Protons as substitutes for sodium and potassium in the sodium pump reaction.
The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.
Polvani
C
C
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Blostein
R
R
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011188:Potassium / Q000378:metabolism*; D011522:Protons; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013327:Strophanthidin / Q000494:pharmacology
D011522:Protons; D000644:Quaternary Ammonium Compounds; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D013327:Strophanthidin; D012964:Sodium; C009405:tetrabutylammonium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium