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Key Roles of Phe¹¹¹² and Ser¹¹¹⁵ in the Pore-Forming IIIS5-S6 Linker of L-Type Ca²⁺ Channel _1C Subunit (Ca_V 1.2) in Binding of Dihydropyridines and Action of Ca²⁺ Channel Agonists

Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan (S.Y., K.Y., H.I., S.A.-A.); Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada (B.S.Z.); and National Institute of Health Sciences, Kamiyoga, Setagaya-ku, Tokyo, Japan (T.N.)

Voltage-dependent L-type Ca²⁺ channels are modulated by the binding of Ca²⁺ channel antagonists and agonists to the pore-forming _1c subunit (Ca_V 1.2). We recently identified Ser¹¹¹⁵ in IIIS5-S6 linker of _1C subunit as a critical determinant of the action of 1,4-dihydropyridine agonists. In this study, we applied alanine-scanning mutational analysis in IIIS5-S6 linker of rat brain _1C subunit (rbCII) to illustrate the role of pore-forming IIIS5-S6 linker in the action of Ca²⁺ channel modulators. Ca²⁺ channel currents through wild-type (rbCII) or mutated _1C subunits, transiently expressed in BHK6 cells with _1a and ₂/ subunits, were analyzed. The replacement of Phe¹¹¹² by Ala (F1112A) significantly impaired the sensitivity to Ca²⁺ channel agonists (S)-(-)-Bay k 8644 and FPL-64176, and modestly to 1,4-dihydropyridine (DHP) antagonists. The low sensitivity of F1112A and S1115A to DHP antagonists was consistent with the reduced binding affinity for [³H](+)PN200-110. The replacement of Phe¹¹¹² by Tyr, but not by Ala, restored the long openings produced by FPL-64176, thus indicating the critical role of aromatic ring of Phe¹¹¹² in the Ca²⁺ channel agonist action. Interestingly, double-mutant Ca²⁺ channel (F1112A/S1115A) failed to discriminate between Ca²⁺ channel agonist (S)-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl] phenyl)-3-pyridine carboxylic acid methyl ester (Bay k 8644) and antagonist (R)-(+)-Bay k 8644 and was blocked by the two enantiomers in an identical manner. These results indicate that both Phe¹¹¹² and Ser¹¹¹⁵ in linker IIIS5-S6 are required for the action of Ca²⁺ channel agonists. A model of the DHP receptor is proposed to visualize possible interactions of Phe¹¹¹², Ser¹¹¹⁵, and other DHP-sensing residues with a typical DHP ligand nifedipine.

Received April 7, 2003; accepted April 28, 2003.

Address correspondence to: Dr. Satomi Adachi-Akahane, Laboratory of Cell Signaling, Graduate school of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: satomiaa{at}mol.f.u-tokyo.ac.jp

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