Effects of phenylalkylamines and benzothiazepines on Ca(v)1.3-mediated Ca2+ currents in neonatal mouse inner hair cells

Calcium currents (I(Ca)) in inner hair cells (IHCs) are carried by the Ca(v)1.3 subtype of L-type calcium channels. They play an important role in synaptic transmission of sound-evoked mechanical stimuli. L-type calcium channels are targets of the organic blocker classes dihydropyridines, phenylalkylamines and benzothiazepines. Previously a low sensitivity of the Ca(v)1.3 subtype towards dihydropyridines has been demonstrated. Therefore, this study evaluates the effect of two phenylalkylamines (verapamil and gallopamil) and the benzothiazepine diltiazem on I(Ca) through Ca(v)1.3 channels in mouse IHCs. Whole-cell I(Ca) was measured using the patch-clamp technique in mouse IHCs aged postnatal day 3-7 with 5 mM calcium as a charge carrier. The phenylalkylamines verapamil and gallopamil and the benzothiazepine diltiazem inhibited I(Ca) in IHCs in a concentration-dependent manner. This block was largely reversible. Dose-response curves revealed IC(50) values of 199+/-19 microM for verapamil, 466+/-151 microM for gallopamil and 326+/-67 microM for diltiazem. The inhibition of peak I(Ca) by phenylalkylamines and benzothiazepines was voltage-independent. Verapamil (300 microM) enhanced current inactivation from -20 to +20 mV while diltiazem (300 microM) did so only at very depolarised potentials (+20 mV). In conclusion, the concentrations of phenylalkylamines and benzothiazepine necessary to inhibit 50% of I(Ca) in IHCs were one order larger compared to concentrations which inhibited I(Ca) through Ca(v)1.2 channels in native cells or expression systems. However, inhibitory concentrations were in the same range as those required for block of I(Ca) in turtle hair cells.