Evidence for proton countertransport by the sarcoplasmic reticulum Ca2(+)-ATPase during calcium transport in reconstituted proteoliposomes with low ionic permeability.
To ascertain the coupling between Ca2+ and H+ fluxes during Ca2+ transport by the Ca2(+)-pumping ATPase of the sarcoplasmic reticulum, we used well characterized reconstituted proteoliposomes. The method for the functional reconstitution of the Ca2(+)-ATPase was an extension of our recently published procedure (Rigaud, J. L., Paternostre, M. T., and Bluzat, A. (1988) Biochemistry, 27, 2677-2688). The reconstituted vesicles which sustained high Ca2+ transport activities in the absence of Ca2+ precipitating anions exhibited low ionic passive permeability. Proton fluxes generated by external acid pulses have been monitored by using the fluorescence of the pH-sensitive probe pyranine trapped inside proteliposomes. When K+ was the only permeant ion, low proton-hydroxyl passive permeability was found (permeability coefficient congruent to 5 x 10(-5) cm s-1). In the presence of Cl-1 ions, a higher proton permeability was observed, presumably due to diffusion of HCl molecules. It was further demonstrated that systematic characterization of the passive permeability is essential for understanding and controlling the ATP-dependent Ca2+ accumulation in the reconstituted liposomes. The first line of evidence for Ca2(+)-H+ countertransport during operation of the Ca2(+)-ATPase came from Ca2+ uptake measurements. The ATP-dependent Ca2+ accumulation into proteoliposomes was shown to be critically dependent upon the ionic composition of the medium and the presence of ionophores. In K2SO4 medium a very low Ca2+ uptake was obtained which was only slightly affected by the presence of valinomycin. On the contrary, Ca2+ accumulation was increased 3-4-fold in the presence of the protonophore carbonyl-cyanide-p-trifluoromethoxy phenylhydrazone, indicating that a transmembrane pH gradient was built up during Ca2+ uptake that inhibited the transport activity of the pump. Accordingly, we found that Ca2+ loading capacity increased with internal buffer capacity. Finally in KCl medium, high Ca2+ accumulation was observed even in the absence of protonophore in agreement with a rapid dissipation of the pH gradient in the presence of chloride ions. Additional evidence that the Ca2+ pump of sarcoplasmic reticulum operated as a Ca2(+)-H+ countertransport was provided by measurements of ATP-dependent intraliposomal alkalinization using entrapped 8-hydroxyl-1,3,6-pyrene trisulfonate (pyranine) and accumulation of the weak acid acetate. In K2SO4 medium, transmembrane pH gradients of about 1 pH unit were generated with kinetics parallel to those of the Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)
Levy
D
D
Département de Biologie and URA (Centre National de la Recherche Scientifique) D 1290, Centre Etudes Nuclearies Saclay, Gif-sur-Yvette, France.
Seigneuret
M
M
Bluzat
A
A
Rigaud
J L
JL
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002412:Cations; D002463:Cell Membrane Permeability / Q000187:drug effects; D004058:Diffusion; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011522:Protons; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014634:Valinomycin / Q000494:pharmacology
D000085:Acetates; D002412:Cations; D008081:Liposomes; D011522:Protons; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000252:Calcium-Transporting ATPases; D019342:Acetic Acid; D011188:Potassium; D002118:Calcium