THE ALPHA-2 DELTA SUBUNIT OF VOLTAGE-SENSITIVE CA2+ CHANNELS IS A SINGLE TRANSMEMBRANE EXTRACELLULAR PROTEIN WHICH IS INVOLVED IN REGULATEDSECRETION/

The membrane topology of alpha 2/delta subunit was investigated utiliz ing electrophysiological functional assay and specific anti-alpha 2 an tibodies, (a) cRNA encoding a deleted alpha 2/delta subunit was coinje cted with alpha 1C subunit of the L-type calcium channel into Xenopus oocytes. The truncated form, lacking the third putative TM domain (alp ha 2/delta TMIII), failed to amplify the expressed inward currents, no rmally induced by alpha(1C) coinjected with intact alpha 2/delta subun it, Western blot analysis of alpha 2/delta Delta TMIII shows the appea rance of a degraded alpha 2 protein and no expression of the full-size two-TM truncated-protein. The improper processing of alpha 2/delta De lta TMIII suggests that the alpha 2/delta is a single TM domain protei n and the TM region is positioned at the delta subunit, (b) External a pplication of anti-alpha 2 antibodies, prepared for an epitope within the alternatively spliced and 'intracellular' region, inhibits depolar ization induced secretion in PC12, further supporting an external loca tion of the alpha 2 subunit and establishing delta subunit as the only membrane anchor for the extracellular alpha 2 subunit.