Different Ca_V1.3 Channel Isoforms Control Distinct Components of the Synaptic Vesicle Cycle in Auditory Inner Hair Cells.

The mechanisms orchestrating transient and sustained exocytosis in auditory inner hair cells (IHCs) remain largely unknown. These exocytotic responses are believed to mobilize sequentially a readily releasable pool of vesicles (RRP) underneath the synaptic ribbons and a slowly releasable pool of vesicles (SRP) at farther distance from them. They are both governed by Ca_v1.3 channels and require otoferlin as Ca²⁺ sensor, but whether they use the same Ca_v1.3 isoforms is still unknown. Using whole-cell patch-clamp recordings in posthearing mice, we show that only a proportion (∼25%) of the total Ca²⁺ current in IHCs displaying fast inactivation and resistance to 20 μm nifedipine, a l-type Ca²⁺ channel blocker, is sufficient to trigger RRP but not SRP exocytosis. This Ca²⁺ current is likely conducted by short C-terminal isoforms of Ca_v1.3 channels, notably Ca_v1.3_42A and Ca_v1.3_43S, because their mRNA is highly expressed in wild-type IHCs but poorly expressed in Otof^-/- IHCs, the latter having Ca²⁺ currents with considerably reduced inactivation. Nifedipine-resistant RRP exocytosis was poorly affected by 5 mm intracellular EGTA, suggesting that the Ca_v1.3 short isoforms are closely associated with the release site at the synaptic ribbons. Conversely, our results suggest that Ca_v1.3 long isoforms, which carry ∼75% of the total IHC Ca²⁺ current with slow inactivation and confer high sensitivity to nifedipine and to internal EGTA, are essentially involved in recruiting SRP vesicles. Intracellular Ca²⁺ imaging showed that Ca_v1.3 long isoforms support a deep intracellular diffusion of Ca²⁺SIGNIFICANCE Auditory inner hair cells (IHCs) encode sounds into nerve impulses through fast and indefatigable Ca²⁺-dependent exocytosis at their ribbon synapses. We show that this synaptic process involves long and short C-terminal isoforms of the Ca_v1.3 Ca²⁺ channel that differ in the kinetics of their Ca²⁺-dependent inactivation and their relative sensitivity to the l-type Ca²⁺ channel blocker nifedipine. The short C-terminal isoforms, having fast inactivation and low sensitivity to nifedipine, mainly control the fast fusion of the readily releasable pool (RRP); that is, they encode the phasic exocytotic component. The long isoforms, with slow inactivation and great sensitivity to nifedipine, mainly regulate the vesicular replenishment of the RRP; that is, the sustained or tonic exocytosis.