A novel plasma membrane Ca2+-pump inhibitor: caloxin 1A1

The plasma membrane Ca2+-Mg2+-ATPase is a Ca2+-pump that expels Ca2+ from cells. Here we report caloxin 1A1-a novel peptide inhibitor (Ki=100 μM) of plasma membrane Ca2+-pump-obtained by screening a cysteine bridge-constrained random peptide library for binding to the first extracellular domain of plasma membrane Ca2+-pump. Dithiothreitol removed the inhibition indicating that the constraint imposed by the cysteine bridge is required for the inhibition. Caloxin 1A1 also inhibited the fast twitch sarcoplasmic reticulum Ca2+-Mg2+-ATPase although weakly. Glutathione dimers (containing a cysteine bridge) inhibited the Ca2+-Mg2+-ATPase activity of sarcoplasmic reticulum Ca2+-Mg2+-ATPase, but not that of plasma membrane Ca2+-pump. Caloxin 1A1 stabilised Ca2+-dependent formation of the acid stable 140-kDa acylphosphate which is a partial reaction of this enzyme. Thus caloxin 1A1 inhibits the plasma membrane Ca2+-pump by perturbing the first extracellular domain indicating that the transmembrane domains 1 and 2 play a role in its reaction cycle. This finding is consistent with rearrangements that occur in transmembrane helices 1 and 2 during reaction cycle of sarcoplasmic reticulum Ca2+-pump. Caloxin 1A1 caused an increase in cytosolic Ca2+ concentration in endothelial cells.