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-ATP^2-, 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.