AJP - Cell Physiology, Vol 260, Issue 6 C1315-C1324, Copyright © 1991 by American Physiological Society

ARTICLES

Passive chloride permeability charge coupled to H(+)-ATPase of avian osteoclast ruffled membrane

H. C. Blair, S. L. Teitelbaum, H. L. Tan, C. M. Koziol and P. H. Schlesinger
Department of Pathology, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri.

We prepared proton-transporting membrane vesicles from the avian osteoclast's ruffled membrane, a specialized region of the cell surface that acidifies the bone resorption space. We demonstrated a unique conductive Cl- permeability that is charge coupled to the vesicle H(+)-ATPase and is required for acidification. Ion replacement indicated an anion selectivity of Br- approximately Cl- greater than SO4(2-) greater than NO3- approximately SCN- in supporting acidification. The anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (10 microM) was a competitive inhibitor of acidification and raised the Michaelis constant for ATP of the proton pump approximately 11-fold in 120 mM KCl. Inhibition was reversed by valinomycin, which provides an alternate path for charge neutralization. The Cl- dependence of acidification was nonlinear and yielded a Hill coefficient of 3-4, showing that it is distinct from a linear Cl- dependence reported for acidification of renal cortical endosomes. The K+ ionophore valinomycin augmented H+ transport in K2SO4, and not in KCl. Dependence of Cl- transport on membrane potential was confirmed by direct measurement of 36Cl- transport. We uncoupled charge transport from proton transport with a large excess of ammonia, which had no effect on 36Cl- accumulation in vesicles, and by measuring 36Cl- accumulation in response to a membrane diffusion potential, produced with a [K+] gradient and valinomycin in the absence of ATP. These experiments demonstrate that the electrogenic proton pump of the osteoclast ruffled membrane is charge coupled to a passive Cl- permeability in the same membrane.

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