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J Biol Chem, Vol. 273, Issue 8, 4635-4641, February 20, 1998

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

Akihiko Kuniyasu, Kiyoshi Itagaki, Toshiro Shibano^§, Minoru Iino^§, Gwen Kraft^¶, Arnold Schwartz^¶, and Hitoshi Nakayama

From the Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto 862, Japan,  Laboratory of Signal Transduction, National Institute of Environmental Health Services, Research Triangle Park, North Carolina 27709, ^§ New Product Research Laboratories II, Daiichi Pharmaceutical Co. Ltd., 1-16-13, Kita-Kasai, Edogawa-Ku, Tokyo 134, Japan, and ^¶ Institute for Molecular Pharmacology and Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0828

To identify the binding domain of a new Ca²⁺ antagonist semotiadil on L-type Ca²⁺ channels from skeletal muscle, photolabeling was carried out by using an azidophenyl derivative of [³H]semotiadil. Photoincorporation was observed in several polypeptides of membrane triad preparations; the only specific photoincorporation was in the ₁ subunit of the Ca²⁺ 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: Tyr¹³⁵⁰-Met¹³⁶⁶ and Leu¹³⁶⁷-Met¹³⁸¹ 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 Ca²⁺ antagonists.

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