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⁺ (K_V) channel subunit participates in very different K_V channels dominating the membrane conductances of type I and 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 (Wersall, 1956; Lysakowski and Goldberg, 2004) and that they express g_K,L (Correia and Lang, 1990; Rennie and Correia, 1994; Rüsch and Eatock, 1996a): a large non-inactivating conductance with an activation range from –100 to –60 mV, far more negative than other ‘low-voltage-activated’ K_V channels. HCs are known for their large outwardly rectifying K⁺ conductances, which repolarize membrane voltage following a mechanically evoked perturbation and in some cases contribute to sharp electrical tuning of the HC membrane. g_K,L is unusually large and unusually negatively activated, and therefore strongly attenuates and speeds up the receptor potentials of type I HCs (Correia et al., 1996; Rüsch and Eatock, 1996b). In addition, g_K,L augments non-quantal transmission from type I HC to afferent calyx by providing open channels for K⁺ flow into the synaptic cleft (Contini et al., 2012; Contini et al., 2017; Contini et al., 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, g_A) and delayed rectifier (g_DR) conductances that are opened by depolarization above resting potential (V_rest).

The unusual properties of g_K,L have long attracted curiosity about its molecular nature. g_K,L has been proposed to include ‘M-like’ K_V channels in the K_V7 and/or erg channel families (Kharkovets et al., 2000; Hurley et al., 2006; Holt et al., 2007). The K_V7.4 subunit was of particular interest because it contributes to the low-voltage-activated conductance, g_K,n, in cochlear outer HCs, but was eventually eliminated as a g_K,L subunit by experiments on K_V7.4-null mice (Spitzmaul et al., 2013).

Several observations suggested the K_V1.8 (KCNA10) subunit as an alternative candidate for g_K,L. K_V1.8 is highly expressed in vestibular sensory epithelia (Carlisle et al., 2012), particularly HCs (Lee et al., 2013; Scheffer et al., 2015; McInturff et al., 2018), with slight expression elsewhere (skeletal muscle, Lee et al., 2013; kidney, Yao et al., 2002). Kcna10^–/– 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 K_V1 channels, K_V1.8 has a cyclic nucleotide-binding domain (Lang et al., 2000) with the potential to explain g_K,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 Kcna10^–/– and Kcna10^+/+,+/– mouse utricles showed that K_V1.8 expression is necessary for g_K,L. More surprisingly, K_V1.8 expression is also required for A-type and delayed rectifier conductances of type II HCs. In both HC types, eliminating the K_V1.8-dependent major conductances revealed a smaller delayed rectifier conductance involving K_V7 channels. Thus, the distinctive outward rectifiers that produce such different receptor potentials in type I and II HCs both include K_V1.8 and K_V7 channels.

We compared whole-cell voltage-activated K⁺ currents in type I and II HCs from homozygous knockout (Kcna10^–/–) animals and their wildtype (Kcna10^+/+) or heterozygote (Kcna10^+/–) littermates. We immunolocalized K_V1.8 subunits in the utricular epithelium and pharmacologically characterized the residual K⁺ currents of Kcna10^–/– animals. Current clamp experiments demonstrated the impact of K_V1.8-dependent currents on passive membrane properties.

We recorded from three utricular zones: lateral extrastriola (LES), striola, and medial extrastriola (MES); 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 K_V channel properties, as reported (González-Garrido et al., 2021).

Kcna10^–/– animals appeared to be healthy and to develop and age normally, as reported (Lee et al., 2013), and HCs were healthy (see Methods for criteria).

K_V1.8 is necessary for g_K,L in type I HCs

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.

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Table 1

Zone	Kcna10	Tail V1/2, mV*	Tail S, mV†	Tail gmax, nS‡	Tail gmax/Cm, nS/pF§	Age (median, range)
Extrastriola	+/+	–85 ± 2 (12)	4.3 ± 0.4 (12)	270 ± 40 (11)	47 ± 8 (11)	22, 14–287
	+/–	–83 ± 1 (40)	5.2 ± 0.3 (40)	210 ± 20 (40)	37 ± 4 (40)	19, 13–259
	–/–	–40.2 ± 0.9 (26)	5.7 ± 0.3 (26)	5.4 ± 0.3 (26)	1.11 ± 0.08 (26)	45, 14–277
Striola	+/+	–87 ± 3 (6)	4.3 ± 0.3 (6)	310 ± 70 (6)	41 ± 7 (6)	40, 15–59
	+/–	–88 ± 2 (3)	4.7 ± 0.9 (3)	270 ± 60 (3)	44 ± 6 (3)	19, 14–20
	–/–	–38 ± 1 (13)	6.2 ± 0.4 (13)	6.5 ± 0.6 (13)	1.5 ± 0.1 (13)	202, 14–370

1. *

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 7.7; –/– vs +/–: two-way ANOVA, p < 1E−9, g 6.8.

2. †

   –/– vs +/+: two-way ANOVA, p = 8.4E−4, g 1.2.

3. ‡

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 3.7; –/– vs +/–: two-way ANOVA, p < 1E−9, g 2.1.

4. §

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 3.6; –/– vs +/–: two-way ANOVA, p < 1E−9, g 2.0.

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.

K_V1.8 strongly affects type I passive properties and responses to current steps

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).

Table 2

Zone	Kcna10	Vrest, mV*, †	Rinput, MΩ‡	${\displaystyle {\tau }_{{\phantom{10}}_{RC}}}$, ms§	Cm, pF¶	Age (median, range)
ES	+/+	–84 ± 3 (6)	44 ± 6 (6)	0.24 ± 0.03 (6)	6.1 ± 0.4 (13)	20, 14–287
	+/–	–88.0 ± 0.7 (28)	55 ± 5 (24)	0.32 ± 0.03 (23)	5.8 ± 0.2 (44)	21, 16–29
	–/–	–63 ± 2 (15)	1400 ± 100 (15)	6.4 ± 0.6 (15)	5.0 ± 0.2 (27)	45, 14–202
S	+/+	–87 ± 2 (4)	50 ± 8 (4)	0.30 ± 0.04 (4)	7.4 ± 0.7 (7)	43, 40–59
	+/–	–87 ± 3 (3)	38 ± 8 (2)	0.21 ± 0.01 (2)	5.9 ± 0.6 (3)	19, 19–20
	–/–	–74 ± 5 (5)	1000 ± 300 (4)	4.2 ± 1.0 (4)	4.4 ± 0.2 (14)	202, 24–370

1. *

   Striolar –/– vs ES –/–: two-way ANOVA, p = 0.006, g 1.2; striolar –/– vs striolar +/+,+/–: two-way ANOVA, p = 0.005, g 1.7.

2. †

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 2.3; –/– vs +/–: two-way ANOVA, p < 1E−9, g 3.4.

3. ‡

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 3.1; –/– vs +/–: two-way ANOVA, p < 1E−9, g 3.9.

4. §†

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 2.7; –/– vs +/–: two-way ANOVA, p < 1E−9, g 3.4.

5. ¶‡

   –/– vs +/+: two-way ANOVA, p = 3E−7, g 1.5; –/– vs +/–: two-way ANOVA, p = 1.3E−4, g 1.0; +/–vs +/+: two-way ANOVA, p = 0.048, g 0.6.

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 (${\displaystyle {\tau }_{RC}}$). 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).

Figure 2

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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.

K_V1.8 is necessary for both inactivating and non-inactivating K_V currents in type II HCs

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 (${\displaystyle {\tau }_{Inact,Fast}}$) of ~30 ms in LES (Figure 3A.2). ${\displaystyle {\tau }_{Inact,Fast}}$ 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).

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Table 3

Zone	Kcna10	${\displaystyle {\tau }_{Act}}$ at 30 mV, ms*, †	${\displaystyle {\tau }_{Inact,Fast}}$ at 30 mV, ms ‡, §	Fast inactivation prominence¶	Inactivation %**,††	N cells	Age (median, range)
LES	+/+	2.11 ± 0.09	23 ± 3	0.46 ± 0.03	45 ± 2	30	46, 14–312
	+/–	1.64 ± 0.09	15 ± 2	0.53 ± 0.03	51 ± 2	27	29, 13–280
	–/–	4.4 ± 0.5	NA	NA	25 ± 3	21	128, 15–355
MES	+/+	2.8 ± 0.5	50 ± 10	0.40 ± 0.04	42 ± 3	9	94, 22–296
	+/–	2.2 ± 0.2	90 ± 60	0.42 ± 0.03	47 ± 2	15	24, 13–52
	–/–	10 ± 7	NA	NA	29 ± 5	10	84, 28–355
Striola	+/+	2.7 ± 0.3	50 ± 10	0.31 ± 0.07	39 ± 3	5	45, 40–287
	+/–	2.9 ± 0.4	300 ± 200	0.3 ± 0.06	28 ± 2	5	19, 14–30
	–/–	7 ± 2	NA	NA	22 ± 2	6	202, 29–298

1. *

   –/– vs +/+: KWA, p = 0.0048, g 0.6; –/– vs +/–: KWA, p = 2.3E−7, g 0.6.

2. †

   Striola vs LES: KWA, p = 5.7E−4, g 1.0.

3. ‡

   +/– vs +/+: KWA, p = 0.027, g 0.2.

4. §

   LES vs MES: KWA, p = 0.0018, g 0.3; LES vs Striola: KWA, p = 1.9E−4, g 0.8.

5. ¶

   LES vs Striola: two-way ANOVA, p = 0.0041, g 0.7.

6. **

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 1.7; –/– vs +/–: two-way ANOVA, p < 1E−9, g 1.8.

7. ††

   Striola vs LES: two-way ANOVA, p = 3.4E−5, g 0.9; Striola vs MES: two-way ANOVA, p = 0.0011, g 1.0; interaction between genotype and zone: two-way ANOVA, p = 0.026.

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.

Table 4

Zone	Kcna10	Peak V1/2, mV**	Peak S, mV††, ‡	A-type gmax/Cm, nS/pF§ §	SS Vhalf, mV¶ ¶	SS S, mV****	SS gmax/Cm, nS/pF †† ‡ ‡	N cells	Age (median, range)
LES	+/+	–19.8 ± 0.6	11.8 ± 0.4	4.0 ± 0.3	–25.0 ± 0.5	8.7 ± 0.3	7.1 ± 0.8	37	46, 14–312
	+/–	–19.8 ± 0.8	12.8 ± 0.4	3.8 ± 0.3	–26.8 ± 0.8	8.7 ± 0.3	4.9 ±0.4	35	29, 13–280
	–/–	–18 ± 1	11.7 ± 0.4	0.37 ± 0.05	–19 ± 1	12.1 ± 0.5	1.8 ±0.2	20	128, 15–355
MES	+/+	–22 ± 1	11 ± 0.7	4.1 ± 0.7	–26 ± 1	8.3 ± 0.5	9 ±1	11	94, 22–296
	+/–	–21 ± 1	11.8 ± 0.4	3.6 ± 0.5	–27 ± 1	9.0 ± 0.3	5.9 ±0.7	16	24, 13–52
	–/–	–19 ± 1	10.8 ± 0.6	0.6 ± 0.1	–20 ± 1	10.7 ± 0.7	2.5 ±0.3	15	84, 28–355
Striola	+/+	–24 ± 1	9.6 ± 0.5	5 ± 1	–26.6 ± 0.9	8.2 ± 0.4	12 ±1	7	45, 40–287
	+/–	–25 ± 2	9.4 ± 0.4	2.6 ± 0.6	–28 ± 2	8.2 ± 0.3	10±2	6	19, 14–30
	–/–	–21.3 ± 0.9	10.3 ± 0.5	0.7 ± 0.1	–21.7 ± 0.8	10.5 ± 0.6	3.9±0.5	8	202, 29–298

1. *

   Striola vs LES: two-way ANOVA, p = 0.00116, g 0.9.

2. †

   Striola vs MES: two-way ANOVA, p = 0.016, g 0.8; Striola vs LES: two-way ANOVA, p = 7.5E−6, g 1.2.

3. ‡

   –/– vs +/–: two-way ANOVA, p = 0.036, g 0.5.

4. §

   –/– vs +/+: Welch ANOVA, p < 1E−9, g 2.3; –/– vs +/–: Welch ANOVA, p < 1E−9, g 2.3.

5. ¶

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 1.4; –/– vs +/–: two-way ANOVA, p < 1E−9, g 1.6.

6. **

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 1.4; –/– vs +/–: two-way ANOVA, p = 4.5E−7, g 1.1.

7. ††

   –/– vs +/+: Welch ANOVA, p < 1E−9, g 1.6; –/– vs +/–: Welch ANOVA, p < 1E−9, g 1.3; +/+vs +/–: Welch ANOVA, p = 0.007, g 1.6.

8. ‡ ‡

   Striola vs LES: one-way ANOVA, p = 0.001, g (0.9); Striola vs MES: one-way ANOVA, p = 0.01, g 0.8.

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.

K_V1.8 affects type II passive properties and responses to current steps

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 ${\displaystyle {\tau }_{RC}}$ (Figure 4A.2 and A.3).

Figure 4

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Table 5

Zone	Kcna10	Vrest, mV	Rinput, GΩ*	${\displaystyle {\tau }_{RC}}$, ms†	Peak height, mV‡	Peak time, ms§	Cm, pF	Age (median, range)
ES	+/+	–71 ± 2 (19)	1.4 ± 0.2 (16)	11 ± 3 (16)	–20 ± 2 (15)	2.5 ± 0.2 (15)	4.7 ± 0.2 (50)	45, 16–312
	+/–	–71 ± 2 (34)	1.2 ± 0.1 (27)	9 ± 1 (27)	–20 ± 1 (30)	2.44 ± 0.08 (30)	4.6 ± 0.1 (52)	27, 13–280
	–/–	–76 ± 2 (9)	2.3 ± 0.3 (7)	16 ± 3 (7)	2 ± 6 (7)	3.6 ± 0.3 (7)	4.6 ± 0.2 (35)	53, 15–154
S	+/+	–73.1 ± 1.0 (6)	1.4 ± 0.1 (6)	9 ± 1 (6)	–20 ± 2 (5)	2.7 ± 0.1 (5)	4.6 ± 0.2 (7)	45, 40–224
	+/–	–71 ± 3 (5)	1.4 ± 0.3 (6)	7 ± 2 (6)	–20 ± 2 (6)	2.3 ± 0.1 (6)	4.8 ± 0.2 (6)	19, 19–30
	–/–	–68 ± 2 (6)	3.0 ± 0.7 (6)	26 ± 10 (6)	2 ± 7 (4)	4 ± 1 (4)	4.4 ± 0.3 (7)	178, 29–298

1. *

   –/– vs +/+: KWA, p = 0.015, g 1.2; –/– vs +/–: KWA, p = 0.002, g 1.5.

2. †

   –/– vs +/+: KWA, p = 0.016, g 0.7; –/– vs +/–: KWA, p = 0.008, g 1.2.

3. ‡

   –/– vs +/+: KWA, p = 0.006, g 2.1; –/– vs +/–: KWA, p = 2E−4, g 2.6.

4. §

   –/– vs +/+: two-way ANOVA, p < 1E−9, g 1.3; –/– vs +/–: two-way ANOVA, p < 1E−9, g 1.9.

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).

K_V1.8 immunolocalized to basolateral membranes of both type I and II HCs

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).

Figure 5

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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.

K_V1.4 may also contribute to g_A

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 (${\displaystyle {\tau }_{Fast,Inact}}$ 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.

Figure 6

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K_V7 channels contribute a small delayed rectifier in type I and II HCs

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).

Figure 7 with 2 supplements see all

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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 ${\displaystyle {\tau }_{deactivation}}$ 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).

We have shown that constitutive knockout of K_V1.8 eliminated g_K,L in type I HCs, and g_A and much of g_DR in type II HCs. K_V1.8 immunolocalized specifically to the basolateral membranes of type I and II HCs. We conclude that K_V1.8 is a pore-forming subunit of g_K,L, g_A, and part of g_DR [g_DR(K_V1.8)]. We suggest that fast inactivation of g_A may arise from heteromultimerization of non-inactivating K_V1.8 subunits and inactivating K_V1.4 subunits. Finally, we showed that a substantial component of the residual delayed rectifier current in both type I and II HCs comprises K_V7 channels.

K_V1.8 is expressed in HCs 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 g_K,L, K_V1.8 contributes to g_A and g_DR, which are widespread in vertebrate HCs (reviewed in Meredith and Rennie, 2016). K_V1.8 expression has not been detected in rodent brain but is reported in the pacemaker nucleus of weakly electric fish (Smith et al., 2018).

Data generated and analyzed in this study are available on Dryad (https://doi.org/10.5061/dryad.37pvmcvrw). Dryad hosts downloadable spreadsheets that are organized as follows: F1_sourcedata, numerical data for Figure 1; F1-fs1_sourcedata, numerical data for Figure 1-figure supplement 1; F2_sourcedata, numerical data for Figure 2; F3_sourcedata, numerical data for Figure 3; F3-fs1_sourcedata, numerical data for Figure 3—figure supplement 1; F3-fs2_sourcedata, numerical data for Figure 3—figure supplement 2; F3-fs3_sourcedata, numerical data for Figure 3—figure supplement 3; F4_sourcedata, numerical data for Figure 4; F6_sourcedata, numerical data for Figure 6; F7_sourcedata, numerical data for Figure 7; F7-fs1_sourcedata, numerical data for Figure 7—figure supplement 1; F7-fs2_sourcedata, numerical data for Figure 7—figure supplement 2.

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Author details

1. Hannah R Martin

   Department of Neurobiology, University of Chicago, Chicago, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2028-5798

2. Anna Lysakowski

   Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, United States

   Contribution

   Funding acquisition, Investigation, Methodology, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6259-0294

3. Ruth Anne Eatock

   Department of Neurobiology, University of Chicago, Chicago, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing – review and editing

   For correspondence

   eatock@uchicago.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7547-2051

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