Caloxin 1c2‐ A Novel Engineered Selective Inhibitor of Plasma Membrane Ca2+ ‐ Pump Isoform 4, Effects on Coronary Artery

Abstract

Coronary artery smooth muscle expresses two isoforms of plasma membrane Ca2+ pumps: PMCA4 and PMCA1. The previously reported peptide inhibitor caloxin 1b1 was obtained by using extracellular domain 1 (amino acids 115–131) of PMCA4 as the target. Here, we report its mutagenesis by creating a phage display library of caloxin 1b1‐like peptides. The library was screened by affinity chromatography with the PMCA purified from the human erythrocyte ghosts that contain mainly PMCA4 isoform. This yielded caloxin 1c2 with the following key properties: a) Ki = 2.3 ± 0.3 μM which corresponds to a 20x higher affinity for PMCA than caloxin 1b1, and b) it is isoform selective since it has 10x higher affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on the coronary artery smooth muscle: a) it increased the basal tone of the de‐endothelialized arteries: the increase being similar at 10, 20 or 50 μM, 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+ homeostasis in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and Ca2+ homeostasis due to its isoform selectivity and ability to act when added extracellularly.Supported by Heart & Stroke Foundation of Ontario.