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.
Bibliographical noteFunding Information:
The authors thank Dr. N. Narayanan (University of Western Ontario, London, Canada) for the sarcoplasmic reticulum, Dr. E.E. Escher (Sherbrooke University, Sherbrooke,Canada) for synthesis of the target peptide and advice during course of the project, Ragika Paramanathan, Sue Samson and Paromita Ghosh for help with some of the experiments. This project was supported by a Grant-in-Aid (T4690)and a Career award to AKG from the Heart and Stroke Foundation of Ontario and a summer studentship to KKM from the Hypertension Society of Canada.
Copyright 2017 Elsevier B.V., All rights reserved.
- Signal transduction