Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca(2+)-pump isoform 4, on coronary artery.
Coronary artery smooth muscle expresses the plasma membrane Ca(2+) 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 microM 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 microM, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca(2+) when Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and the sarco/endoplasmic reticulum Ca(2+) pump were inhibited. We conclude that PMCA4 is pivotal to Ca(2+) 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.
Pande
Jyoti
J
Department of Biology, McMaster University, Hamilton, ON, Canada.
Szewczyk
Magdalena M
MM
Kuszczak
Iwona
I
Grover
Shawn
S
Escher
E
E
Grover
Ashok K
AK
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors* / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D003331:Coronary Vessels / Q000187:drug effects / Q000502:physiology*; D016296:Mutagenesis; D019151:Peptide Library; D010455:Peptides / Q000008:administration & dosage* / Q000138:chemical synthesis / Q000737:chemistry; D015202:Protein Engineering; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D013552:Swine
D019151:Peptide Library; D010455:Peptides; D020033:Protein Isoforms; C531373:caloxin 1c2; D017301:Ca(2+) Mg(2+)-ATPase