Molecular identification of an N-type Ca 2+ channel in saccular hair cells

Abstract

We report new molecular evidence for the presence of an N-type (Ca v2.2, α1B) voltage-gated Ca 2+ 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 Ca 2+ 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 Ca 2+-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.