The pore-forming α1 subunit of L-type voltage-gated Ca2+ channels contains a Ca2+-binding site that is allosterically coupled to the receptor site for dihydropyridine (DHP) Ca2+ antagonists. Site-directed mutations of conserved Phe and Glu residues in the pore-lining SS1/SS2 segments greatly reduced Ca2+ enhancement of DHP binding. Substitution of Phe-1013 in the α1 subunit from rabbit skeletal muscle (α(1S)) with Gly (F1013G) as in DHP-insensitive Ca2+ channels caused a 4-fold decrease in sensitivity to Ca2+. Mutation of the Ca2+-binding residues Glu-1014 in domain III and Glu-1323 in domain IV to Gln (E1014Q and E1323Q) caused 11- and 35-fold decreases in sensitivity to Ca2+, respectively, as well as decreases in the maximal DHP binding affinities attained at optimal concentrations of Ca2+. DHP binding to the charge-reversal mutation, E1014K, had no sensitivity to Ca2+. Our results demonstrate that high affinity Ca2+ binding to the Glu residues in the SS1/SS2 segments of domains III and IV of α(1s) stabilizes the DHP receptor site in its high affinity state. We propose a three-state model in which the affinity for DHPs is dependent on the presence of 0, 1, or 2 bound Ca2+ ions at sites in the pore.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology