Fig. 4.
PTXa-expressing IHCs show an increased and hyperpolarized Ca2+ influx at the whole-cell level. (A) IV relationship of the whole-cell Ca2+ current (ruptured-patch configuration, 5 mM [Ca2+]e) shows a significantly increased current amplitude in the PTXa mutant IHCs (n = 40 IHCs, N = 13 in the PTXa mutant; n = 40 IHCs, N = 14 in the PTXa control; P < 0.0001, t test) (Ai). The protocol, consisting of 20-ms steps of 5 mV from −82 to +63 mV, as well as the resulting currents of a representative control IHC, are shown in the Bottom Right. Mean (line) ± SEM (shaded areas) are displayed; the box plots show 10th, 25th, 50th, 75th, and 90th percentiles with individual data points overlaid, and means are shown as crosses, as for B. (B) Fractional activation of the whole-cell Ca2+ current derived from the IV relationships (A) was fitted to a Boltzmann function. (Bi) Box plots of the voltage for half-maximal activation Vh and Vh estimates of individual IHCs show a significant hyperpolarized shift of the fractional activation (Vh) of the CaV1.3 Ca2+ channels in the PTXa mutant condition (n = 40 IHCs, N = 13 in the PTXa mutant; n = 40 IHCs, N = 14 in the PTXa control; P < 0.0001, t test). (Bii) Box plots of the voltage sensitivity or slope factor k and k estimates of individual IHCs illustrate increased voltage sensitivity in the PTXa mutant condition (n = 40 IHCs, n = 13 in the mutant; n = 40 IHCs, N = 14 in the PTXa control; P < 0.0001, t test). (C) Representative Ca2+ currents (Top) and corresponding low-pass filtered capacitance (Bottom) traces recorded from PTXa mutant and control IHCs in response to 20-ms step depolarization to −17 mV from the holding potential of −87 mV (perforated-patch configuration, 1.3 mM [Ca2+]e). The PTXa mutant IHCs showed bigger Ca2+ currents than the control IHCs, while the capacitance jumps (ΔCm) were comparable. (D) Mean exocytic ΔCm (Top) and Ca2+ current integrals (QCa) (Bottom) as a function of depolarization duration (mean ± SEM, n = 10 IHCs, N = 7 in the PTXa mutant; n = 10 IHCs, N = 6 in the PTXa control; for QCa at 2 ms, P = 0.002; for QCa at 5 ms, P = 0.005; for QCa at 10 ms, P = 0.003; for QCa at 20 ms, P = 0.007; for QCa at 50 ms, P = 0.007, t test; for ΔCm at 2 ms, P = 0.02, t test; at 10 ms, P = 0.03, Mann–Whitney–Wilcoxon test). (E) Relation between exocytic ΔCm and QCa from PTXa mutant and control IHCs (mean ± SEM; fill color of the mean points darkens with increasing depolarization duration). PTXa mutant IHCs showed significantly lower efficiency of Ca2+ influx to drive exocytosis for every depolarization duration. (Ei) Ratio of ΔCm and QCa from PTXa mutant and control IHCs upon 20-ms step depolarization (n = 10 IHCs, N = 7 in the PTXa mutant; n = 10 IHCs, N = 6 in the PTXa control; for ratio at 2 ms, P = 0.02; at 5 ms, P = 0.01; at 10 ms, P = 0.006; at 20 ms, P = 0.001; QCa at 50 ms, P = 0.005, t test). (F) Representative Ca2+ currents (Middle) and corresponding low-pass filtered capacitance (Bottom) traces recorded from PTXa mutant and control IHCs upon 100-ms step depolarization to −41 mV from the holding potential (perforated-patch configuration, 1.3 mM [Ca2+]e). Stimulus template (Top) illustrates the 2-mV steps starting from −53 to −37 mV. (G) Mean exocytic ΔCm (Top) and Ca2+ current integrals (QCa) (Bottom) as a function of depolarization voltage (mean ± SEM, n = 8 IHCs, N = 4 in the PTXa mutant; n = 9 IHCs, N = 5 in the PTXa control; for QCa at −37 mV, P = 0.002; for QCa at −39 mV, P = 0.005; for QCa at −41 mV, P = 0.01, t test; for QCa at −43 mV, P = 0.02; for QCa at −45 mV, P = 0.016; for QCa at −47 mV, P = 0.05; for QCa at −49 mV, P = 0.046, Mann–Whitney–Wilcoxon test). (H) Relation between exocytic ΔCm and QCa from PTXa mutant and control IHCs (mean ± SEM; fill color of the mean points darkens with increasing depolarization voltage). The ratio between the ΔCm and QCa was comparable throughout the different voltage range (−53 to −37 mV, with 2-mV increments). (Hi) Ratio of ΔCm and QCa from PTXa mutant and control IHCs upon 100-ms step depolarization to −39 mV.