Calcium binding to extracellular sites of skeletal muscle calcium channels regulates dihydropyridine binding.
The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding.
Ebata
H
H
Department of Physiological Chemistry, Ohio State University, College of Medicine, Columbus 43210.
Mills
J S
JS
Nemcek
K
K
Johnson
J D
JD
D000818:Animals; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000378:metabolism*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D004095:Dihydropyridines / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D017275:Isradipine; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D009132:Muscles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009206:Myocardium / Q000378:metabolism; D010069:Oxadiazoles / Q000378:metabolism; D011817:Rabbits; D012508:Sarcolemma / Q000378:metabolism
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002413:Cations, Divalent; D004095:Dihydropyridines; D010069:Oxadiazoles; D000001:Calcimycin; D004533:Egtazic Acid; C038806:1,4-dihydropyridine; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium; D017275:Isradipine