Few Ca_V1.3 Channels Regulate the Exocytosis of a Synaptic Vesicle at the Hair Cell Ribbon Synapse

Effects of changes in (apparent) single channel Ca²⁺ domain amplitude. A, Representative Ca²⁺ currents (bottom) and exocytic capacitance changes (ΔC_{m, 20 ms}; top) in response to 20 ms depolarization to the peak Ca²⁺ current potential. The Ca²⁺ current was large and the ΔC_m sizable in the presence of 10 mm [Ca²⁺]_e, but the reduced Ca²⁺ current at 0.25 mm [Ca²⁺]_e failed to elicit an obvious ΔC_m. B, Scatter plot of ΔC_{m, 20 ms} versus the corresponding Ca²⁺ current integrals (Q_Ca) for three experiments in which [Ca²⁺]_e was gradually changed (10, 4, 2, 1, 0.5, and 0.25 mm). The solid line is a least-squares fit of a power of exponent function to the data approximating the plateau of RRP exocytosis, the specific Q_Ca, and the apparent cooperativity. C, The time course of ΔC_{m, 20 ms} (top trace) and the Ca²⁺ current (bottom trace; 10 mm [Ca²⁺]_e) for a typical perforated-patch experiment, in which Zn²⁺ (1 mm) was applied after a control steady state had been established. An obvious ΔC_{m, 20 ms} drop occurred only after the Ca²⁺ current had already substantially declined. D, ΔC_{m, 20 ms} and Q_Ca of five such experiments (circles). The solid line presents a power of exponent fit (as in B).

Effects of primary changes in open Ca²⁺ channel number. A, The time course of exocytic ΔC_{m, 20 ms} (top) and Q_Ca (bottom) in response to repetitive depolarization (20 ms; to the peak Ca²⁺ current potential; 10 mm [Ca²⁺]_e). In these representative perforated-patch experiments, nifedipine (10μm; ×) or BayK8644 (10μm; ○) were applied by slow bath perfusion as indicated by the arrow. B, ΔC_{m, 20 ms} and Q_Ca for a total of 15 such experiments (10μm isradipine, +; 10 μm nifedipine, ×; and 5 μm BayK8644, ○; n = 5 for each condition). DHP were washed in after achieving a control steady state as illustrated in A. The lines correspond to the fit functions provided in the graph. Solid and dotted lines, Line and power of exponent fits to the data acquired with inhibitory DHP (the fit interval from lowest integral up to 5 pC was chosen as in Fig. 2 for better comparison); dashed line, line fit to BayK8644 data. C, The logarithms of exocytic ΔC_{m, 20 ms} (in farads) and Q_Ca (in coulombs) for both manipulations (DHP, black circles; Zn²⁺ and Δ[Ca²⁺]_e, gray asterisks). The lines represent line fits to both data sets (along with 99% confidence intervals) yielding Ca²⁺ cooperativities of 1.4 and 2.3 for DHP and Zn²⁺/Δ[Ca²⁺]_e, respectively. D, Steady-state effects of 10 μm BayK8644, 10 μm nifedipine, 10 μm isradipine, or 0.5–1 mm Zn²⁺ (n = 5 for each condition) on ΔC_{m, 20 ms} (top) and Q_Ca (bottom). The gray filling indicates the average ΔC_{m, 20 ms} and Q_Ca estimates after drug application after normalization to the grand averages obtained before drug. Error bars represent SEM. Q_Ca, Ca²⁺ current integrals.

Direct comparison of the effects of changes in single-channel current and open-channel number on RRP exocytosis. Scatter plot of ΔC_{m, 20 ms} versus the corresponding Ca²⁺ current integrals of a representative perforated-patch experiment during (1) slow change of [Ca²⁺]_e from 3 to 10 mm, (2) application of 10 μm nifedipine at 10 mm [Ca²⁺]_e, and (3) wash out of nifedipine in the continued presence of 10 mm [Ca²⁺]_e are shown. Data were smoothed by nonoverlapping two-point box car averaging. The time course of the experiment is indicated by the black-to-gray gradient.

Effects of depolarization strength on RRP exocytosis. A, B, The steady-state IV (black symbols) of the experiments used to study the voltage dependence of RRP exocytosis (10 mm [Ca²⁺]_e). The circles indicate the potentials for which the kinetics of exocytosis was studied. The instantaneous IV (A; gray symbols) was approximated as the ratio of the steady-state IV (A; black symbols) and the channel activation curve (B) in the range of 5–50 mV and then linearly extrapolated toward hyperpolarized potentials. C, Example currents for depolarization to –26, –11, and 19 mV, using the color coding of A and B. D, Summary of the exocytic ΔC_m achieved for the three depolarization levels and different pulse durations. Pulses were applied to 33 IHCs in random order with an interval of at least 30 s and 1073 sweeps were used for analysis. Of those, a smaller number of sweeps was acquired at –41 mV (n = 158; data not shown), which did not provide a sufficiently resolved kinetics plot. E, The first 2 ms of exocytosis kinetics in better resolution. F, The exocytic responses (ΔC_{m, 10 ms}) to 10-ms-long depolarizations to four potentials (–41, –28, –13, and 17 mV) versus the corresponding Ca²⁺ current integrals. The first point corresponds to a 20 ms blank (–86 mV). The gray scale codes for depolarization strength as in the other panels: from light gray (–86 mV) to black (17 mV). Note the different amounts of exocytosis despite similar Ca²⁺ current integrals for –28 mV [larger single-channel current (see A) but lower open probability (see B)] and 17 mV [smaller i (see A) but larger p (see B)]. Error bars represent SEM.