Permeation in the Dihydropyridine-sensitive Calcium Channel

We investigated the mechanism whereby ions cross dihydropyridinesensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the singlechannel level, we found no evidence o f an anomalous mole-fraction effect like that reported previously for whole-cell currents in mixtures o f Ba and Ca. With the total concentration o f Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi-ion nature o f the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions o f models with only one binding site in the permeation pathway, single-channel conductance does not follow MichaelisMenten kinetics as Ba activity is increased over three orders of magnitude. Twofold variation in the Debye length o f permeant ion solutions has litde effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis-Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence o f three sites helps explain both a continued rise in conductance as [Ba 2+ ] is increased above 1 10 mM, and the high single-channel conductance ( -7 pS) with 1 mM [Ba ~§ ] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence o f an anomalous mole-fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism. Address reprint requests to Dr. Eduardo Marban, Hunterian 116, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. J. G~:N. PHYSIOL ~) The Rockefeller University Press 9 0022-1295/90/05/0911/99 $2.00 Volume 95 May 1990 911-939 911 on Jne 1, 2017 D ow nladed fom Published May 1, 1990