Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca2+-pump isoform 4, on coronary artery

Coronary artery smooth muscle expresses the plasma membrane Ca2+ pump (PMCA) isoforms PMCA4 and PMCA1. We previously reported the peptide inhibitor caloxin 1b1 that was obtained by using extracellular domain 1 of PMCA4 as the target (Am J Physiol Cell.290 [2006] C1341). To engineer inhibitors with greater affinity and isoform selectivity, we have now created a phage display library of caloxin 1b1-like peptides. We screened this library by affinity chromatography with PMCA from erythrocyte ghosts that contain mainly PMCA4 to obtain caloxin 1c2. Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 mu M which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on coronary artery smooth muscle: (a) it increased basal tone of the de-endothelialized arteries; the increase being similar at 10, 20 or 50 mu M, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca2+ when Ca2+ extrusion via the Na+-Ca2+ exchanger and the sarco/endoplasmic reticulum Ca2+ pump were inhibited. We conclude that PMCA4 is pivotal to Ca2+ extrusion in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.