Differences between the negatively activating potassium conductances of Mammalian cochlear and vestibular hair cells

Abstract

Cochlear and type I vestibular hair cells of mammals express negatively activating potassium (K(+)) conductances, called g(K,n) and g(K,L) respectively, which are important in setting the hair cells' resting potentials and input conductances. It has been suggested that the channels underlying both conductances include KCNQ4 subunits from the KCNQ family of K(+) channels. In whole-cell recordings from rat hair cells, we found substantial differences between g(K,n) and g(K,L) in voltage dependence, kinetics, ionic permeability, and stability during whole-cell recording. Relative to g(K,L), g(K,n) had a significantly broader and more negative voltage range of activation and activated with less delay and faster principal time constants over the negative part of the activation range. Deactivation of g(K,n) had an unusual sigmoidal time course, while g(K,L) deactivated with a double-exponential decay. g(K,L), but not g(K,n), had appreciable permeability to Cs(+). Unlike g(K,L), g(K,n)'s properties did not change ("wash out") during the replacement of cytoplasmic solution with pipette solution during ruptured-patch recordings. These differences in the functional expression of g(K,n) and g(K,L) channels suggest that there are substantial differences in their molecular structure as well.

Figure 1

g_K,n and other voltage-dependent currents in a rat outer hair cell, revealed by their voltage dependence and sensitivity to the KCNQ blocker, linopirdine. Whole-cell currents were recorded from an outer hair cell isolated from the apical turn of a P17 rat cochlea. A–C Whole-cell currents evoked by the voltage protocol shown below, in control solution (high-Ca²⁺ L15) (A), control + 10 μM linopirdine (B) and wash (high-Ca²⁺ L15) (C). Voltage protocol shown at the bottom. From the holding potential of −64 mV, voltage was stepped to −124 mV, which in control solutions deactivated g_K,n (A,C) (arrow in A), and then to a series of steps between −134 and + 36 mV; g_K,n was reactivated by steps positive to −124 mV. At the offset of the iterated voltage steps, the voltage was stepped to −34 mV and peak tail currents were measured. These currents, which reflect the activation state of the channels at the end of the preceding voltage steps, are plotted as functions of the prepulse voltage in E. Linopirdine (B) blocked the current component that deactivated during the hyperpolarizing step (arrow in A), and much of the current during the iterated voltage steps. D. The linopirdine-blocked current (through g_K,n), obtained by subtracting records in B from records in A. E. Activation curves generated from tail currents at the offset of the iterated voltage steps; the tail current voltage was −34 mV. In control and wash, the curves are fit by the sum of two Boltzmanns [Eq.(2)], with V_1/2 and S values of −91.3 mV and 11.9 mV, respectively, and −12.0 mV and 5.3 mV (control data). Linopirdine fully blocked g_K,n, partly blocked (see text) an inwardly rectifying conductance (g_K1 ; V_1/2 = −73.5 mV, S = 11.3 mV), and did not affect the more positively activating outward rectifier (g_K,neo, V_1/2 = −13.25 mV, S = 3.7 mV). The linopirdine-sensitive activation curve (g_K,n, from D) is well fit by a single Boltzmann with V_1/2 = −89.4 mV and S = 12.2 mV.

Figure 2

Differences in the voltage dependences of g_K,n and g_K,L. Whole-cell currents were recorded from a P16 outer hair cell (Aa) and a P17 type I cell (Ba) with 3-s voltage step protocols (top panels). Aa. Steps to −124 mV from a holding potential of −64 mV deactivated g_K,L. Ba. Steps to −129 mV from a holding potential of −69 mV deactivated g_K,L. Depolarizing steps reactivated the conductances. Ab, Bb. At the offset of the iterated voltage steps, the voltage was stepped to −34 mV (Aa) and −39 mV (Ba) and tail currents were measured (at the arrows) for plotting against prepulse voltage in c. Ac, Bc. Activation curves for the tail current data shown in Ab,Bb (filled circles) are fit with a Boltzmann function [Eq.(1) thick lines, yielding V_1/2 and S values of −93. 6 mV and 12.4 mV (AC, g_K,n) and −82.4 mV and 5.0 mV (Bc, g_K,L), near the mean values for our samples of outer hair cells and type I hair cells, respectively. In this outer hair cell, unlike the cell in Figure 1, the activation curve is well fit by a single Boltzmann, with no indication of a second outward rectifier at depolarized potentials. This is not related to the long-duration iterated voltage steps, as a similar activation curve was obtained with 300-ms voltage steps (Ac, (triangles and thin line: V_1/2 and S values of −92.8 mV and 12.3 mV). In the type I cell, in contrast, the activation curve taken after 300-ms voltage steps (triangles) was positively shifted (thin line): V_1/2 = −68.1 mV and S = 8.19 mV.

Figure 3

Differences in the activation kinetics of g_K,n and g_K,L. A,B. Fitting of activation kinetics with Eq.(3) for (A) g_K,n from a P16 outer hair cell and (B) g_K,L from a P19 type I hair cell. Voltage was first stepped to −124 mV for 500 ms (A) and −129 mV for 200 ms (B) to fully deactivate the conductances, then stepped to the values indicated by each trace. g_K,n: V_1/2= −87.5, S = 14.1 mV, no second outward rectifier, average of 3 traces at each voltage; g_K,L: V_1/2 = −83.6, S = 5.0 mV, not averaged. C. Currents from A and B at two similar command voltages, normalized to the current at 3 s for comparison of kinetics. g_K,L activated much more slowly at voltages near −90 mV and with a longer delay at voltages near −50 mV. Note that the reversal potentials for these K⁺ conductances were approximately −80 mV, so that current was inward at −90 mV and outward at −50 mV. D,E. Fast (D) and slow (E) time constants as functions of voltage. Triangles values for g_K,n averaged from 5 OHCs. Circles, values for g_K,L averaged from 4–5 type I hair cells, except for the data points at −29 mV, which are averages of fits from two cells (τ_slow = 13.4, 27.2 ms; τ_fast = 13.4, 11.9ms).

Figure 4

Differences in the deactivation kinetics of g_K,n and g_K,L. g_K,L deactivation followed a single- or double-exponential decay function, while g_K,n deactivation had a sigmoidal onset. A. Steps from −64 to −124 mV (g_K,n) or -69 to −129 mV (g_K,L) caused deactivation of currents through both g_K,n, and g_K,L ; thin traces are for g_K,n from 5 outer hair cells, thick traces are for g_K,L from 5 type I cells. Currents are normalized to the peak current at the beginning of the step. B. Fits (thick lines) to the deactivation of two of the data traces in A (thin lines; obscured by fits except at the step onset). The type I data were fit by a double-exponential function [eq.(4)]: A₁ = −264 pA, τ₁ = 2.5 ms, A₂ = −684 pA, τ₂ = 26.8 ms. The OHC data were fit by Eq.(3): τ₁ = 7.8 ms, τ₂ = 33.1 ms.

Figure 5

g_K,L , but not g_K,n, is Cs⁺ permeable. Left column: Currents in control conditions, with high internal K⁺ and 5.8-mM external K⁺. Middle column (different cells): With an internal Cs⁺ solution, g_K,n (B) carried only inward current but g_K,L (E) still passed both inward and outward current. Right column (same cells as middle column): When, in addition, external K⁺ was replaced with Cs⁺, all current through g_K,n was blocked (C; residual slope conductance = 600 pS) but outward current still flowed in the type I cell (F). Current and voltage axes on the left apply to all panels in a row. In standard solutions, relatively small voltage steps applied to the type I cell (D) elicited very large currents through g_K,L. Currents are from a P16 OHC (A), a P17 OHC (B,C), a P24 type I cell (D), and a P23 type I cell (E,F).

Figure 6

Changes in the reversal potential of g_K,L during long voltage steps may result from significant K⁺ flux through g_K,L. Standard vestibular external medium and Cs⁺ pipette solution for perforated patch recordings. Same cell as in Figure 5B,C. After a 200-ms step to −129 mV, the peak currents (b, arrow) reversed at approximately -65 mV. With time during 3-s steps positive to −69 mV, however, the reversal potential shifted positively, reaching approximately −40 mV at the end of the steps (c). The change in reversal potential is shown clearly by the reversal of current polarity during the steps at −59 and −49 mV (thick traces).