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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 PMCA1, 2, 3 and 4 Ca2+–Mg2+–ATPases by caloxins 1b1 (B) and 1c2 (C). Sequences of the extra-cellular domain 1a of PMCA4 that was used as synthetic target for screening are also compared with the domains 1a of PMCA1, 2 and 3 (A). Rabbit duodenal muscosal PM rich fraction was used as source of PMCA1, human erythrocyte ghosts as source of PMCA4 and microsomes from overexpressing insect cells sf9 for PMCA2 and 3. Each value is 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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fig02: Inhibition of PMCA1, 2, 3 and 4 Ca2+–Mg2+–ATPases by caloxins 1b1 (B) and 1c2 (C). Sequences of the extra-cellular domain 1a of PMCA4 that was used as synthetic target for screening are also compared with the domains 1a of PMCA1, 2 and 3 (A). Rabbit duodenal muscosal PM rich fraction was used as source of PMCA1, human erythrocyte ghosts as source of PMCA4 and microsomes from overexpressing insect cells sf9 for PMCA2 and 3. Each value is 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: Caloxin 1c2 was obtained by mutagenesis of caloxin 1b1, which had been selected with the extracellular domain 1a of PMCA4 (residues 115–131) as a target. However, sequences of the extracellular domain 1a between PMCA genes contain several similar residues but are not identical (Fig. 2A). Therefore, we compared the effects of caloxins 1b1 and 1c2 on the Ca2+–Mg2+–ATPase activities of PMCA1–4 (Fig. 2B and Fig. 2C). The values of Ki in μM for caloxin 1b1 were PMCA1 (105 ±11), PMCA2 (167 ±67), PMCA3 (274 ± 40) and PMCA4 (45 ± 4). Thus caloxin 1b1 inhibited PMCA4 with slightly higher affinity than for PMCA1, 2 or 3. In contrast, caloxin 1c2 had much greater selectivity for PMCA4: Ki in μM for caloxin 1c2 were: PMCA1 (21 ± 6), PMCA2 (40 ± 10), PMCA3 (67 ±8) and PMCA4 (2.3 ±0.3). Thus, when compared to caloxin 1b1, caloxin 1c2 had a much greater affinity and higher selectivity for PMCA4.


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 PMCA1, 2, 3 and 4 Ca2+–Mg2+–ATPases by caloxins 1b1 (B) and 1c2 (C). Sequences of the extra-cellular domain 1a of PMCA4 that was used as synthetic target for screening are also compared with the domains 1a of PMCA1, 2 and 3 (A). Rabbit duodenal muscosal PM rich fraction was used as source of PMCA1, human erythrocyte ghosts as source of PMCA4 and microsomes from overexpressing insect cells sf9 for PMCA2 and 3. Each value is 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

fig02: Inhibition of PMCA1, 2, 3 and 4 Ca2+–Mg2+–ATPases by caloxins 1b1 (B) and 1c2 (C). Sequences of the extra-cellular domain 1a of PMCA4 that was used as synthetic target for screening are also compared with the domains 1a of PMCA1, 2 and 3 (A). Rabbit duodenal muscosal PM rich fraction was used as source of PMCA1, human erythrocyte ghosts as source of PMCA4 and microsomes from overexpressing insect cells sf9 for PMCA2 and 3. Each value is 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: Caloxin 1c2 was obtained by mutagenesis of caloxin 1b1, which had been selected with the extracellular domain 1a of PMCA4 (residues 115–131) as a target. However, sequences of the extracellular domain 1a between PMCA genes contain several similar residues but are not identical (Fig. 2A). Therefore, we compared the effects of caloxins 1b1 and 1c2 on the Ca2+–Mg2+–ATPase activities of PMCA1–4 (Fig. 2B and Fig. 2C). The values of Ki in μM for caloxin 1b1 were PMCA1 (105 ±11), PMCA2 (167 ±67), PMCA3 (274 ± 40) and PMCA4 (45 ± 4). Thus caloxin 1b1 inhibited PMCA4 with slightly higher affinity than for PMCA1, 2 or 3. In contrast, caloxin 1c2 had much greater selectivity for PMCA4: Ki in μM for caloxin 1c2 were: PMCA1 (21 ± 6), PMCA2 (40 ± 10), PMCA3 (67 ±8) and PMCA4 (2.3 ±0.3). Thus, when compared to caloxin 1b1, caloxin 1c2 had a much greater affinity and higher selectivity for PMCA4.

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