title: Different CaV1.3 Channel Isoforms Control Distinct Components of the Synaptic Vesicle Cycle in Auditory Inner Hair Cells.
abstract: 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 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 Cav1.3 channels, notably Cav1.342Aand Cav1.343S, because their mRNA is highly expressed in wild-type IHCs but poorly expressed inOtof-/-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 confer 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+SIGNIFICANCE STATEMENTAuditory 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); 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.
authors.affiliation: Université de Bordeaux, Institut des Neurosciences de Bordeaux, Equipe Neurophysiologie de la Synapse Auditive, Inserm, Unité Mixte de Recherche en Santé 1120, Centre Hospitalier Universitaire Hôpital Pellegrin, 33076 Bordeaux, France.
authors.forename: Philippe F Y
authors.identifier: 0000-0001-7712-7198
authors.initials: PF
authors.lastname: Vincent
authors.affiliation: Université de Bordeaux, Institut des Neurosciences de Bordeaux, Equipe Neurophysiologie de la Synapse Auditive, Inserm, Unité Mixte de Recherche en Santé 1120, Centre Hospitalier Universitaire Hôpital Pellegrin, 33076 Bordeaux, France.
authors.forename: Yohan
authors.initials: Y
authors.lastname: Bouleau
authors.affiliation: Université de Bordeaux, Institut des Neurosciences de Bordeaux, Equipe Neurophysiologie de la Synapse Auditive, Inserm, Unité Mixte de Recherche en Santé 1120, Centre Hospitalier Universitaire Hôpital Pellegrin, 33076 Bordeaux, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Charpentier
authors.affiliation: Institut Pasteur et Université Pierre et Marie Curie, Unité de Génétique et Physiologie de l'Audition, Inserm, Unité Mixte de Recherche en Santé 1120, 75015 Paris, France, and.
authors.forename: Alice
authors.identifier: 0000-0002-5919-751X
authors.initials: A
authors.lastname: Emptoz
authors.affiliation: Institut Pasteur et Université Pierre et Marie Curie, Unité de Génétique et Physiologie de l'Audition, Inserm, Unité Mixte de Recherche en Santé 1120, 75015 Paris, France, and.
authors.forename: Saaid
authors.identifier: 0000-0002-6159-0572
authors.initials: S
authors.lastname: Safieddine
authors.affiliation: Institut Pasteur et Université Pierre et Marie Curie, Unité de Génétique et Physiologie de l'Audition, Inserm, Unité Mixte de Recherche en Santé 1120, 75015 Paris, France, and.|Collège de France, 75005 Paris, France.
authors.forename: Christine
authors.identifier: 0000-0002-9069-002X
authors.initials: C
authors.lastname: Petit
authors.affiliation: Université de Bordeaux, Institut des Neurosciences de Bordeaux, Equipe Neurophysiologie de la Synapse Auditive, Inserm, Unité Mixte de Recherche en Santé 1120, Centre Hospitalier Universitaire Hôpital Pellegrin, 33076 Bordeaux, France, didier.dulon@inserm.fr.
authors.forename: Didier
authors.identifier: 0000-0003-1541-6861
authors.initials: D
authors.lastname: Dulon
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000145:classification / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D006199:Hair Cells, Auditory, Inner / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020033:Protein Isoforms / Q000145:classification / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism*
chemical_list: C485995:Cacna1d protein, mouse; D020746:Calcium Channels, L-Type; D020033:Protein Isoforms; D002118:Calcium
keywords: auditory; calcium channels; hair cells; synaptic exocytosis