Coordination of Ca2+ by the pore region glutamates is essential for high-affinity dihydropyridine binding to the cardiac Ca2+ channel alpha 1 subunit.
The molecular determinants for Ca2+ modulation of dihydropyridine (DHP) binding to cardiac Ca2+ channels were identified by mutational neutralization of the glutamate residues that comprise the Ca2+ channel selectivity filter. The binding activity of the DHP (+)-[3H]isradipine, monitored after expression of wild-type and mutant alpha 1 subunits in COS-7 cells, was markedly reduced in four single mutants and a double mutant. Evidence for decreased Ca2+ affinity was obtained for two single mutants in kinetic and equilibrium binding studies. Mutational destabilization of Ca2+ binding resulted in a concomitant decrease of (+)-[3H]isradipine binding affinity. Recovery of (+)-[3H]isradipine binding activity by the allosteric modulator (+)-tetrandrine in two single mutants was associated with a recovery of Ca2+ and DHP binding kinetics to wild-type values. Our findings demonstrate that high-affinity DHP binding is dependent on Ca2+ coordination by glutamate residues which form the selectivity filter of the channel pore.
Mitterdorfer
J
J
Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
Sinnegger
M J
MJ
Grabner
M
M
Striessnig
J
J
Glossmann
H
H
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D044182:Benzylisoquinolines; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002347:Carps; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004095:Dihydropyridines / Q000378:metabolism*; D005260:Female; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials; D008961:Models, Structural; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis / Q000378:metabolism
D000470:Alkaloids; D044182:Benzylisoquinolines; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D017931:DNA Primers; D004095:Dihydropyridines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C009438:tetrandrine; C038806:1,4-dihydropyridine