PHOTOCHEMICAL IDENTIFICATION OF TRANSMEMBRANE SEGMENT IVS6 AS THE BINDING REGION OF SEMOTIADIL, A NEW MODULATOR FOR THE L-TYPE VOLTAGE-DEPENDENT CA2+ CHANNEL

To identify the binding domain of a new Ca2+ antagonist semotiadil on L-type Ca2+ channels from skeletal muscle, photolabeling was carried o ut by using an azi dophenyl derivative of [H-3]semotiadil, Photoincorp oration was observed in several polypeptides of membrane triad prepara tions; the only specific photoincorporation was in the alpha 1 subunit of the Ca2+ channel, After solubilization and purification, the photo labeled alpha(1) subunit was subjected to proteolytic and CNBr cleavag e followed by antibody mapping, Specific labeling was associated solel y with the region of transmembrane segment S6 in repeat IV. Quantitati ve immunoprecipitation was found in the tryptic and the Lys-C/Glu-C fr agments of 6.6 and 6.1 kDa, respectively, Further CNBr cleavage of the Lys-C digests produced two smaller fragments of 3.4 and 1.8 kDa that were included in the tryptic and Lys-C/Glu-C fragments. The smallest l abeled fragments were: Tyr(1350)-Met(1366) and Leu(1367)-Met(1381) con taining IVS6, a possible pore forming region, The data suggest that se motiadil binds to a region that is overlapped with but not identical t o those for phenylalkylamines, dihydropyridines and benzothiazepines. The present study also provides evidence that region IV represents an important component of a binding pocket for Ca2+ antagonists.