Rapidly inactivating and non-inactivating calcium-activated potassium currents in frog saccular hair cells

Abstract

1. Using a semi-intact epithelial preparation we examined the Ca(2+)-activated K(+) (K(Ca)) currents of frog (Rana pipiens) saccular hair cells. After blocking voltage-dependent K(+) (K(V)) currents with 4-aminopyridine (4-AP) an outward current containing inactivating (I(transient)) and non-inactivating (I(steady)) components remained. 2. The contribution of each varied greatly from cell to cell, with I(transient) contributing from 14 to 90 % of the total outward current. Inactivation of I(transient) was rapid (tau approximately 2-3 ms) and occurred within the physiological range of membrane potentials (V(1/2) = -63 mV). Recovery from inactivation was also rapid (tau approximately 10 ms). 3. Suppression of both I(transient) and I(steady) by depolarizations that approached the Ca(2+) equilibrium potential and by treatments that blocked Ca(2+) influx (application Ca(2+)-free saline or Cd(2+)), suggest both are Ca(2+) dependent. Both were blocked by iberiotoxin, a specific blocker of large-conductance K(Ca) channels (BK), but not by apamin, a specific blocker of small-conductance K(Ca) channels. 4. Ensemble-variance analysis showed that I(transient) and I(steady) flow through two distinct populations of channels, both of which have a large single-channel conductance (~100 pS in non-symmetrical conditions). Together, these data indicate that both I(transient) and I(steady) are carried through BK channels, one of which undergoes rapid inactivation while the other does not. 5. Inactivation of I(transient) could be removed by extracellular papain and could later be restored by intracellular application of the 'ball' domain of the auxiliary subunit (beta2) thought to mediate BK channel inactivation in rat chromaffin cells. We hypothesize that I(transient) results from the association of a similar beta subunit with some of the BK channels and that papain removes inactivation by cleaving extracellular sites required for this association.

Figure 1. Separating currents into I_Ca, I_transient and I_steady

A, I_Ca (thin trace) was estimated by fitting the initial inward component of the recorded current using the known activation kinetics (eqn (1) with τ_m= 0.13 ms). The size of I_Ca (a_Ca) was measured to be the steady-state value of the fitted current. Peak amplitudes of I_transient (a_transient) and I_steady (a_steady) were measured as shown. B, in order to separate the two currents for ensemble-variance analysis, the amplitudes of the transient and steady-state components were estimated from m(t). K_s-s was defined as the steady-state current at the end of the second voltage step minus the baseline current before the step. K_trans was determined by fitting a single exponential function (thick grey trace) to the declining phase of the transient current, extrapolating back to the beginning of the voltage step (t = 0), and subtracting K_s-s. The dotted vertical lines mark the limits of the exponential fit. C, I_trans(t) and I_s-s(t) determined from m(t), K_s-s, K_trans and τ_D using eqns (5) and (6). A typical estimated I_Ca(t) waveform is shown to illustrate that by 0.4 ms after the beginning of the voltage step (dotted vertical line) I_Ca is essentially fully activated. The horizontal line in each panel denotes 2 ms.

Figure 10. External application of papain removes inactivation

Perforated-patch recording from a single hair cell bathed in 10 mm 4-AP before (thick trace) and after (thin trace) bath application of 8 units ml⁻¹ of twice crystallized papain for approximately 10 min. The current at −20 mV was estimated by linear interpolation of currents recorded from adjacent steps.

Figure 8. Ensemble-variance analysis of I_transient and I_steady in two hair cells

A, mean current (m(t)) and variance (σ²(t)) calculated from 500 voltage steps to −5 mV (R_S corrected). B, plot of σ²(t) vs. m(t) of the cell in A. Only data points between 0.44 ms after the beginning of the voltage step (after the capacitative transients) and the end of the 10 ms step are plotted and were used for fitting. The fitted line (fit using eqn (8)) is a single, continuous curve that doubles back on itself, to form a double parabola. The values for this fit are: i = 3.6 pA, N_steady= 162, N_transient= 484 and x_offset= 229 pA. C, m(t) and σ²(t) calculated from a second hair cell in response to 500 voltage steps to 21 mV (R_S corrected). D, plot of σ²(t) vs. m(t) of the cell in C. Data points between 0.4 ms after the beginning of the voltage step and the end of the 10 ms step are plotted and were used for fitting. The line is a single, continuous curve fitted to the data with values of: i = 5.3 pA, N_steady= 799, N_transient= 647 and x_offset= 148 pA. In both σ²(t) vs. m(t) plots, data points corresponding to the rising phase of m(t) are marked with open squares (□) and points corresponding to the falling phase are marked with filled triangles (▴). Intermediate points are marked with open circles (○). Leak subtraction was not used in this protocol. Recordings were made using the whole-cell configuration and in the presence of 10 mm 4-AP.

Figure 2. Blocking K_V with 4-AP reveals a rapidly activating, partially inactivating outward current

A, a perforated-patch recording from a hair cell bathed in 10 mm 4-AP. Currents were elicited by depolarizing voltage steps (command potentials between +90 and −60 mV, in 10 mV increments) from a holding potential of −70 mV. The largest amplitude voltage reached (R_S corrected) is indicated next to the step protocol. B, plot of the peak I–V relationship for the cell in A.

Figure 3. Variability in the ratio of a_transient to a_steady

Perforated-patch recordings from hair cells bathed in 10 mm 4-AP that exhibited large (A), intermediate (B), and small (C) amounts of I_transient relative to I_steady. The thick grey lines are exponential fits showing the time constant of decay (τ_D) of I_transient. All currents are interpolated to −20 mV.

Figure 6. Ca²⁺ dependence of I_transient and I_steady

A, application of 100 μm Cd²⁺ reversibly eliminated both I_transient and I_steady, leaving a small residual outward current. B, the effect of removing external Ca²⁺ was similar to that of Cd²⁺, although a larger outward current remained. The panels on the right show the peak I–V relationship for each cell before (□), during (•) and after (♦) these treatments. The stimulus protocol was the same as in Fig. 2. The largest amplitude voltage reached (R_S corrected) for each family of traces is indicated on the right. Recordings were made using the whole-cell configuration and 10 mm 4-AP was present throughout.

Figure 4. Rapid inactivation is not an odd effect of 4-AP

A, perforated-patch recordings from a single hair cell bathed sequentially in normal extracellular saline, 6 mm TEA and 10 mm 4-AP. The thin trace below the recording in 6 mm TEA shows the estimated I_Ca. The currents at −30 mV were estimated by linear interpolation of the current from adjacent steps. B, the TEA-sensitive current was obtained by subtracting the current recorded in 6 mm TEA from the current recorded in normal extracellular saline. For comparison with the recordings in A, the estimated I_Ca was added to the trace in B. The larger size of the TEA-sensitive current compared to the current recorded in 4-AP is likely to be the result of current run-down during the time course of these recordings.

Figure 5. Recovery from inactivation and steady-state inactivation of I_transient

A, I_transient recovers rapidly from inactivation. I_transient inactivated during the first 10 ms voltage step to −11 mV (R_S corrected; command potential was 0 mV). After stepping back to the holding potential (−70 mV) for between 2 and 30 ms (tested in 2 ms intervals), a second 10 ms voltage step was applied. The dotted line is an exponential fit of the recovery from inactivation, with time constant τ_R. For clarity, stimulus artefacts present at the beginning of the voltage steps were blanked in making this figure. This recording was made in 10 mm 4-AP using the whole-cell configuration. B, steady-state inactivation of I_transient. Following a 50 ms prepulse to voltages between −100 and −30 mV (tested in 5 mV increments), a 10 ms voltage step to −20 mV (command potential) was applied. The values of a_transient were measured, normalized to the value after the most negative prepulse (−100 mV), averaged across all nine cells and plotted as a function of prepulse potential. The line represents the fitted h_∞ curve (eqn (9)) with V_1/2=−63 mV and k = 2.0 mV. The values plotted are means ±s.d. (n = 9). Series resistance errors were ≤1 mV during the prepulse step and were therefore not considered in the making of this figure.

Figure 7. The effects of IbTX and apamin on I_transient and I_steady

Application of 100 nm IbTX eliminated both I_transient and I_steady in this hair cell (A), while in another hair cell 1 μm apamin had no effect (B). The right-hand panel in A shows the peak I–V relationship for this cell before (□) and during (•) IbTX application. The voltage protocol used to elicit these currents was the same as in Figs 2 and 6. The largest amplitude voltage reached (R_S corrected) for each family of traces is indicated on the right. Recordings were made using the perforated-patch configuration and 10 mm 4-AP was present throughout. Note that the currents in A were elicited by 10 ms duration voltage steps while those in B are in response to 15 ms duration steps.

Figure 9. Single-channel currents estimated from ensemble-variance analysis vs. membrane potential

The line fitted to the data indicates that the g for I_transient and I_steady is 97 pS and that i = 3.6 pA at 0 mV. The filled squares (▪) are estimates of i obtained from a single cell, tested at two potentials. The g for this cell is 79 pS. The data in this plot are from 12 hair cells.

Figure 11. Internally applied ‘ball’ peptide confers inactivation to the non-inactivating BK current in enzymatically dissociated hair cells

A, whole-cell voltage-clamp recordings made over time from an enzymatically dissociated hair cell to which a 300 μm‘ball’ peptide was added internally (via the pipette). Voltage steps to −39 mV (R_S corrected) were presented every 10 s. For clarity only the response to every other step is shown. The first recording (arrow) was made roughly 30 s after achieving the whole-cell configuration. B, plot of the time constant of decay (τ_D) of the outward current vs. recording number from the cell in A. The line represents the single exponential fit with τ_D-MAX of 8.0 ms.