Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2.
Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 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 Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) 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 Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). 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 Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.
Graduate Program in Biochemistry and Molecular Biology, Purdue University, West Lafayette, Indiana, USA.
Nejmi
N
Dilmac
Nathan
N
Hilliard
Gregory H
GH
Hockerman
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004357:Drug Synergism; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010539:Permeability / Q000187:drug effects; D010649:Phenylalanine / Q000235:genetics; D013912:Threonine / Q000235:genetics; D014700:Verapamil / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D005973:Glutamine; D001464:Barium; D013912:Threonine; D018698:Glutamic Acid; D010649:Phenylalanine; D014700:Verapamil; D000409:Alanine; D002118:Calcium; D005998:Glycine