Calmodulin regulation of Ca2+ transport in human erythrocytes.
Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000097:blood*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D014268:Trifluoperazine / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D014268:Trifluoperazine; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium