Abstract

Dihydropyridines (DHPs) are an important class of drugs, used extensively in the treatment of angina pectoris, hypertension, and arrhythmia. The molecular mechanism by which DHPs modulate Ca²⁺ channel function is not known in detail. We have found that DHP binding is allosterically coupled to Ca²⁺ binding to the selectivity filter of the skeletal muscle Ca²⁺ channel Ca_V1.1, which initiates excitation-contraction coupling and conducts L-type Ca²⁺ currents. Increasing Ca²⁺ concentrations from approximately 10 nM to 1 mM causes the DHP receptor site to shift from a low-affinity state to a high-affinity state with an EC₅₀ for Ca²⁺ of 300 nM. Substituting each of the four negatively charged glutamate residues that form the ion selectivity filter with neutral glutamine or positively charged lysine residues results in mutant channels whose DHP binding affinities are decreased up to 10-fold and are up to 150-fold less sensitive to Ca²⁺ than wild-type channels. Analysis of mutations of amino acid residues adjacent to the selectivity filter led to identification of Phe-1013 and Tyr-1021, whose mutation causes substantial changes in DHP binding. Thermo-dynamic mutant cycle analysis of these mutants demonstrates that Phe-1013 and Tyr-1021 are energetically coupled when a single Ca²⁺ ion is bound to the channel pore. We propose that DHP binding stabilizes a nonconducting state containing a single Ca²⁺ ion in the pore through which Phe-1013 and Tyr-1021 are energetically coupled. The selectivity filter in this energetically coupled high-affinity state is blocked by bound Ca²⁺, which is responsible for the high-affinity inhibition of Ca²⁺ channels by DHP antagonists.