Sequence differences between α1C and α1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor

The molecular basis of the Ca2+ channel block by (+)cis-diltiazem was studied in class A/L-type chimeras and mutant ore. Ca2+ channels. Chimeras consisted of either rabbit heart (alpha(1C-a)) or carp skeletal muscle (alpha(1S)) sequence in transmembrane segments IILS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha(1C-a) were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha(1) subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu(1383), located at the extracellular mouth of the pore) from IVS6 alpha(1C-a) to IVS6 of alpha(1S) Significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha(1) subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.