Probing the extracellular release site of the plasma membrane calcium pump.
The plasma membrane Ca(2+) pump is known to mediate Ca(2+)/H(+) exchange. Extracellular protons activated (45)Ca(2+) efflux from human red blood cells with a half-maximal inhibition constant of 2 nM when the intracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreased the IC(50) for extracellular Ca(2+) from approximately 33 to approximately 6 mM. Changing the membrane potential by >54 mV had no effect on the IC(50) for extracellular Ca(2+). This argues against Ca(2+) release through a high-field access channel. Extracellular Ni(2+) inhibited Ca(2+) efflux with an IC(50) of 11 mM. Extracellular Cd(2+) inhibited with an IC(50) of 1. 5 mM, >10 times better than Ca(2+). The Cd(2+) IC(50) also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca(2+), Cd(2+), and H(+) competing for the same site. The higher affinity for inhibition by Ni(2+) and Cd(2+) is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca(2+) efflux. Our results are consistent with the notion that the release site contains a histidine.
Xu
W
W
Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65212, USA.
Wilson
B J
BJ
Huang
L
L
Parkinson
E L
EL
Hill
B J
BJ
Milanick
M A
MA
D001665:Binding Sites; D002104:Cadmium / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000097:blood*; D003545:Cysteine / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D008564:Membrane Potentials; D008698:Mesylates / Q000494:pharmacology; D008954:Models, Biological; D011522:Protons
D008698:Mesylates; D011522:Protons; C093171:methanethiosulfonate ethylammonium; D002104:Cadmium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D006639:Histidine; D005020:Ethyl Methanesulfonate; D000252:Calcium-Transporting ATPases; D003545:Cysteine