RT Journal Article
SR Electronic
T1 Molecular Determinants of Ca<sup>2</sup><sup>+</sup> Potentiation of
 Diltiazem Block and Ca<sup>2</sup><sup>+</sup>-Dependent Inactivation in the
 Pore Region of Ca<sub>v</sub>1.2
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 491
OP 501
DO 10.1124/mol.64.2.491
VO 64
IS 2
A1 Nejmi Dilmac
A1 Nathan Hilliard
A1 Gregory H. Hockerman
YR 2003
UL http://molpharm.aspetjournals.org/content/64/2/491.abstract
AB Diltiazem block of Cav1.2 is frequency-dependent and potentiated by Ca2+. We examined the molecular determinants of these characteristics using mutations that affect Ca2+ interactions with Cav1.2. Mutant and wild-type (WT) 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 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 Cav1.2 channels in Ca2+ was abolished in the E1118Q, F1117G (domain III), and E1419Q mutants. Frequency-dependent block in Ca2+ was reduced compared with WT Cav1.2 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 Cav1.2 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 Cav1.2 channels. Furthermore, Ca2+-dependent inactivation, which is impaired in E709Q, E1118Q, E1419Q, and I1627A, is not required for Ca2+ potentiation of diltiazem block.