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Molecular Determinants of Ca²⁺ Potentiation of Diltiazem Block and Ca²⁺-Dependent Inactivation in the Pore Region of Ca_v1.2

Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences (N.H., G.H.H.), and Graduate Program in Biochemistry and Molecular Biology (N.D.), Purdue University, West Lafayette, Indiana

Diltiazem block of Ca_v1.2 is frequency-dependent and potentiated by Ca²⁺. We examined the molecular determinants of these characteristics using mutations that affect Ca²⁺ interactions with Ca_v1.2. Mutant and wild-type (WT) Ca_v1.2 channels were transiently expressed in tsA 201 cells with _1b and ₂ subunits. The four conserved glutamates that compose the Ca²⁺ selectivity filter in Ca_v1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q), and each single mutant was assayed for block by diltiazem using whole-cell voltage-clamp recordings in either 10 mM Ba²⁺ or 10 mM Ca²⁺. In Ba²⁺, none of the mutations affected the potency of diltiazem block of closed channels (0.05 Hz stimulation). However, frequency-dependent block (1Hz stimulation) was eliminated in the mutant E1419Q (domain IV), which recovered more rapidly than WT channels from inactivated channel block. Potentiation of diltiazem block of closed Ca_v1.2 channels in Ca²⁺ was abolished in the E1118Q, F1117G (domain III), and E1419Q mutants. Frequency-dependent block in Ca²⁺ was reduced compared with WT Ca_v1.2 in the F1117G, E1118Q, and E1419Q mutants. The C-terminal tail IQ domain mutation I1627A, which disrupts Ca²⁺ dependent inactivation, enhanced diltiazem block of closed channels in Ba²⁺. We conclude that, in Ba²⁺, E1419 slows recovery from diltiazem block of depolarized Ca_v1.2 channels, but in Ca²⁺, E1118, E1419, and F1117 form a Ca²⁺ binding site that mediates the potentiation of diltiazem block of both closed and inactivated Ca_v1.2 channels. Furthermore, Ca²⁺-dependent inactivation, which is impaired in E709Q, E1118Q, E1419Q, and I1627A, is not required for Ca²⁺ potentiation of diltiazem block.

Address correspondence to: Gregory Hockerman, 575 Stadium Mall Dr., West Lafayette, IN 47907-2091. E-mail: gregh{at}pharmacy.purdue.edu

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