Binding Ca2+ to intracellular or to extracellular sites of dihydropyridine receptor of rabbit skeletal muscle discriminates between in vitro binding of Ca2+-channel agonist and antagonist.
Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an insideout orientation. Digitonin-solubilized, purified dihydropyridine receptor is embedded in digitonin vesicles in an outside-out orientation. Ca2+ selectively stimulates binding of the Ca2+-channel antagonist [3H]PN200-110 to dihydropyridine receptor in the outside-out but not the inside-out orientation. The dissociation constant for binding Ca2+ to the extracellular Ca2+-specific binding site of dihydropyridine receptor is 2-3 microM. The data demonstrate that binding Ca2+ to the extracellular high-affinity Ca2+-binding site is required for binding dihydropyridines to dihydropyridine receptor. This binding is inhibited, however, by 1-10 mM concentrations of any divalent cation tested (Ba2+, Mn2+, Mg2+). Also, Ca2+ selectively stimulates binding of the Ca2+-channel agonist [3H]BayK8644 to dihydropyridine receptor in the inside-out orientation. The titration of this Ca2+ dependence indicates that the dissociation constant for binding Ca2+ to the intracellular Ca2+-specific binding site of dihydropyridine receptor is in the millimolar range. Thus, binding Ca2+-channel agonist or antagonist to dihydropyridine receptor is modulated by binding Ca2+ to different sites of the receptor. Measurements of dissociation rate constants for binding [3H]PN200-110 to dihydropyridine receptor in the presence of diltiazem, verapamil and/or Ca2+ indicate that Ca2+, like diltiazem or verapamil, is an allosteric effector of this receptor.
Kanngiesser
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U
Ruhr-Universität Bochum, Lehrstuhl für Biochemie, Federal Republic of Germany.
Pongs
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O
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002413:Cations, Divalent; D007425:Intracellular Membranes / Q000378:metabolism; D017275:Isradipine; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism*; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000378:metabolism*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002413:Cations, Divalent; D010069:Oxadiazoles; D011978:Receptors, Nicotinic; D002118:Calcium; D017275:Isradipine