PASSIVE CHLORIDE PERMEABILITY CHARGE COUPLED TO H+-ATPASE OF AVIAN OSTEOCLAST RUFFLED MEMBRANE

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- > SO4(2-) > NO3- approximately SCN- in supporting acidification. The anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (10-mu-M) 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 Cl--36 transport. We uncoupled charge transport from proton transport with a large excess of ammonia, which had no effect on Cl--36 accumulation in vesicles, and by measuring Cl--36 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.