A pK change of acidic residues contributes to cation countertransport in the Ca-ATPase of sarcoplasmic reticulum. Role of H+ in Ca(2+)-ATPase countertransport.
Proteoliposomal vesicles reconstituted with sarcoplasmic reticulum ATPase and exogenous lipids sustain ATP-dependent Ca2+ uptake and H+ ejection, as well as net charge displacement by Ca2+. We have studied the effect of lumenal (inner) and medium (extravesicular) pH variations on the countertransport ratios of H+ and Ca2+. We find that the Ca2+/H+ molar ratio is approximately 1 when the lumenal and medium pH is near neutrality, but changes with a specific pattern when the medium pH is varied in the presence of a constant lumenal pH and when the lumenal pH is varied in the presence of a constant medium pH. Empirical analysis of the experimental data shows that the apparent pK of the residue(s) releasing H+ into the medium is approximately 6.1, whereas the apparent pK of the residue(s) binding lumenal H+ is approximately 7.7. Assuming that the same acidic residues are involved in H+ and Ca2+ countertransport, our findings suggest a lower affinity for H+ in their outward orientation (prevalent in the ground state of the enzyme) and a higher affinity for H+ in lumenal orientation (prevalent in the phosphorylated state of the enzyme). Cyclic pK changes, coupled to ATP utilization, promote cation exchange, Ca2+ uptake, and H+ ejection by the vesicles. The stoichiometry of countertransport and net charge displacement is matched by a corresponding electrogenic behavior. A calculation of voltage development related to initial rates of charge transfer (dV/dt = (dQ/dt)/Cm) is given as a corrective replacement of a previous steady state calculation.
Yu
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X
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Hao
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L
Inesi
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G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D008954:Models, Biological; D009132:Muscles / Q000201:enzymology; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium