_K,L), the voltage response of the type I HC is smaller but better reproduces the time course of the input current.

Type II HCs also express K_V1.8 mRNA (McInturff et al., 2018; Orvis et al., 2021). Although their outwardly rectifying conductances (g_A and g_DR) differ substantially in voltage dependence and size from g_K,L, both conductances were strongly affected by the null mutation: g_A was eliminated and the delayed rectifier was substantially smaller. Below we describe g_A and g_DR in Kcna10^+/+,+/– type II HCs and the residual outward-rectifying current in Kcna10^–/– type II HCs.

Kcna10^+/+,+/– type II HCs. Most (81/84) Kcna10^+/+,+/– type II HCs expressed a rapidly activating, rapidly inactivating A-type conductance (g_A). We define A current as the outwardly rectifying current that inactivates by over 30% within 200 ms. g_A 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 to ~30 mV. Outward currents fit with Equation 3 yielded fast inactivation time constants (τInact,Fast) of ~30 ms in LES (Figure 3A.2). τInact,Fast was faster in LES than in MES or striola (Figure 3A.3) and fast inactivation was a larger fraction of the total inactivation in LES than striola (~0.5 vs 0.3, Figure 3A.4).

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 g_A has mostly inactivated (Figure 3D.2). Kcna10^+/– HCs had smaller currents than Kcna10^+/+ HCs, reflecting a smaller g_DR (Figure 3D) and faster fast inactivation (Figure 3A.3). As discussed later, these effects may relate to effects of the Kcna10 gene dosage on the relative numbers of different K_V1.8 heteromers.

For Kcna10^+/+ and Kcna10^+/– HCs, the voltage dependence as summarized by V_half and slope factor (S) was similar. Relative to g_SS, g_Peak had a more positive V_half (~–21 vs ~–26) and greater S (~12 vs ~9, Figure 3D, Table 4). Because g_Peak includes channels with and without fast inactivation, the shallower g_Peak–V curve may reflect a more heterogeneous channel population. Only g_Peak showed zonal variation, with more positive V_half in LES than striola (~–20 vs ~–24 mV, Figure 3D, Table 4). We later suggest that variable subunit composition may drive zonal variation in g_Peak.

Kcna10^–/– type II HCs. Kcna10^–/– type II HCs from all zones were missing g_A and 30–50% of g_DR (Figure 3B–D). The residual delayed rectifier (1.3 nS/pF) had a more positive V_half than g_DR in Kcna10^+/+,+/– HCs (~–20 vs ~–26 mV, Figure 3D.2). We refer to the K_V1.8-dependent delayed rectifier component as g_DR(K_V1.8) and to the residual, K_V1.8-independent delayed rectifier component as g_DR(K_v7) because, as we show later, it includes K_V7 channels.

Figure 3—figure supplement 2 shows the development of K_V1.8-dependent and -independent K_V currents in type II HCs with age from P5 to over P300. In Kcna10^+/+,+/– type II HCs, g_A was present at all ages with a higher % inactivation after P18 than at P5–P10 (Figure 3—figure supplement 2A.4). g_Peak 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, Figure 3—figure supplement 2A.3).

We saw small rapidly inactivating outward currents in a minority of Kcna10^–/– type II HCs (23%, 7/30), all >P12 and extrastriolar (Figure 3—figure supplement 3). These currents overlapped with g_A 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 K_V subunits that in control HCs join with K_V1.8 subunits and confer inactivation on the heteromeric conductance.

In type II HCs, absence of K_V1.8 did not change V_rest (Figure 4A.1) because g_A and g_DR both activate positive to rest, but significantly increased R_in and τRC (Figure 4A.2 and A.3).

Positive current steps evoked an initial depolarizing transient in both Kcna10^+/+ and Kcna10^–/– type II HCs, but the detailed time course differed (Figure 4B). Both transient and steady-state responses were larger in Kcna10^–/–, consistent with their larger R_in values.

Absence of K_V1.8 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 R_in values made Kcna10^–/– type II HCs more prone to electrical resonance; Figure 4C.1 shows a striking example. Median resonance quality (Q_e, sharpness of tuning) was greater in Kcna10^–/– (1.33, n=26) than Kcna10^+/+ (0.66, n = 23) or Kcna10^+/– (0.59, n = 44) type II HCs.

K_V1.8 affected the time course of the initial peak in response to much larger current injections (Figure 4D, E). Fast activation of g_A 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 (Figure 4D.2). This behavior has the potential to counter mechanotranduction adaptation (Vollrath and Eatock, 2003).

If K_V1.8 is a pore-forming subunit in the K_V1.8-dependent conductances g_K,L, g_A, and g_DR, it should localize to HC membranes. Figure 5 compares K_V1.8 immunoreactivity in Kcna10^+/+ and Kcna10^–/– utricles, showing specific immunoreactivity along the basolateral membranes of both HC types in Kcna10^+/+ utricles. To identify HC type and localize the HC membrane, we used antibodies against K_V7.4 (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 (Figure 5A). K_V1.8 immunoreactivity was present in HC membrane apposing K_V7.4-stained calyx inner face in Kcna10^+/+ utricles (Figure 5A.1 and A.2) and not in Kcna10^–/– utricles (Figure 5A.3).

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, Figure 5B). An HC is type II if it is calretinin-positive (Figure 5B.1) or if it lacks a K_V7.4- or calretinin-positive calyceal cup (Figure 5A.2 and B.3, rightmost cells). HC 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 HCs.

Previously, Carlisle et al., 2012 reported K_V1.8-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 HCs 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-specific.

Results with the K_V1.8 knockout suggest that type II HCs have an inactivating K_V1 conductance that includes K_V1.8 subunits. K_V1.8, like most K_V1 subunits, does not show fast inactivation as a heterologously expressed homomer (Lang et al., 2000; Ranjan et al., 2019; Dierich et al., 2020), nor do the K_V1.8-dependent channels in type I HCs, as we show, and in cochlear inner HCs (Dierich et al., 2020). K_V1 subunits without intrinsic inactivation can produce rapidly inactivating currents by associating with K_Vβ1 or K_Vβ3 subunits. K_Vβ1 (Kcnb1) is present in type II HCs alongside K_Vβ2 (Kcnb2) (McInturff et al., 2018; Jan et al., 2021; Orvis et al., 2021), which does not confer rapid inactivation (Dwenger et al., 2022).

Another possibility is that in type II HCs, K_V1.8 subunits heteromultimerize with K_V1.4 subunits—the only K_V1 subunits which, when expressed as a homomer, have complete N-type (fast) inactivation (Stühmer et al., 1989). Multiple observations support this possibility. K_V1.4 has been linked to g_A in pigeon type II HCs (Correia et al., 2008) and is the second-most abundant K_V1 transcript in mammalian vestibular HCs, after K_V1.8 (Scheffer et al., 2015). K_V1.4 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 (Figure 3A).

Functional heteromers form between K_V1.4 and other K_V1.x and/or K_Vβ1 (Imbrici et al., 2006; Correia et al., 2008; Al Sabi et al., 2011). Although K_V1.4 and K_V1.8 heteromers have not been studied directly, g_A’s inactivation time course (τFast,Inact of ~30 ms +30 mV, Figure 3A) and voltage dependence (V_half –41 mV, Figure 6B) are consistent with these other K_V1.4-containing heteromers.

In Kcna10^–/– HCs, absence of I_K,L and I_A revealed smaller delayed rectifier K⁺ currents that, unlike I_K,L, activated positive to resting potential and, unlike I_A, lacked fast inactivation. Candidate channels include members of the K_V7 (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 tested for K_V7 contributions in Kcna10^–/– type I HCs, Kcna10^–/– type II HCs, and Kcna10^+/+,+/– type II HCs of multiple ages by applying XE991 at 10 µM (Figure 7A), a dose selective for K_V7 channels (Brown et al., 2002) and close to the IC₅₀ (Alexander et al., 2019). In Kcna10^–/– HCs of both types, 10 µM XE991 blocked about half of the residual K_V conductance (Figure 7B.1), consistent with K_V7 channels conducting most or all of the non-K_V1.8 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 K_V7.2, 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 g_DR(K_V7). The voltage dependence and g_max density (g_max/C_m) of g_DR(K_V7) were comparable across HC types and genotypes (Figure 7B.2–4). Although K_V7.4 was not detectable in HCs during immunostaining (Figure 5), K_V7.4 has been shown in type I HCs with immunogold labeling (Kharkovets et al., 2000; Hurley et al., 2006).

These results are consistent with similar K_V7 channels contributing a relatively small delayed rectifier in both HC types. In addition, the similarity of XE991-sensitive currents of Kcna10^+/+ and Kcna10^–/– type II HCs indicates that knocking out K_V1.8 did not cause general effects on ion channel expression. We did not test XE991 on Kcna10^+/+,+/– type I HCs because g_K,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 Kcna10^–/– type I HC, XE991 also blocked a small conductance that activated negative to rest (Figure 7—figure supplement 1A, B). This conductance (V_half ~ = –100 mV, Figure 7—figure supplement 1C) was detected only in Kcna10^–/– type I HCs from the striola (5/23 vs 0/45 extrastriolar). The V_half and τdeactivation were similar to values reported for K_V7.4 channels in cochlear HCs (Wong et al., 2004; Dierich et al., 2020). This very negatively activating K_V7 conductance coexisted with the larger more positively activating K_V7 conductance (Figure 7—figure supplement 1C) and was too small (<0.5 nS/pF) to contribute significantly to g_K,L (~10–100 nS/pF, Figure 1D).

While our data are consistent with K_V1.8- and K_V7-containing channels carrying most of the outward-rectifying current in mouse utricular HCs, there is evidence in other preparations for additional channels, including K_V11 (KCNH, Erg) channels in rat utricular type I HCs (Hurley et al., 2006) and BK (KCNMA1) channels in rat utricle and rat and turtle semicircular canal HCs (Schweizer et al., 2009; Contini et al., 2020).

BK is expressed in mouse utricular HCs (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 Kcna10^–/– type I HCs); 1 ± 5% (5 Kcna10^+/+,+/– type II HCs); 7% and 14% (2 Kcna10^–/– 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 HCs’ small Ca_V currents (Bao et al., 2003) or cytoplasmic second messengers (Hurley et al., 2006).

To check whether the constitutive K_V1.8 knockout has strong non-specific effects on channel trafficking, we examined the summed HCN and fast inward rectifier currents (I_H and I_Kir) at –124 mV, and found them similar across genotypes (Figure 7—figure supplement 2). The g_K,L knockout allowed identification of zonal differences in I_H and I_Kir in type I HCs, previously examined in type II HCs (Masetto and Correia, 1997; Levin and Holt, 2012). In type I HCs from both control and null utricles, I_H and I_Kir were less prevalent in striola than extrastriola, and, when present, the combined inward current was smaller (Figure 7—figure supplement 2).

Recent single-cell expression studies on mouse utricles (McInturff et al., 2018; Jan et al., 2021; Orvis et al., 2021) have detected just one K_V1 subunit, K_V1.8, in mouse type I HCs. Given that K_V1.8 can only form multimers with K_V1 family members, and given that g_K,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 K_V1.8 homomers. Other evidence is consistent with this proposal. g_K,L (Rüsch and Eatock, 1996a) and heterologously expressed K_V1.8 homomers in oocytes (Lang et al., 2000) are non-inactivating and blocked by millimolar Ba²⁺ and 4-aminopyridine and >10 mM tetraethyl ammonium. Unlike channels with K_V1.1, K_V1.2, and K_V1.6 subunits, g_K,L is not sensitive to 10 nM α-dendrotoxin (Rüsch and Eatock, 1996a). g_K,L and heterologously expressed K_V1.8 channels have similar single-channel conductances (~20 pS for g_K,L at positive potentials, Chen and Eatock, 2000; 11 pS in oocytes, Lang et al., 2000). g_K,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 K_V1.8.

A major novel property of g_K,L is that it activates 30–60 mV negative to type II K_V1.8 conductances and most other low-voltage-activated K_V channels (Ranjan et al., 2019). The very negative activation range is a striking difference between g_K,L and known homomeric K_V1.8 channels. Heterologously expressed homomeric K_V1.8 channels have an activation V_half of –10 to 0 mV (X. laevis oocytes, Lang et al., 2000; Chinese hamster ovary cells, Dierich et al., 2020). In cochlear inner HCs, currents attributed to K_V1.8 (by subtraction of other candidates) have a near-zero activation V_half (–4 mV, Dierich et al., 2020).

Possible factors in the unusually negative voltage dependence of g_K,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 g_K,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 g_K,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 g_K,L (~50 nS/pF in striolar Kcna10^+/+ 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 g_K,L, as proposed for K_V7.4 channels in outer HCs (Perez-Flores et al., 2020). Type I HC-specific partners that may facilitate this close packing include ADAM11 (McInturff et al., 2018), which clusters presynaptic K_V1.1 and K_V1.2 to enable ephaptic coupling at a cerebellar synapse (Kole et al., 2015).

(3) Modulation by accessory subunits. Type I HCs express K_Vβ1 (McInturff et al., 2018; Orvis et al., 2021), an accessory subunit that can confer fast inactivation and hyperpolarize activation V_half by ~10 mV. K_Vβ1 might interact with K_V1.8 to shift voltage dependence negatively. Arguments against this possibility include that g_K,L lacks fast inactivation (Rüsch and Eatock, 1996a; Hurley et al., 2006; Spaiardi et al., 2017) and that cochlear inner HCs co-express K_V1.8 and K_Vβ1 (Liu et al., 2018) but their K_V1.8 conductance has a near-0 V_half (Dierich et al., 2020).

The K_V1.8-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 K_V1.8 subunits resemble most other K_V1 family subunits, with the exception of K_V1.4 (for comprehensive review, see Ranjan et al., 2019). K_V1.4 is a good candidate to provide fast inactivation based on immunolocalization and voltage dependence (Figures 4 and 6). We suggest that g_A and g_DR(K_V1.8) are K_V1.8-containing channels that may include a variable number of K_V1.4 subunits and K_Vβ2 and K_Vβ1 accessory subunits.

K_V1.4–K_V1.8 heteromeric assembly could account for several related observations. The faster τInact,Fast in Kcna10^+/– relative to Kcna10^+/+ type II HCs (Figure 3A.3, Figure 3—figure supplement 1A.1) could reflect an increased ratio of K_V1.4–K_V1.8 subunits and therefore more N-terminal inactivation domains per heteromeric channel. Zonal variation in the extent and speed of N-type inactivation (Figure 3A) might arise from differential expression of K_V1.4. The small fast-inactivating conductance in ~20% of extrastriolar Kcna10^–/– type II HCs (Figure 3—figure supplement 3) might flow through K_V1.4 homomers.

Fast inactivation may also receive contribution from K_Vβ subunits. K_Vβ1 is expressed in type II HCs (McInturff et al., 2018; Jan et al., 2021; Orvis et al., 2021), and, together with K_V1.4, has been linked to g_A in pigeon vestibular HCs (Correia et al., 2008). K_Vβ2, also expressed in type II HCs (McInturff et al., 2018; Orvis et al., 2021), accelerates but does not confer fast inactivation.

We speculate that g_A and g_DR(K_V1.8) have different subunit composition: g_A may include heteromers of K_V1.8 with other subunits that confer rapid inactivation, while g_DR(K_V1.8) may comprise homomeric K_V1.8 channels, given that they do not have N-type inactivation.

In both type I and II utricular HCs, K_V1.8-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 (Figure 2).

g_K,Ldominates passive membrane properties in mature Kcna10^+/+,+/– type I HCs such that Kcna10^–/– 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 Kcna10^–/– 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 HCs, 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 HC 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 K_V1.8-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 HCs 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 biophysically distinct and functionally different ion channels, presenting a natural experiment in functional differentiation of sensory receptor cells.

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 (Animal Care and Use Procedure #72360) and the Office of Animal Care and Institutional Biosafety at the University of Illinois Chicago (Protocol for Animal Use #17106). Most mice belonged to a transgenic line with a knockout allele of Kcna10 (referred to here as Kcna10^–/–). 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 12:12 hr 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. HC K_V channel data were pooled across sexes as most results did not appear to differ by sex; an exception was that g_K,L had a more negative V_half in males (Supplementary file 1a), 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 min, 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.

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 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES), 5 ethylene glycol tetraacetic acid (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).

Kcna10^–/– HCs appeared healthy in that cells had resting potentials negative to –50 mV, cells lasted a long time (20–30 min) in ruptured patch recordings, membranes were not fragile, and extensive blebbing was not seen. Type I HCs with g_K,L were transiently hyperpolarized to ~–90 mV to close g_K,L enough to increase R_input above 100 MΩ, as needed to estimate series resistance and cell capacitance. The average resting potential, V_rest, was –87 mV ±1 (41), similar to the calculated E_K of –86.1 mV, which is not surprising given the large K⁺ conductance of these cells. V_rest is likely more positive in vivo, where lower endolymphatic Ca²⁺ increases standing inward current through MET channels.

Voltage protocols to characterize K_V 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 g_K,L and 50 ms in type II HCs in order to remove baseline inactivation of g_A. The subsequent iterated step depolarizations lasted 500 ms in type I HCs because g_K,L activates slowly (Wong et al., 2004) and 200 ms in type II HCs, where K_V conductances activate faster. The 50 ms tail voltage was near the reversal potential of HCN channels (–44 mV in mouse utricular HCs, 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 HC. Nevertheless, the values we obtained for type I and II HCs resemble values recorded elsewhere, including experiments in which extra care was taken to avoid extracellular K⁺ accumulation (Spaiardi et al., 2017; Spaiardi et al., 2020). The effects of K⁺ accumulation on g_K,L’s steady-state activation curves are small because the operating range is centered on E and can be characterized with relatively small currents (Figure 1A).

Drug-containing solutions were locally 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.

G–V curves. Current was converted to conductance (G) by dividing by driving force (V – E_K; E_K 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. We fit G–V curves to the first-order Boltzmann equation (Equation 1) using a custom MATLAB function (fitzmann.m, Source code 2).(1)G(V)=Gmin+Gmax1+exp(Vhalf−VS)

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

Activation time course of type II HCs. We fit current traces using a custom MATLAB function (fitkin.m, Source code 1). For type II HCs lacking fast inactivation, outward current activation was fit with Equation 2.(2)I(t)=ISS∗(1−exp(−tτw))n+I0

I_SS is steady-state current; τw is activation time constant (referred to elsewhere as τAct); n is the state factor related to the number of closed states (typically constrained to 3); and I_o is baseline current.

To measure activation and inactivation time course of g_A, we used Equation 3 to fit outward K⁺ currents evoked by steps from –125 mV to above –50 mV (Rothman and Manis, 2003).(3)I(t)=Imax∗(1−exp(−tτw))n∗[1−Z∗(f∗(1−exp(−tτzf))+(1−f)∗(1−exp(−tτzs)))]+I0

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; I_max is maximal current; τzf (referred to elsewhere as τInact,Fast) 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 g_A was maximized. In most Kcna10^–/– and some striolar Kcna10^+/+,+/– cells, where fast inactivation was absent and adjusted R² did not improve on a single-exponential fit by >0.01, we constrained f in Equation 3 to 0 to avoid overfitting.

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

Percent inactivation was calculated at 30 mV with Equation 4:(4)%Inactivation=(IPeak−ISS)/IPeak

I_Peak is maximal current, and I_SS 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 Equation 5, a damped sinusoid, to the voltage trace from half-maximum of the initial depolarizing peak until the end of the current step.(5)V(t)=Vss+Vp∗exp(−tτe)∗sin⁡(2πfe−θ)

V_SS is steady-state voltage; V_p is the voltage of the peak response; τe is the decay time constant; f_e is the fundamental frequency; and θ is the phase angle shift.

Quality factor, Q_e, was calculated with Equation 6 (Crawford and Fettiplace, 1981).(6)Qe=[(πfeτe)2+0.25]1/2

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 post hoc 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 40 ml 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 phosphate-buffered saline (PBS) for 1 hr at room temperature, then incubated with 0.5% Triton X-100 in a blocking solution of 0.5% fish gelatin and 1% bovine serum albumin for 1 hr at room temperature. Sections were incubated with two to three primary antibodies for 72 hr at 4°C and with two to three secondary antibodies. Sections were rinsed with PBS between and after incubations and mounted on slides in Mowiol (Calbiochem).