Modulation of cardiac Ca(V)1.2 channels by dihydropyridine and phosphatase inhibitor requires Ser-1142 in the domain III pore loop.
Dihydropyridine-sensitive, voltage-activated calcium channels respond to membrane depolarization with two distinct modes of activity: short bursts of very short openings (mode 1) or repetitive openings of much longer duration (mode 2). Here we show that both the dihydropyridine, BayK8644 (BayK), and the inhibitor of SerThr protein phosphatases, okadaic acid, have identical effects on the gating of the recombinant cardiac calcium channel, Ca(V)1.2 (alpha(1)C). Each produced identical mode 2 gating in cell-attached patches, and each prevented rundown of channel activity when the membrane patch was excised into ATP-free solutions. These effects required Ser or Thr at position 1142 in the domain III pore loop between transmembrane segments S5 and S6, where dihydropyridines bind to the channel. Mutation of Ser-1142 to Ala or Cys produced channels with very low activity that could not be modulated by either BayK or okadaic acid. A molecular model of Ca(V)1.2 indicates that Ser-1142 is unlikely to be phosphorylated, and thus we conclude that BayK binding stabilizes mode 2 gating allosterically by either protecting a phospho Ser/Thr on the alpha(1)C subunit or mimicking phosphorylation at that site.
Erxleben
Christian
C
Laboratory of Signal Transduction and Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
Gomez-Alegria
Claudio
C
Darden
Thomas
T
Mori
Yasuo
Y
Birnbaumer
Lutz
L
Armstrong
David L
DL
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002120:Calcium Channel Agonists / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004095:Dihydropyridines / Q000494:pharmacology*; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D002120:Calcium Channel Agonists; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D004791:Enzyme Inhibitors; C419385:L-type calcium channel alpha(1C); D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C038806:1,4-dihydropyridine; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases