RT Journal Article SR Electronic T1 Molecular Determinants of Frequency Dependence and Ca2+ Potentiation of Verapamil Block in the Pore Region of Cav1.2 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1236 OP 1247 DO 10.1124/mol.104.000893 VO 66 IS 5 A1 Nejmi Dilmac A1 Nathan Hilliard A1 Gregory H. Hockerman YR 2004 UL http://molpharm.aspetjournals.org/content/66/5/1236.abstract AB Verapamil block of Cav1.2 is frequency-dependent and potentiated by Ca2+. We examined the molecular determinants of these characteristics using mutations that effect Ca2+ interactions with Cav1.2. 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 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.