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 Cav1.3 channels and require otoferlin as Ca2+ sensor but whether they use the same Cav1.3 isoforms is still unknown. Using whole cell patch clamp recordings in post-hearing 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 Cav1.3 channels, notably Cav1.342A and Cav1.343S, since their mRNA are 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 Cav1.3 short isoforms are closely associated with the release site at the synaptic ribbons. Conversely, our results suggest that Cav1.3 long isoforms, which carry ∼75 % of the total IHC Ca2+ current with slow inactivation, and confers high sensitivity to nifedipine and to internal EGTA, are essentially involved in recruiting SRP vesicles. Intracellular Ca2+ imaging showed that Cav1.3 long isoforms support a deep intracellular diffusion of Ca2+.
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 Cav1.3 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), i.e. encode the phasic exocytotic component. The long isoforms, with slow inactivation and great sensitivity to nifedipine, mainly regulate the vesicular replenishment of the RRP, i.e. the sustained or tonic exocytosis.
We thank the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging where the confocal high resolution immuno-microscopy of Cav1.3 channels and ribbons was done. This work was in part supported by the Fondation Agir Pour l'Audition (2015-APA Research Grant “Deciphering the role of otoferlin and Usher proteins at the auditory hair cell ribbon synapse”) to Didier Dulon.