Key roles of Phe¹¹¹² and Ser¹¹¹⁵ in the pore-forming IIIS5-S6 linker of L-type Ca²⁺ channel α_1C subunit (Ca_v1.2) in binding of dihydropyridines and action of Ca²⁺ channel agonists

Voltage-dependent L-type Ca²⁺ channels are modulated by the binding of Ca²⁺ channel antagonists and agonists to the pore-forming α_1c subunit (Ca_v1.2). We recently identified Ser¹¹¹⁵ in IIIS5-S6 linker of α_1c subunitas acritical 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 α_1c subunit (rbCII) to illustrate the role of poreforming IIIS5-S6 linker in the action of Ca²⁺ channel modulators. Ca²⁺ channel currents through wild-type (rbCII) or mutated α_1c subunits, transiently expressed in BHK6 cells with β_1a and α₂/δ 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.