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

Abstract

Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an inside‐out 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 2–3μM. The data demonstrate that binding Ca2+ to the extracellular high‐affinity Ca2+‐binding site is required for binding dihydropyridines to dihydropyridine recepptor. 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. Copyright © 1989, Wiley Blackwell. All rights reserved