Cochlear outer hair cell (OHC) forward and reverse transduction is regulated by intracellular Ca²⁺ concentration ([Ca²⁺]_i). Here we describe a capacitative calcium entry (CCE) mechanism in mammalian cochlear OHC. OHC were isolated from adult guinea-pigs and rats overdosed with pentobarbitone as previously described (Raybould et al. 1997). Using the whole-cell patch clamp technique, repeated voltage ramps were used to monitor membrane currents. In some experiments OHC [Ca²⁺]_i was measured using confocal microscopy. TRPC3 mRNA expression was assessed using RT-PCR analysis. Confocal immunofluorescence microscopy was used to localise TRPC3 protein in whole-mount rat and guinea-pig organ of Corti.

CCE was evident as a biphasic overshoot in OHC membrane current following transient lowering of [Ca²⁺]_i. Application of the K_Ca channel blocker charybdotoxin (1 µM) almost completely blocked activation of the outward current and revealed a Ca²⁺-permeable inward current. This inward current reversed at approximately +25 mV and was dependent on extracellular Ca²⁺ concentration with a threshold of approximately 600 µM.

The primary candidates for the ion channels that mediate CCE are the transient receptor potential (TRP) superfamily of proteins (Minke & Cook, 2002). External application of 2-aminoethyl diphenylborate (2APB, 250 µM-1 mM), an antagonist of TRPC3-mediated currents, produced a differential block of the inward component of the CCE-mediated overshoot in membrane current (Trebak et al. 2002). Inclusion of 2APB in the recording solution had no effect. Bath superfusion of 1-oleoyl-2-acetyl-sn-glycerol (OAG, a synthetic analogue of diacylglycerol; 100 µM-1 mM) produced an increase in membrane current consistent with PLC/DAG-mediated activation of TRPC subunit proteins (Hofman, 1999). These findings were confirmed by RT-PCR detection of TRPC3 mRNA and immunolocalisation of the TRPC3 protein in OHC to known regions of IP₃R expression (Mammano, 1999). CCE was not dependent on stored Ca²⁺, as dumping of intracellular stores with the SERCA blocker cyclopiazonic acid (10 µM) had no effect on the CCE-mediated biphasic current response.

These findings suggest that OHC express TRPC3 subunit protein associated with a CCE pathway that provides a mechanism for the refilling of stores, and also complements Ca²⁺ signalling linked to IP₃R pathways activated by P2YR and AChR.

This work was funded by the Health Research Council, Marsden Fund and Deafness Research Foundation and approved by the University of Auckland Animal Ethics Committee.