Direct measurement of single‐channel Ca2+ currents in bullfrog hair cells reveals two distinct channel subtypes

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Abstract
1. To confer their acute sensitivity to mechanical stimuli, hair cells employ Ca(2+) ions to mediate sharp electrical tuning and neurotransmitter release. We examined the diversity and properties of voltage-gated Ca(2+) channels in bullfrog saccular hair cells by means of perforated and cell-attached patch-clamp techniques. Whole-cell Ca(2+) current records provided hints that hair cells express L-type as well as dihydropyridine-insensitive Ca(2+) currents. 2. Single Ca(2+) channel records confirmed the presence of L-type channels, and a distinct Ca(2+) channel, which has sensitivity towards omega-conotoxin GVIA. Despite its sensitivity towards omega-conotoxin GVIA, the non-L-type channel cannot necessarily be considered as an N-type channel because of its distinct voltage-dependent gating properties. 3. Using 65 mM Ca(2+) as the charge carrier, the L-type channels were recruited at about -40 mV and showed a single-channel conductance of 13 pS. Under similar recording conditions, the non-L-type channels were activated at approximately -60 mV and had a single-channel conductance of approximately 16 pS. 4. The non-L-type channel exhibited at least two fast open time constants (tau(o) = 0.2 and 5 ms). In contrast, the L-type channels showed long openings (tau(o) = approximately 23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (tau(o) = 0.3 and 10 ms). 5. The number of functional channels observed in patches of similar sizes suggests that Ca(2+) channels are expressed singly, in low-density clusters (2-15 channels) and in high-density clusters (20-80 channels). Co-localization of the two channel subtypes was observed in patches containing low-density clusters, but was rare in patches containing high-density clusters. 6. Finally, we confirmed the existence of two distinct Ca(2+) channel subtypes by using immunoblot and immunohistochemical techniques.