CaV1.3 channels are essential for development and presynaptic activity of cochlear inner hair cells

Abstract

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, afferent synaptic transmission is triggered by spontaneous Ca2+ spikes of IHCs, which are under efferent cholinergic control. Around the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired, and Ca2+ spikes as well as the cholinergic innervation are lost. Here, we performed patch-clamp measurements in IHCs of mice lacking the CaV1.3 channel (CaV1.3-/-) to investigate the role of this prevailing voltage-gated Ca2+ channel in IHC development and synaptic function. The small Ca2+ current remaining in IHCs from 3-week-old CaV1.3-/- mice was mainly mediated by L-type Ca2+ channels, because it was sensitive to dihydropyridines but resistant to inhibitors of non-L-type Ca2+ channels such as omega-conotoxins GVIA and MVIIC and SNX-482. Depolarization induced only marginal exocytosis in CaV1.3-/- IHC, which was solely mediated by L-type Ca2+ channels, whereas robust exocytic responses were elicited by photolysis of caged Ca2+. Secretion triggered by short depolarizations was reduced proportionally to the Ca2+ current, suggesting that the coupling of the remaining channels to exocytosis was unchanged. CaV1.3-/- IHCs lacked the Ca2+ action potentials and displayed a complex developmental failure. Most strikingly, we observed a continued presence of efferent cholinergic synaptic transmission and a lack of functional large-conductance Ca2+-activated K+ channels up to 4 weeks after birth. We conclude that CaV1.3 channels are essential for normal hair cell development and synaptic transmission.

Figure 4.

Lack of functional large-conductance Ca²⁺-activated K⁺ channels in Ca_V1.3^-/- IHC. A compares representative current traces of wt (P25) and mutant (P30) IHCs evoked by 150-msec-long step depolarizations to the indicated potentials. In B example traces of wt (-23 mV), wt + 5 mm TEA (-23 mV), and Ca_V1.3^-/- (-25 mV) IHCs are shown on expanded time scale after normalizing them to their steady-state values reached at the end of the depolarization (outside axis, steady-state values were not reached within the displayed time frame, but see Fig. 4A) as well as in absolute numbers (inside axis). Normalized and absolute currents of wt overlap. C shows averaged current traces (mean ± SEM) obtained from Ca_V1.3^-/- IHCs after depolarization to -8 mV, which had either low (no added Ca²⁺; 0.1 mm free EGTA in the pipette; n = 3 IHCs) or high (1 mm added Ca²⁺; no EGTA in the pipette; n = 5 IHCs) basal [Ca²⁺]_i. For the latter, the change of intracellular [Ca²⁺]_i was qualitatively confirmed by fura-2 fluorimetry (data not shown). D displays average I-V relationships for wt (n = 5 IHCs; P24-P29), wt + 5 mm TEA (n = 2 IHCs; P24, P29), and Ca_V1.3^-/- (high Ca²⁺; n = 5 IHCs; P25) IHCs. “Early” and “late” designate the averaging time windows (shown as gray bars in A) of 1.2 msec for 100 μsec (mainly representing I_K,f in wt) and 140 msec for 9.5 msec (representing the sum of all outward currents), respectively. E plots representative current density traces obtained from wt and mutant IHCs during a hyperpolarizing voltage step from -65 to -125 mV. The mean series resistance of all cells included in Figure 4 was 5.6 ± 0.3 MΩ after R_s compensation and data were off-line-corrected for the remaining voltage error.

Figure 1.

Strongly reduced Ca²⁺ current and exocytosis in Ca_V1.3^-/- IHCs. A plots representative Ca²⁺ current traces for wt (P31) and Ca_V1.3^-/- (P32) IHCs, which were elicited by step depolarization to the peak Ca²⁺ current potential (Solutions I_i and I_e were used for A-D). Data were leak-subtracted using a p/6 protocol. In B average current-voltage relationships (I-V) recorded from wt (n = 6 cells) and a Ca_V1.3^-/- (n = 7 cells) IHCs are displayed. The dashed line represents a scaled version of the Ca_V1.3^-/-I-V (scaling factor of 12.8 to match peak Ca²⁺ currents). C, D, Capacitance responses (ΔC_m) to paired depolarizations to the peak Ca²⁺ current potential, spaced 100 msec apart and having variable duration of 10, 20, 50, and 100 msec were recorded for wt (n = 6 cells; P20-P32) and Ca_V1.3^-/- (n = 9 cells; P14-P32) IHCs. C shows representative examples of wt and Ca_V1.3^-/- ΔC_m in response to a 2 ^* 20 msec paired pulse (low-pass filtered at 100 Hz). The bars indicate averages of baseline-subtracted Ca_V1.3^-/-C_m over 400 msec before and 40-100 msec after the first and second depolarization. Intervals between the pairs were at least 30 sec to allow for complete recovery of the readily releasable pool (Moser and Beutner, 2000). D plots mean wt and mutant ΔC_m responses to the first depolarization versus the respective duration of the stimuli. E shows the average ΔC_m response of Ca_V1.3^-/- IHCs (n = 9 recordings) to flash-photolysis of caged Ca²⁺. The mean Ca_V1.3^-/- ΔC_m is displayed along with its SE.

Figure 2.

Developmental changes of Ca²⁺ current density: lack of Ca²⁺ spikes in Ca_V1.3^-/- IHCs. A shows mean absolute (top panel) and normalized (bottom panel, normalized to P6 values) Ca²⁺ current densities for Ca_V1.3^-/-, C57BL/6J, and NMRI IHCs (the NMRI data set was taken from Beutner and Moser, 2001) at P6 (n_NMRI = 10; n_C57BL/6J = 2; n_Cav1.3^-/- = 5 IHCs), P10 (n_NMRI = 9; n_Cav1.3^-/- = 7 IHCs), and P14-P32 (n_NMRI = 28; n_C57BL/6J = 8; n_Cav1.3^-/- = 8 IHCs). The top panel highlights the dramatic reduction in Ca²⁺ current density in Ca_V1.3^-/- IHCs. The bottom panel emphasizes the developmental current density reduction in all strains investigated. B, Whereas current injection readily triggered APs in wt IHCs (left panel, example is P5), even strong current injections failed to elicit APs in Ca_V1.3^-/- IHCs (right panel, example is P7). The inset compares the initial rise of membrane potential of representative wt and Ca_V1.3^-/- IHCs after injection of 300 pA.

Figure 3.

Identity and function of the Ca²⁺ channels in Ca_V1.3^-/- IHCs. A plots average Ba²⁺ current IVs (mean ± SEM) of mutant IHCs under control conditions and during application of the DHP agonist BayK8644 (5 μm; n = 4 cells), the DHP antagonist isradipine (10 μm; n = 7 cells), or nickel (5 mm; n = 2 cells). Control I-V relationships before application of DHP agonists and antagonists were pooled. Individual I-V relationships were leak-corrected (see Materials and Methods). B compares leak-corrected Ca²⁺ currents and corresponding ΔC_m recorded in the perforated-patch configuration in response to pairs of 500 msec depolarizations to -6 mV during subsequent treatment with 200 nm and 5 μm of isradipine. C plots average Ba²⁺ current I-V relationships of a representative perforated-patch experiment in which application of 1 μm of ω-conotoxin (ctx) GVIA was followed by wash-in of 3 μm of ω-conotoxin MVIIC without any obvious effects on current amplitude or voltage dependence. D summarizes the mean effects of the specified drugs on the Ca_V1.3^-/- IHCs peak Ba²⁺ current. Each group of drugs was compared with their own control data acquired in perforated-patch experiments before the wash-in of the drug. The error probabilities for assuming statistically different mean amplitudes are provided underneath the plot.

Figure 5.

Continued presence of postsynaptic responses in Ca_V1.3^-/- IHCs. A compares representative spontaneous postsynaptic currents of wt and Ca_V1.3^-/- IHCs before and after the onset of hearing in wt (potentials are indicated next to the traces). I-V relationships of the slow component of the postsynaptic currents are plotted in B. C displays biphasic postsynaptic currents of a P30 Ca_V1.3^-/- IHC (at -75 mV) in more detail. D shows a stretch of a current-clamp V_m recording from the same cell as in C, illustrating the depolarizing onset and hyperpolarizing main component of these postsynaptic potentials. Extracellular application of 300 nm strychnine (P35; E) and dequalinium (1 μm; F; same cell as in E) abolished the postsynaptic currents of Ca_V1.3^-/- IHCs. The inhibitory effects of both drugs were reversible.