Photochemical Identification of Transmembrane Segment IVS6 as the Binding Region of Semotiadil, a New Modulator for the L-type Voltage-dependent Ca2+ Channel

Abstract

To identify the binding domain of a new Ca2؉ antagonist semotiadil on L-type Ca 2؉ channels from skeletal muscle, photolabeling was carried out by using an azidophenyl derivative of [ 3 H]semotiadil. Photoincorporation was observed in several polypeptides of membrane triad preparations; the only specific photoincorporation was in the ␣ 1 subunit of the Ca 2؉ channel. After solubilization and purification, the photolabeled ␣ 1 subunit was subjected to proteolytic and CNBr cleavage followed by antibody mapping. Specific labeling was associated solely with the region of transmembrane segment S6 in repeat IV. Quantitative immunoprecipitation was found in the tryptic and the Lys-C/Glu-C fragments 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 labeled fragments were: Tyr 1350 -Met 1366 and Leu 1367 -Met 1381 containing IVS6, a possible pore-forming region. The data suggest that semotiadil binds to a region that is overlapped with but not identical to those for phenylalkylamines, dihydropyridines and benzothiazepines. The present study also provides evidence that region IV represents an important component of a binding pocket for Ca 2؉

Voltage-dependent calcium channels: From structure to function

“…Different techniques have been used to localize potential binding sites of these drugs on the calcium channel complex. Earlier experimental observations from photoaffinity labelling and peptide mapping studies on the skeletal muscle channel revealed that all three classes bind to the transmembrane region of repeat IV of the al subunit Catterall & Striessnig 1992;Kuniyasu et al 1998) with additional sites on repeat III (Catterall and Striessnig 1992;Kalasz et al 1993) and repeat I for the DHPs. These localizations were refined by the use of chimeric cqc/c~i^ and C~c/a~ channels and site-directed mutagenesis of single amino acids in the Ct~s or ct~c subunit (Fig.…”

Stoichiometry and Binding Site Analysis of Ligand-Human Serum Albumin Complex by Photoaffinity Labeling and Mass Spectrometry

“…Radioactivity photo-incorporated into HSA was analyzed by Radioluminography. 11) In brief, photolabelled HSA (2,000 cpm per lane) was separated by SDS῍PAGE (10῏ gels), transferred onto PVDF membrane, and the membrane was placed in contact with an imaging plate, BAS-TR2040S (Fuji Photo Film Co., Tokyo, Japan). After 24 h, the plate was scanned and analyzed by a Bio-Imaging Analyzer BAS 1,000 model (Fuji Photo Film Co.).…”

Binding between the Niemann–Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain

“…The immunoprecipitates were subjected to 4-14% gradient SDS͞PAGE. To detect the labeled protein bands, a gel-slicing method (38) was used (instead of the radioluminography method). In brief, after electrophoresis, individual gel lanes were manually cut into 6-mm slices.…”