McPharmNet: Molecular Pharmacology at McMaster

Plasma membrane Ca²⁺pump(PMCA) structure
This model was constructed by Dr. H. Young (University of Alberta) based on the PMCA homology model of Dr. J. Penniston.
Transmembrane view Extracellular surface view

Caloxins are specific extracellular inhibitors of PMCA.
Page index: overview, caloxin nomenclature and information on our latest caloxin (1c2)and other individual caloxins (1b1, 1b2, 3a1, 3a2, 1a1, 1a2 and 2a1). Click here for a recent review on caloxins.
This site is hosted by Dr. A. K. Grover. Please, click here to e-mail for questions or suggestions.
To find out about Dr. A. K. Grover please click A.K. Grover at McMaster University, Hamilton, Ontario, Canada

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Overview: Plasma membrane Ca²⁺pumps (PMCA) are Ca²⁺-Mg²⁺-ATPases that expel Ca²⁺ from cells. They are encoded by alternative splices of 4 genes PMCA 1, 2, 3 or 4. Genes PMCA1 and 4 are most widely expressed. All functional PMCA have 5 extracellular domains which are short sequences and have not been previously associated with any of the PMCA functions. Our aim is to invent caloxins selective for PMCA1, 2, 3 and 4.
However, the roles of PMCA in cell function are not known because the previously used non-selective inhibitors which directly or indirectly affected other Ca²⁺ extrusion pathways also. We hypothesized that a peptide which bind a PMCA extracellular domains could act as a caloxin . Caloxins could then be used to understand the physiology of PMCA in cells in healthy and diseased states and potentially also therapeutically. Go to index.
Caloxin nomenclature: The first caloxin we reported was caloxin 2a1. In this name, the number 2 (first number) denotes that it was obtained as a peptide binding to PED2. "a" indicates that the screening method consisted of using a synthetic peptide based on PED2 as a target to screen a random peptide phage display library. The 1 (second number) indicates that this is the first peptide in this series. Similarly, in caloxin 1b2, the 1 indicates that the target to screen is based on PED. "b" indicates that the screening was conducted in a two step method: the first using a synthetic peptide based on PED1 as a target to screen a random peptide phage display library and the second step was based on affinity chromatography on a Sepharose-calmodulin-PMCA. The 2 indicates that this is the second peptide obtained in this series. C series are obtained by mutagenesis of B series.Go to index.
Individual caloxins:
Caloxin 1b1:(click here for publication). Synthetic target peptide: PMCA4-115 (CISLVLSFYRPAGEENEL)
Caloxin 1b1: TAWSEVLHLLSRGGG-amide. It dissolves better in 10-25 % ethanol than in water. Prepare 10 mM stock solution in 10% ethanol. The stock can be kept at -80^oC (we have not tested it beyond several weeks).
Ki for Ca²⁺-Mg²⁺-ATPase in erythrocytes ghosts (PMCA4): 46µM, Ki in HEK293 cells (PMCA1): 107 µM, works better on aortic smooth muscle (PMCA4 + some PMCA1) than on endothelium (predominantly PMCA1).
Specificity: No effect on SERCA1 Ca²⁺-Mg²⁺-ATPase, Na⁺-K⁺-ATPase, Mg²⁺-ATPase, K⁺-activated or Mg²⁺-activated phosphatase. Randomized control RP1b1 (GAETLSHGLRLGSVW-amide) caused no inhibition.
Caloxins 1c2 is a PMCA4 selective caloxin. Ki = 2.3 uM for PMCA4 and greater than 20 uM for PMCA1, 2 or 3 (submitted for publication to Journal of Cellular and Molecular Medicine) obtained by mutagensis of caloxin 1b1. Click here to see properties properties of caloxin 1c2 and other key caloxins. Go to index.
Caloxin 1b2: (new, sequence not yet published), Synthetic target peptide: PMCA4-133 (GQVATTPEDENEAQAC).
Ki for Ca²⁺-Mg²⁺-ATPase in erythrocytes ghosts (PMCA4): 33 µM, Ki in HEK293 cells: 100 µM. Not yet tested for specificity. Go to index.
Caloxin 3a1:(click here for publication). Synthetic target peptide based on PMCA4 874-883 (CITQDSPLKA). Note that the PED3 sequence (DSPLKA) is conserved in all PMCA isoforms and in species from man to Drosophilia.
Caloxin 3a1: WSSTSSVSAPLFGGGGSAK (Ki for Ca²⁺-Mg²⁺-ATPase in erythrocytes ghosts (PMCA4) = 200 µM, forms micelles, not useful for full inhibition of PMCA). Specificity: does not inhibit SERCA1 Ca²⁺-Mg²⁺-ATPase. Go to index.
Caloxin 3a2: (will not be published). Synthetic target peptide based on PMCA4 874-883 (CITQDSPLKA).
Caloxin 3a2: DSHINNEPSRRKGGGK ( Ki = 700 µM for Ca²⁺-Mg²⁺-ATPase in erythrocytes ghosts (PMCA4), whether N-acetylated or not). Specificity: does not inhibit SERCA1 Ca²⁺-Mg²⁺-ATPase. Go to index.
Caloxins 1a1 (click here for publication) and 1a2:
Synthetic target peptide: PMCA4-133 (GQVATTPEDENEAQAC).
Caloxin 1a1: ACPWWSPHACGGG. The two cysteines form an internal disulfide bridge.
Ki for Ca²⁺-Mg²⁺-ATPase in erythrocytes ghosts (PMCA4) = 86 µM. DTT increases Ki to 2 mM.
Specificity: Also inhibits SERCA1 in microsomes due to the disulfide bridge.
Caloxin 1a2: ACPIWQPHYCGGG (PMCA4 inhibition similar to caloxin 2a1, not fully characterized, not to be published). Go to index.
Caloxin 2a1 (First caloxin reported) (click here for publication)
Synthetic target peptide: PMCA1-398 (WVQKRPWLAESTPIYIQYFVKC)
Caloxin 1b1: VSNSNWPSFPSSGGG-amide.
Ki for Ca²⁺-Mg²⁺-ATPase in erythrocytes ghosts (PMCA4): 500 µM.
Specificity: No effect on SERCA1 Ca²⁺-Mg²⁺-ATPase, Na⁺-K⁺-ATPase, Mg²⁺-ATPase. Randomized control RP2a1 (SWSSFPGSGGVSNPN-amide) caused no inhibition.
Inhibition is not competitive with respect to Ca²⁺, calmodulin or ATP (click here for publication). Inhibits formation of acylphosphate in the forward direction (from ATP) but not in the reverse direction (from orthophosphate) (click here for publication)
Go to index.

All the peptides for this work were synthesized by Dalton Pharma Services.