The alpha 2/delta subunit of voltage sensitive Ca2+ channels is a single transmembrane extracellular protein which is involved in regulated secretion.
The membrane topology of alpha 2/delta subunit was investigated utilizing electrophysiological functional assay and specific anti-alpha 2 antibodies. (a) cRNA encoding a deleted alpha 2/delta subunit was coinjected with alpha 1C subunit of the L-type calcium channel into Xenopus oocytes. The truncated form, lacking the third putative TM domain (alpha 2/delta delta TMIII), failed to amplify the expressed inward currents, normally induced by alpha 1C coinjected with intact alpha 2/delta subunit. Western blot analysis of alpha 2/delta delta TMIII shows the appearance of a degraded alpha 2 protein and no expression of the full-size two-TM truncated-protein. The improper processing of alpha 2/delta delta TMIII suggests that the alpha 2/delta is a single TM domain protein and the TM region is positioned at the delta subunit. (b) External application of anti-alpha 2 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 alpha 2 subunit and establishing delta subunit as the only membrane anchor for the extracellular alpha 2 subunit.
Wiser
O
O
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Trus
M
M
Tobi
D
D
Halevi
S
S
Giladi
E
E
Atlas
D
D
D000818:Animals; D000906:Antibodies; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D008564:Membrane Potentials; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D016716:PC12 Cells; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018075:RNA, Complementary / Q000235:genetics; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D014982:Xenopus laevis
D000906:Antibodies; D015220:Calcium Channels; D018075:RNA, Complementary; D011994:Recombinant Proteins