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Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca(2+)-pump isoform 4, on coronary artery.

Pande J, Szewczyk MM, Kuszczak I, Grover S, Escher E, Grover AK - J. Cell. Mol. Med. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, ON, Canada.

ABSTRACT
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.

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Inhibition of Ca2+–Mg2+–ATPase in human erythrocyte ghosts at different concentrations of caloxin 1b1 and its mutants 1c1, 1c2, 1c3. Each value is the mean from 3–4 measurements carried out on 2–4 different days. On each day, the mean value of the Ca2+–Mg2+–ATPase was taken as 100% to compute the percent inhibition.
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fig01: Inhibition of Ca2+–Mg2+–ATPase in human erythrocyte ghosts at different concentrations of caloxin 1b1 and its mutants 1c1, 1c2, 1c3. Each value is the mean from 3–4 measurements carried out on 2–4 different days. On each day, the mean value of the Ca2+–Mg2+–ATPase was taken as 100% to compute the percent inhibition.

Mentions: In the phage, the variable 12 amino acid sequence is followed by the amino acid sequence GGGSK and then the remainder of the gIII protein. Therefore, caloxins 1c1 (TTWSEVVHRLSRGGGSK-amide), 1c2 (TAWSEVLDLLRRGGGSK-amide) and 1c3 (ASWSEVLHLLSRGGGSK-amide) were synthesized. Difference in Mg2+–ATPase activity in erythrocyte ghosts (express mainly PMCA4) with and without Ca2+ was defined as the PMCA Ca2+–Mg2+–ATPase [42]. The assay solution contained inhibitors of SERCA (thapsigargin), Na+–K+–ATPase (ouabain) and mitochondrial Ca2+–ATPase (azide). As reported previously for caloxin 1b1, none of the peptides inhibited the ATP hydrolysis in the absence of Ca2+[42]. Figure 1 shows the effects of the parent peptide caloxin 1b1, its randomized control peptide RP1b1 and the three mutant peptides. The control peptide RP1b1 had no effect on the PMCA Ca2+–Mg2+–ATPase activity. We have previously shown that caloxin inhibition is noncompetitive [50]. Therefore, the Ki values were determined at saturating substrate concentrations as described in the Methods. Using non-linear regression, the Ki values of the various caloxins were 1b1: 45 ± 4, 1c1: 20 ± 3, 1c2: 2.3 ± 0.3 and 1c3: 18 ± 3 μM. Analysis of the same data by linear regression as described in the Methods gave Ki values similar to those using non-linear regression: 1b1: 55 ±14, 1c1: 12 ± 3, 1c2: 2.0 ± 0.1 and 1c3: 20 ± 6 μM. In all subsequent experiments, only the non-linear regression was used. Thus, the selected caloxin 1b1-like mutants inhibited the erythrocyte ghost Ca2+–Mg2+–ATPase with greater affinity than that of the parent caloxin 1b1. Caloxin 1c2 had the highest affinity with its Ki value (2.3 ± 0.3 μM) being one-twentieth that of caloxin 1b1 (45 ± 4 μM).


Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca(2+)-pump isoform 4, on coronary artery.

Pande J, Szewczyk MM, Kuszczak I, Grover S, Escher E, Grover AK - J. Cell. Mol. Med. (2008)

Inhibition of Ca2+–Mg2+–ATPase in human erythrocyte ghosts at different concentrations of caloxin 1b1 and its mutants 1c1, 1c2, 1c3. Each value is the mean from 3–4 measurements carried out on 2–4 different days. On each day, the mean value of the Ca2+–Mg2+–ATPase was taken as 100% to compute the percent inhibition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401146&req=5

fig01: Inhibition of Ca2+–Mg2+–ATPase in human erythrocyte ghosts at different concentrations of caloxin 1b1 and its mutants 1c1, 1c2, 1c3. Each value is the mean from 3–4 measurements carried out on 2–4 different days. On each day, the mean value of the Ca2+–Mg2+–ATPase was taken as 100% to compute the percent inhibition.
Mentions: In the phage, the variable 12 amino acid sequence is followed by the amino acid sequence GGGSK and then the remainder of the gIII protein. Therefore, caloxins 1c1 (TTWSEVVHRLSRGGGSK-amide), 1c2 (TAWSEVLDLLRRGGGSK-amide) and 1c3 (ASWSEVLHLLSRGGGSK-amide) were synthesized. Difference in Mg2+–ATPase activity in erythrocyte ghosts (express mainly PMCA4) with and without Ca2+ was defined as the PMCA Ca2+–Mg2+–ATPase [42]. The assay solution contained inhibitors of SERCA (thapsigargin), Na+–K+–ATPase (ouabain) and mitochondrial Ca2+–ATPase (azide). As reported previously for caloxin 1b1, none of the peptides inhibited the ATP hydrolysis in the absence of Ca2+[42]. Figure 1 shows the effects of the parent peptide caloxin 1b1, its randomized control peptide RP1b1 and the three mutant peptides. The control peptide RP1b1 had no effect on the PMCA Ca2+–Mg2+–ATPase activity. We have previously shown that caloxin inhibition is noncompetitive [50]. Therefore, the Ki values were determined at saturating substrate concentrations as described in the Methods. Using non-linear regression, the Ki values of the various caloxins were 1b1: 45 ± 4, 1c1: 20 ± 3, 1c2: 2.3 ± 0.3 and 1c3: 18 ± 3 μM. Analysis of the same data by linear regression as described in the Methods gave Ki values similar to those using non-linear regression: 1b1: 55 ±14, 1c1: 12 ± 3, 1c2: 2.0 ± 0.1 and 1c3: 20 ± 6 μM. In all subsequent experiments, only the non-linear regression was used. Thus, the selected caloxin 1b1-like mutants inhibited the erythrocyte ghost Ca2+–Mg2+–ATPase with greater affinity than that of the parent caloxin 1b1. Caloxin 1c2 had the highest affinity with its Ki value (2.3 ± 0.3 μM) being one-twentieth that of caloxin 1b1 (45 ± 4 μM).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, ON, Canada.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus