Role of third extracellular domain of plasma membrane Ca2+-Mg2+-ATPase based on the novel inhibitor caloxin 3A1.
The plasma membrane Ca2+ pump (PMCA) is a Ca2+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+ ([Ca2+]i). It contains five putative extracellular domains (PEDs). Earlier we had reported that binding to PED2 leads to PMCA inhibition. Mutagenesis of residues in transmembrane domain 6 leads to loss of PMCA activity. PED3 connects transmembrane domains 5 and 6. PED3 is only five amino acid residues long. By screening a phage display library, we obtained a peptide sequence that binds this target. After examining a number of peptides related to this original sequence, we selected one that inhibits the PMCA pump (caloxin 3A1). Caloxin 3A1 inhibits PMCA but not the sarcoplasmic reticulum Ca2+-pump. Caloxin 3A1 did not inhibit formation of the 140 kDa acylphosphate intermediate from ATP or its degradation. Thus, PEDs play a role in the reaction cycle of PMCA even though sites for binding to the substrates Ca2+ and Mg-ATP2-, and the activator calmodulin are all in the cytosolic domains of PMCA. In endothelial cells exposed to low concentration of a Ca2+-ionophore, caloxin 3A1 caused a further increase in [Ca2+]i proving its ability to inhibit PMCA pump extracellularly. Thus, even though PED3 is the shortest PED, it plays key role in the PMCA function.
Pande
Jyoti
J
Department of Medicine, McMaster University, 1200 Main Street West, Hamilton, Ont., L8N 3Z5, Canada.
Mallhi
Kanwaldeep K
KK
Grover
Ashok K
AK
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors* / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003331:Coronary Vessels / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D034421:Sus scrofa
D009842:Oligopeptides; D010455:Peptides; C501841:caloxin 3A1; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium