Drug modulation of L-type calcium channels: Role of pore residues on calcium-potentiation of closed-channel block and frequency-dependent block by diltiazem and verapamil

Nejmi Dilmac, Purdue University

Abstract

Diltiazem and verapamil block of Cav1.2 channels is frequency-dependent and potentiated by Ca2+. The purpose of this study was to examined the molecular determinants of these characteristics using mutations that effect Ca2+ interactions with Ca v1.2. Mutant and wild type Cav1.2 channels were transiently expressed in tsA 201 cells with β1b and α2δ subunits. The four conserved glutamates that compose the Ca2+ selectivity filter in Cav1.2 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 diltiazem or verapamil using whole-cell voltage-clamp recordings in 10 mM Ba2+ or 10 mM Ca2+. In Ba 2+, none of the pore-region mutations reduced the fraction of current blocked by 50 μM diltiazem or 30 μM verapamil at 0.05 Hz stimulation. However, the pore mutations showed distinct effect on frequency-dependent block and Ca2+-potentiation. All of the pore-region mutations abolished Ca2+-potentiation of verapamil block at 0.05 Hz, while only F1117G, E1118Q, and E1419Q mutations disrupted Ca2+-potentiation of diltiazem. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1 Hz stimulation) in both Ba 2+ and Ca2+, whereas E1118Q and E1419Q drastically reduced frequency-dependent diltiazem block in Ba2+. The I1627A mutation, which disrupts Ca2+-dependent inactivation, increased the fraction of closed-channels blocked by diltiazem and verapamil in Ba 2+, but did not affect frequency-dependent block in Ba2+ or Ca2+. Our data suggest that F1117G and E1118Q (domain III) and E1419Q (domain IV) may regulate both frequency-dependent block and Ca 2+-potentiation of closed-channel block by diltiazem. In the case of verapamil, the pore region of domain III may contribute to a high-affinity binding site accessed during 1 Hz stimulation, and that Ca2+ binding to multiple sites may be required for potentiation of closed-channel block.

Degree

Ph.D.

Advisors

Hockerman, Purdue University.

Subject Area

Pharmacology

COinS