Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of PMCA Ca2+–Mg2+–ATPase in human erythrocyte ghosts by caloxin 1c2 derivatives.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4401146&req=5

fig04: Inhibition of PMCA Ca2+–Mg2+–ATPase in human erythrocyte ghosts by caloxin 1c2 derivatives.

Mentions: 3Bpa1c2biotin (TA[Bpa]SEVLDLLRRGGGSK (biotin)-amide) and 16Bpa1c2biotin (TAWSEVLDLL-RRGGG[Bpa]K[biotin]-amide). If the C-terminal domain were to play a role in the inhibition, the 16Bpa substitution and the change in the positive charge of K at 17 by adding biotin to the epsilon amino group would result in weaker inhibition by the 16Bpa1c2biotin. However, if the moiety WS(E/D)-V(L/V) were to be key to the inhibition, 3Bpa1c2biotin would be a weak inhibitor. The effects of both the derivatives were examined on the human erythrocyte ghost Ca2+–Mg2+–ATPase as the Ca2+-stimulated hydrolysis of [33P]-γ-ATP (Fig. 4). The Ki value for 16Bpa1c2biotin derivative (5.1 ± 0.8 μM) was only slightly greater than that of caloxin 1c2 (2.3 ± 0.3 μM). Thus the substitution of 16S with Bpa and biotinylation of K altered the PMCA inhibition only marginally, consistent with these residues being outside the selected 12 amino acid domain of caloxin 1c2. In contrast, the Ki value for the 3Bpa1c2biotin (50 ± 6 μM) was much greater. This experiment confirmed the hypothesis that the moiety WS(E/D)-V(L/V) in caloxin 1b1 and its mutants is important in inhibiting PMCA4 Ca2+–Mg2+–ATPase. This observation may be useful in further studies on caloxin 1c2-membrane interactions.


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 PMCA Ca2+–Mg2+–ATPase in human erythrocyte ghosts by caloxin 1c2 derivatives.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: Inhibition of PMCA Ca2+–Mg2+–ATPase in human erythrocyte ghosts by caloxin 1c2 derivatives.
Mentions: 3Bpa1c2biotin (TA[Bpa]SEVLDLLRRGGGSK (biotin)-amide) and 16Bpa1c2biotin (TAWSEVLDLL-RRGGG[Bpa]K[biotin]-amide). If the C-terminal domain were to play a role in the inhibition, the 16Bpa substitution and the change in the positive charge of K at 17 by adding biotin to the epsilon amino group would result in weaker inhibition by the 16Bpa1c2biotin. However, if the moiety WS(E/D)-V(L/V) were to be key to the inhibition, 3Bpa1c2biotin would be a weak inhibitor. The effects of both the derivatives were examined on the human erythrocyte ghost Ca2+–Mg2+–ATPase as the Ca2+-stimulated hydrolysis of [33P]-γ-ATP (Fig. 4). The Ki value for 16Bpa1c2biotin derivative (5.1 ± 0.8 μM) was only slightly greater than that of caloxin 1c2 (2.3 ± 0.3 μM). Thus the substitution of 16S with Bpa and biotinylation of K altered the PMCA inhibition only marginally, consistent with these residues being outside the selected 12 amino acid domain of caloxin 1c2. In contrast, the Ki value for the 3Bpa1c2biotin (50 ± 6 μM) was much greater. This experiment confirmed the hypothesis that the moiety WS(E/D)-V(L/V) in caloxin 1b1 and its mutants is important in inhibiting PMCA4 Ca2+–Mg2+–ATPase. This observation may be useful in further studies on caloxin 1c2-membrane interactions.

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