CALCIUM-BINDING IN THE PORE OF L-TYPE CALCIUM CHANNELS MODULATES HIGH-AFFINITY DIHYDROPYRIDINE BINDING

The pore forming alpha(1) subunit of L-type voltage-gated Ca2+ channel s contains a Ca2+-binding site that is allosterically coupled to the r eceptor site for dihydropyridine (DHP) Ca2+ antagonists. Site-directed mutations of conserved Phe and Glu residues in the pore-lining SS1/SS 2 segments greatly reduced Ca2+ enhancement of DHP binding. Substituti on of Phe-1O13 in the alpha(1) subunit from rabbit skeletal muscle (al pha(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 (E10 14Q and E1323Q) caused 11- and 35-fold decreases in sensitivity to Ca2 +, respectively, as well as decreases in the maximal DHP binding affin ities attained at optimal concentrations of Ca2+, DHP binding to the c harge-reversal mutation, E1014K, had no sensitivity to Ca2+. Our resul ts demonstrate that high affinity Ca2+ binding to the Glu residues in the SS1/SS2 segments of domains III and TV of alpha(1S) stabilizes the DHP receptor site in its high affinity state. We propose a three-stat e 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.