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.
Bibliographical noteFunding Information:
The authors thank Dr. N. Narayanan (University of Western Ontario, London, Canada) for the sarcoplasmic reticulum, Dr. E. Escher (Sherbrooke University, Sherbrooke, Canada) for synthesis of the target peptide and advice during course of the project, Ragika Paramanathan 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. Patent pending.
Copyright 2008 Elsevier B.V., All rights reserved.
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