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- 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.
Blair
H C
HC
Department of Pathology, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri.
Teitelbaum
S L
SL
Tan
H L
HL
Koziol
C M
CM
Schlesinger
P H
PH
D000165:Acridine Orange; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D002645:Chickens; D002712:Chlorides / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008854:Microscopy, Electron; D008954:Models, Biological; D010010:Osteoclasts / Q000378:metabolism* / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000838:Anions; D002712:Chlorides; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000165:Acridine Orange