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

The plasma membrane Ca²⁺-Mg²⁺-ATPase is a Ca ²⁺-pump that expels Ca²⁺ from cells. Here we report caloxin 1A1-a novel peptide inhibitor (Ki=100 μM) of plasma membrane Ca ²⁺-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 Ca ²⁺-Mg²⁺-ATPase although weakly. Glutathione dimers (containing a cysteine bridge) inhibited the Ca²⁺-Mg ²⁺-ATPase activity of sarcoplasmic reticulum Ca²⁺-Mg ²⁺-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.