The α2/δ subunit of voltage sensitive Ca2+ channels is a single transmembrane extracellular protein which is involved in regulated secretion

摘要

>The membrane topology of α2/δ subunit was investigated utilizing electrophysiological functional assay and specific anti-α2 antibodies. (a) cRNA encoding a deleted α2/δ subunit was coinjected with α1C subunit of the L-type calcium channel into Xenopus oocytes. The truncated form, lacking the third putative TM domain (α2/δΔTMIII), failed to amplify the expressed inward currents, normally induced by α _1c coinjected with intact α2/δ subunit. Western blot analysis of α2/δΔTMIII shows the appearance of a degraded α2 protein and no expression of the full-size two-TM truncated-protein. The improper processing of α2/δΔTMIII suggests that the α2/δ is a single TM domain protein and the TM region is positioned at the δ subunit. (b) External application of anti-α2 antibodies, prepared for an epitope within the alternatively spliced and ‘intracellular’ region, inhibits depolarization induced secretion in PC12, further supporting an external location of the α2 subunit and establishing δ subunit as the only membrane anchor for the extracellular α2 subunit.