Evidence for a 95 kDa Short Form of the a1A Subunit Associated with the v-Conotoxin MVIIC Receptor of the P/Q-type Ca Channels

Neuronal voltage-dependent Ca channels have been isolated previously and shown to contain a primary a1 poreforming subunit as well as auxiliary a2d and b subunits, in addition to an uncharacterized 95 kDa protein. In the present study, using multiple approaches, we have extensively characterized the molecular structure of the 95 kDa protein. Separation of the P/Qand N-type neuronal Ca channels showed that the 95 kDa protein is associated exclusively with the v-Conotoxin MVIIC receptor of the P/Q-type channels. Analysis of purified synaptic plasma membranes and the isolated P/Qtype channels, using a1A-specific antibodies, suggested a structural relationship between the a1A subunit and the 95 kDa protein. This finding was supported by protein–protein interaction data, which revealed that the b subunit can associate with the 95 kDa protein in addition to the a1A subunit. Changes in electrophoretic mobility after enzymatic treatment with Endo F indicated that the 95 kDa protein is glycosylated. Furthermore, microsequencing of the 95 kDa protein yielded 13 peptide sequences, all of which are present in the first half of the a1A subunit up to amino acid 829 of the cytoplasmic linker between repeats II and III. Taken together, our results strongly suggest that the 95 kDa glycoprotein associated with the P/Q-type Ca channels is a short form of the a1A subunit.