Molecular identification of an N-type Ca²⁺ channel in saccular hair cells

We report new molecular evidence for the presence of an N-type (Ca_v2.2, α1B) voltage-gated Ca²⁺ channel in hair cells of the saccular epithelium of the rainbow trout. The Ca_v2.2 amino-acid sequence shows 68% and 63% identity compared with chick and human Ca_v2.2, respectively. This channel reveals features that are characteristic of an N-type Ca²⁺ channel: an ω-conotoxin GVIA binding domain, G_βγ binding regions, and a synaptic protein interaction site. Immunohistochemical studies with a custom antibody show that immunoreactivity for the Ca_v2.2 is concentrated in the basolateral and apical regions of hair cells. Whereas trout brain and saccular macula express an 11-amino-acid insert in the second G_βγ binding domain of the Ca_v2.2 I-II loop, isolated hair cells appear not to express this variant. We constructed fusion polypeptides representing portions of the I-II loop, β1 and β2a auxiliary subunits, the II-III loop, and syntaxin, and examined their intermolecular interactions via immunoprecipitation and surface plasmon resonance. The I-II loop polypeptides bound both β1 and β2a subunits with a preference for β1, and the II-III loop exhibited Ca²⁺-dependent syntaxin binding. We demonstrated syntaxin immunoreactivity near afferent endings in hair cells, at hair-cell apices, and in efferent endings on hair cells, the former two sites consistent with binding of syntaxin to Ca_v2.2. The present molecular characterization of the Ca_v2.2 channel provides novel biochemical evidence for an N-type channel in hair cells, and details molecular interactions of this channel that reflect hair-cell function, such as spontaneous activity and vesicular trafficking. The current work, to our knowledge, represents the first demonstration of a putative N-type channel in hair cells as documented by tissue-specific antibody immunoreactivity and hair-cell-specific cDNA sequence.