Serine residue in the IIIS5-S6 linker of the L-type Ca2+ channel α1C subunit is the critical determinant of the action of dihydropyridine Ca2+ channel agonists

The dihydropyridine (DHP)-binding site has been identified within L-type Ca2+ channel alpha (1C) subunit. However, the molecular mechanism underlying modulation of Ca2+ channel gating by DHPs has not been clarified. To search for novel determinants of high affinity DHP binding, we introduced point mutations in the rat brain Ca2+ channel alpha (1C) subunit (rbCII or Ca(v)1.2c) based on the comparison of amino acid sequences between rbCII and the ascidian L-type Ca2+ channel alpha (1) subunit, which is insensitive to DHPs. The alpha (1C) mutants (S1115A, S1146A, and A1420S) and rbCII were transiently expressed in BHK6 cells with beta (1a) and alpha (2)/delta subunits. The mutation did not affect the electrophysiological properties of the Ca2+ channel, or the voltage- and concentration-dependent block of Ca2+ channel currents produced by diltiazem and verapamil. However, the S1115A channel was significantly less sensitive to DHP antagonists. Interestingly, in the S1115A channel, DHP agonists failed to enhance whole-cell Ca2+ channel currents and the prolongation of mean open time, as well as the increment of NPo. Responsiveness to the non-DHP agonist FPL-64176 was also markedly reduced in the S1115A channel. When S1115 was replaced by other amino acids (S1115D, S1115T, or S1115V), only S1115T was slightly sensitive to S-(-)-Bay K 8644. These results indicate that the hydroxyl group of Ser(1115) in IIIS5-S6 linker of the L-type Ca2+ channel alpha (1C) subunit plays a critical role in DHP binding and in the action of DHP Ca2+ channel agonists.