Persistence of Cav1.3 Ca2+ Channels in Mature Outer Hair Cells Supports Outer Hair Cell Afferent Signaling

Abstract

Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca²⁺ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca²⁺ channel currents (charge carrier, 10 mm Ba²⁺) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from ∼170 to ∼50 pA at approximately the onset of hearing. Ba²⁺ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba²⁺ current amplitude of apical OHCs was 58 ± 9 pA (n = 20, P19–P30) compared with that of the inner hair cells (IHCs) of 181 ± 50 pA (n = 24, P17–P30). Properties of Ba²⁺ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca_v1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca_v1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca_v1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca_v1.3 channels in exocytosis of mature OHCs.