Direct measurement of single‐channel Ca 2+ currents in bullfrog hair cells reveals two distinct channel subtypes

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 ω‐conotoxin GVIA. Despite its sensitivity towards ω‐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 m m 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 ∼–60 mV and had a single‐channel conductance of ∼16 pS. 4 The non‐L‐type channel exhibited at least two fast open time constants (τ o = 0.2 and 5 ms). In contrast, the L‐type channels showed long openings (τ o =∼23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (τ 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.