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Molecular Determinants of Frequency Dependence and Ca²⁺ Potentiation of Verapamil Block 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

Verapamil block of Ca_v1.2 is frequency-dependent and potentiated by Ca²⁺. We examined the molecular determinants of these characteristics using mutations that effect Ca²⁺ interactions with Ca_v1.2. Mutant and wild-type 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 the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba²⁺ or 10 mM Ca²⁺. In Ba²⁺, none of the pore-region mutations reduced the fraction of current blocked by 30 µM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca²⁺ potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba²⁺ and Ca²⁺. The I1627A mutation, which disrupts Ca²⁺-dependent inactivation, increased the fraction of closed channels blocked by 30 µM verapamil in Ba²⁺ but did not affect frequency-dependent block in Ba²⁺ or Ca²⁺. Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca²⁺ binding to multiple sites may be required for potentiation of verapamil block of closed channels.

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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