title: Key roles of Phe1112 and Ser1115 in the pore-forming IIIS5-S6 linker of L-type Ca2+ channel alpha1C subunit (CaV 1.2) in binding of dihydropyridines and action of Ca2+ channel agonists.
abstract: Voltage-dependent L-type Ca2+ channels are modulated by the binding of Ca2+ channel antagonists and agonists to the pore-forming alpha1c subunit (CaV 1.2). We recently identified Ser1115 in IIIS5-S6 linker of alpha1C 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 alpha1C 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 alpha1C subunits, transiently expressed in BHK6 cells with beta1a and alpha2/delta 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.
authors.affiliation: Laboratory of Cell Signaling, Graduate school of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Boris S
authors.initials: BS
authors.lastname: Zhorov
authors.forename: Katsuro
authors.initials: K
authors.lastname: Yoshioka
authors.forename: Taku
authors.initials: T
authors.lastname: Nagao
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ichijo
authors.forename: Satomi
authors.initials: S
authors.lastname: Adachi-Akahane
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004095:Dihydropyridines / Q000494:pharmacology*; D017275:Isradipine / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism*; D017354:Point Mutation; D021122:Protein Subunits / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D012694:Serine / Q000378:metabolism*
chemical_list: D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D011758:Pyrroles; C071658:FPL 64176; D012694:Serine; D010649:Phenylalanine; C038806:1,4-dihydropyridine; D000409:Alanine; D017275:Isradipine