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

Abstract

Diltiazem block of Ca v 1.2 is frequency-dependent and potentiated by Ca 2ϩ . We examined the molecular determinants of these characteristics using mutations that affect Ca 2ϩ interactions with Ca v 1.2. Mutant and wild-type (WT) Ca v 1.2 channels were transiently expressed in tsA 201 cells with ␤ 1b and ␣ 2

Molecular Determinants of Frequency Dependence and Ca2+ Potentiation of Verapamil Block in the Pore Region of Cav1.2

“…The loss of Ca 2ϩ potentiation of closed-channel block in all of the EQ mutations suggests that Ca 2ϩ potentiation also requires Ca 2ϩ binding in the channel pore. Taken together with our previous observation that Ca 2ϩ -dependent inactivation is disrupted in the EQ mutations (Dilmac et al, 2003), these results also suggest that the consequences of Ca 2ϩ -calmodulin binding to the C-terminal tail of Ca v 1.2 may include conformational changes in the pore region of the channel. Ca 2ϩ potentiation of closed-channel verapamil block was disrupted by mutation of any of the Glu-to-Gln mutants, F1117G, and all of the Thr-to-Ala mutants.…”

“…However, as we previously observed with diltiazem (Dilmac et al, 2003), I1627A increased the sensitivity of closed Ca v 1.2 channels to verapamil block in Ba 2ϩ , whereas frequency-dependent block was not different from WT in Ba 2ϩ or Ca 2ϩ . The I1627A mutation shifted the voltage dependence of inactivation by approximately Ϫ20 mV compared with WT.…”

“…However, the Ba 2ϩ conductance of Ca v 1.2 is higher than that for Ca 2ϩ because Ca 2ϩ binds more tightly to the pore Glu residues (Almers and McCleskey, 1984 (Lee and Tsien, 1983). The conserved Glu residues in domains III and IV, as well as an adjacent Phe residue in the pore region of domain III in Ca v 1.1, mediate the Ca 2ϩ potentiation of DHP (Peterson and Catterall, 1995) and diltiazem affinity (Dilmac et al, 2003). Furthermore, the IQ domain mutant I1627A potentiates diltiazem block of closed channels in Ba 2ϩ , and Ca 2ϩ -dependent inactivation is not required for Ca 2ϩ potentiation of diltiazem block (Dilmac et al, 2003).…”

“…In addition, we studied a mutant in which the L-type-specific residue in domain III, Phe1117, was mutated to Gly (F1117G). We previously demonstrated that the F1117G mutant disrupts frequency dependence and Ca 2ϩ potentiation of Ca v 1.2 by diltiazem (Dilmac et al, 2003). Finally we studied the IQ motif mutant, I1627A, in the C-terminal tail of Ca v 1.2, because we previously reported that this mutant increases the affinity of the channel for diltiazem.…”

“…The conserved Glu residues in domains III and IV, as well as an adjacent Phe residue in the pore region of domain III in Ca v 1.1, mediate the Ca 2ϩ potentiation of DHP (Peterson and Catterall, 1995) and diltiazem affinity (Dilmac et al, 2003). Furthermore, the IQ domain mutant I1627A potentiates diltiazem block of closed channels in Ba 2ϩ , and Ca 2ϩ -dependent inactivation is not required for Ca 2ϩ potentiation of diltiazem block (Dilmac et al, 2003). We report here that Ca 2ϩ potentiation of verapamil block of closed Ca v 1.2 channels is disrupted by any of the EQ or TA mutations.…”