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. 2017 Nov 7;6:e31013. doi: 10.7554/eLife.31013

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© 2017, Michalski et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A1) Protocol used to depolarize IHCs from −95 mV to potentials between −65 to +35 mV (top). Examples of Ca²⁺ currents (I_Ca) (middle) and corresponding C_m traces (bottom) for P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs after 20 ms of depolarization to −10 mV. (A2) Mean Ca²⁺ current amplitudes (I_Ca) (top) and ΔC_m (bottom) for P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs after 20 ms of depolarization to potentials between −65 mV to +35 mV. The vertical dashed line indicates the −30 mV voltage point. (A3) Mean ΔC_m values plotted against the Ca²⁺ currents elicited by depolarizing steps to potentials underlying the falling segment of the I_Ca/V_m curve (−65 mV to −10 mV), corresponding to increasing Ca²⁺ currents. The vertical dashed line indicates the −30 mV voltage point. The Otof ^C2C/+ and Otof ^C2C/C2CΔC_m data were fitted with a power function, yielding an exponent of 0.94 and 0.83, respectively. (B1) Protocol used to depolarize IHCs from −95 mV to −10 mV for voltage steps of different durations from 2 ms to 50 ms (top). Corresponding example C_m traces from P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs (bottom). The example traces for each genotype come from the same patch-clamped IHC. (B2) Kinetics of Ca²⁺-dependent exocytosis in P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs for voltage steps of 2 ms to 50 ms. Mean ΔC_m is plotted against the duration of the depolarization to −10 mV (Δt). The inset shows the detail for Δt values between 2 ms and 10 ms. For the 2 ms and 5 ms depolarizations, five repetitions of the recordings were averaged, to increase the signal-to-noise ratio. The decrease in Ca²⁺-sensitivity of RRP vesicle fusion was evaluated by fitting the ΔC_m versus Δt plots with a line for Δt between 2 and 10 ms in Otof ^C2C/+ IHCs and for Δt between 2 and 20 ms in Otof ^C2C/C2C IHCs. The Otof ^C2C/+ fit was plotted for durations greater than 10 ms, to illustrate the onset of the second component of release corresponding to the initiation of vesicle pool replenishment. (B3) We evaluated the coupling of voltage-gated Ca²⁺ channels to RRP vesicles, by setting the intracellular EGTA concentration to 5 mM in Otof ^C2C/+ IHCs (gray, n = 9) and in Otof ^C2C/C2C IHCs (light blue, n = 10). The data for an intracellular EGTA concentration of 0.5 mM are as in (B2). (C1) Protocol used to depolarize IHCs from −95 mV to −10 mV for 20 ms with different extracellular Ca²⁺ concentrations (top). Example C_m traces from P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs for different extracellular Ca²⁺ concentrations (bottom). Each example C_m trace for a given genotype was obtained from a different IHC. (C2) ΔC_m values plotted against the Ca²⁺ currents elicited at different extracellular Ca²⁺ concentrations ([Ca²⁺]_e) in Otof ^C2C/+ and Otof ^C2C/C2C P15-P18 IHCs. Dashed lines show linear fits to the data. Data information: In (A2, B2–B3), data are presented as the mean ± SEM. ***p<0.001, ns not significant (two-way-ANOVA). In (B2, inset), *p<0.05 (Student's t-test with Welch correction). In A1, example Ca²⁺ traces are corrected for linear leak conductance, leading to a subtraction of the sinusoidal signal. In (A1, B1, C1), the raw C_m traces are shown.

Figure 5—figure supplement 1. — (A1) Protocol used to depolarize IHCs from −95 mV to potentials between −65 to +35 mV (top). Examples of Ca²⁺ currents (I_Ca) (middle) and corresponding C_m traces (bottom) for P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs after 20 ms of depolarization to −10 mV. (A2) Mean Ca²⁺ current amplitudes (I_Ca) (top) and ΔC_m (bottom) for P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs after 20 ms of depolarization to potentials between −65 mV to +35 mV. The vertical dashed line indicates the −30 mV voltage point. (A3) Mean ΔC_m values plotted against the Ca²⁺ currents elicited by depolarizing steps to potentials underlying the falling segment of the I_Ca/V_m curve (−65 mV to −10 mV), corresponding to increasing Ca²⁺ currents. The vertical dashed line indicates the −30 mV voltage point. The Otof ^C2C/+ and Otof ^C2C/C2CΔC_m data were fitted with a power function, yielding an exponent of 0.94 and 0.83, respectively. (B1) Protocol used to depolarize IHCs from −95 mV to −10 mV for voltage steps of different durations from 2 ms to 50 ms (top). Corresponding example C_m traces from P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs (bottom). The example traces for each genotype come from the same patch-clamped IHC. (B2) Kinetics of Ca²⁺-dependent exocytosis in P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs for voltage steps of 2 ms to 50 ms. Mean ΔC_m is plotted against the duration of the depolarization to −10 mV (Δt). The inset shows the detail for Δt values between 2 ms and 10 ms. For the 2 ms and 5 ms depolarizations, five repetitions of the recordings were averaged, to increase the signal-to-noise ratio. The decrease in Ca²⁺-sensitivity of RRP vesicle fusion was evaluated by fitting the ΔC_m versus Δt plots with a line for Δt between 2 and 10 ms in Otof ^C2C/+ IHCs and for Δt between 2 and 20 ms in Otof ^C2C/C2C IHCs. The Otof ^C2C/+ fit was plotted for durations greater than 10 ms, to illustrate the onset of the second component of release corresponding to the initiation of vesicle pool replenishment. (B3) We evaluated the coupling of voltage-gated Ca²⁺ channels to RRP vesicles, by setting the intracellular EGTA concentration to 5 mM in Otof ^C2C/+ IHCs (gray, n = 9) and in Otof ^C2C/C2C IHCs (light blue, n = 10). The data for an intracellular EGTA concentration of 0.5 mM are as in (B2). (C1) Protocol used to depolarize IHCs from −95 mV to −10 mV for 20 ms with different extracellular Ca²⁺ concentrations (top). Example C_m traces from P15-P18 Otof ^C2C/+ and Otof ^C2C/C2C IHCs for different extracellular Ca²⁺ concentrations (bottom). Each example C_m trace for a given genotype was obtained from a different IHC. (C2) ΔC_m values plotted against the Ca²⁺ currents elicited at different extracellular Ca²⁺ concentrations ([Ca²⁺]_e) in Otof ^C2C/+ and Otof ^C2C/C2C P15-P18 IHCs. Dashed lines show linear fits to the data. Data information: In (A2, B2–B3), data are presented as the mean ± SEM. ***p<0.001, ns not significant (two-way-ANOVA). In (B2, inset), *p<0.05 (Student's t-test with Welch correction). In A1, example Ca²⁺ traces are corrected for linear leak conductance, leading to a subtraction of the sinusoidal signal. In (A1, B1, C1), the raw C_m traces are shown.