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.

Pig coronary artery smooth muscle expresses the Na(+)-Ca(2+)-exchanger NCX1 and the sarco/endoplasmic reticulum (SER) Ca(2+) pump SERCA2. NCX has been proposed to play a role in refilling the SER Ca(2+) pool. Caveolae may also direct Ca(2+) traffic during cell signaling. Here, we use immunofluorescence microscopy to determine if there is proximity between NCX1, SERCA2, and the caveolar protein caveolin-1. Stacks of images of cell surface domains were analyzed. Image stacks for one protein were analyzed for overlap with another protein, with and without randomization or image shifting. Within the resolution of light microscopy, there is significant overlap in the distributions of NCX1, SERCA2, and caveolin-1 but the three proteins are not always co-localized. The proximity between NCX1, SERCA2 is consistent with the assertion that NCX may supply Ca(2+) for refilling the SER but this relationship is only partial. Similarly, caveolae may direct traffic in some Ca(2+) signaling pathways but not others.

Pig coronary artery smooth muscle expresses, among many other proteins, Na+-Ca²+-exchanger NCX1 and sarcoplasmic reticulum Ca²+ pump SERCA2. NCX1 has been proposed to play a role in refilling the sarcoplasmic reticulum Ca²+ pool suggesting a functional linkage between the two proteins. We hypothesized that this functional linkage may require close apposition of SERCA2 and NCX1 involving regions of plasma membrane like lipid rafts. Lipid rafts are specialized membrane microdomains that appear as platforms to co-localize proteins. To determine the distribution of NCX1, SERCA2 and lipid rafts, we isolated microsomes from the smooth muscle tissue, treated them with non-ionic detergent and obtained fractions of different densities by sucrose density gradient centrifugal flotation. We examined the distribution of NCX1; SERCA2; non-lipid raft plasma membrane marker transferrin receptor protein; lipid raft markers caveolin-1, flotillin-2, prion protein, GM1-gangliosides and cholesterol; and cytoskeletal markers clathrin, actin and myosin. Distribution of markers identified two subsets of lipid rafts that differ in their components. One subset is rich in caveolin-1 and flotillin-2 and the other in GM1-gangliosides, prion protein and cholesterol. NCX1 distribution correlated strongly with SERCA2, caveolin-1 and flotillin-2, less strongly with the other membrane markers and negatively with the cytoskeletal markers. These experiments were repeated with a non-detergent method of treating microsomes with sonication at high pH and similar results were obtained. These observations are consistent with the observed functional linkage between NCX1 and SERCA2 and suggest a role for NCX1 in supplying Ca²+ for refilling the sarcoplasmic reticulum.

We tested the hypothesis that the de-endothelialized artery rings from the left anterior descending (LAD) coronary artery and its left ventricular branch (LVB) differ in their contractile responses to Na+–Ca2+-exchanger (NCX) mediated Ca2+-entry, muscarinic receptor activation with carbachol, and sarco/endoplasmic reticulum Ca2+ pump (SERCA) inhibition with thapsigargin. In LVB, the force of contraction (in N/g tissue) produced by the NCX mediated Ca2+-entry (17.5 ± 1.4) and carbachol (18 ± 1.5) was only slightly smaller than that due to membrane depolarization with KCl (24.0 ± 1.0). In contrast, in LAD the force of contraction produced with NCX (8.7 ± 0.7) and carbachol (6.1 ± 1.1) was much smaller than with KCl (15.7 ± 0.7). Thapsigargin also contracted LVB with greater force than LAD. When isolated microsomes were used, the binding to the muscarinic receptor antagonist quinuclidinyl benzilate was greater in LVB than in LAD. Microsomes were also used for Western blots. The intensities of signals for both SERCA and NCX were greater in LVB than in LAD. These biochemical observations were consistent with the contractile experiments. Thus, it appears that the differences between LAD and the resistance arteries may begin as early as LVB.

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.