No abstract

Plasma membrane Ca(2+) pumps (PMCA) that expel Ca(2+) from cells are encoded by four genes (PMCA1-4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca(2+)-Mg(2+)-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K(i) value of 46 +/- 5 microM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca(2+)-Mg(2+)-ATPase in human embryonic kidney-293 cells with a higher K(i) value (105 +/- 11 microM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In de-endothelialized aortic rings, caloxin 1b1 (200 microM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca(2+)] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.

Plasma membrane Ca2+ pumps (PMCA) extrude cellular Ca2+ with a high affinity and hence play a major role in Ca2+ homeostasis and signaling. Caloxins (selective extracellular PMCA inhibitors) would aid in elucidating the physiology of PMCA. PMCA proteins have five extracellular domains (exdoms). Our hypotheses are: 1) peptides that bind selectively to each exdom can be invented by screening a random peptide library, and 2) a peptide can modulate PMCA activity by binding to one of the exdoms. The first caloxin 2a1, selected for binding exdom 2 was selective for PMCA (Ki=529 microM). It has been used to examine the physiological role of PMCA. PMCA isoforms are encoded by four genes. PMCA isoform expression differs in various cell types, with PMCA1 and 4 being the most widely distributed. There are differences between PMCA1-4 exdom 1 sequences, which may be exploited for inventing isoform selective caloxins. Using exdom 1 of PMCA4 as a target, modified screening procedures and mutagenesis led to the high-affinity caloxin 1c2 (Ki=2.3 microM for PMCA4). It is selective for PMCA4 over PMCA1, 2, or 3. We hope that caloxins can be used to discern the roles of individual PMCA isoforms in Ca2+ homeostasis and signaling. Caloxins may also become clinically useful in cardiovascular diseases, neurological disorders, retinopathy, cancer, and contraception.

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 μM which corresponds to a 20× 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 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+ 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.