Mechanism of action of the novel plasma membrane Ca2+-pump inhibitor caloxin

Caloxin 2A1 is a novel inhibitor of the plasma membrane (PM) Ca2+-pump [Am. J. Physiol. Cell Physiol. 280 (2001) C1027]. The PM Ca2+-pump is a Ca2+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+. Caloxin 2A1 inhibits Ca2+-Mg2+-ATPase in human erythrocyte leaky ghosts. Here we report that this inhibition is non-competitive with respect to the substrates Ca2+ and ATP and the activator calmodulin. This was anticipated since the high affinity binding site for Ca2+ and sites for ATP and calmodulin are intracellular whereas caloxin 2A1 is a peptide selected for binding to the second extracellular domain of the pump. Caloxin 2A1 also inhibited the Ca2+-dependent formation of the acid stable 140 kDa acylphosphate intermediate from P-32-gamma-ATP However, it did not inhibit the formation of the acylphosphate intermediate in the reverse direction-from P-32-orthophosphate. Consistent with results on mutagenesis of transmembrane residues in the pump protein, we suggest that caloxin 2A1 inhibits conformational changes required during the reaction cycle of the pump. (C) 2002 Elsevier Science Ltd. All rights reserved.