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.

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

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.

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.

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 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

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).

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.

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).

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.