A Model for the Activation of Plasma Membrane Calcium Pump Isoform 4b by Calmodulin

Alan R. Penheiter, Željko ̌ Bajzer, Adelaida G. Filoteo, Richard Thorogate, Katalin Török, Ariel J. Caride

Research output: Contribution to journalArticle

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Abstract

Overexpression of the plasma membrane calcium pump (PMCA) isoform 4b by means of the baculovirus system enabled us, for the first time, to study the kinetics of calmodulin binding to this pump. This was done by stopped-flow fluorescence measurements using 2-chloro-(amino-Lys 75)-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin (TA-calmodulin). Upon mixing with PMCA, the fluorescence of TA-calmodulin changed along a biphasic curve: a rapid and small increase in fluorescence was followed by a slow and large decrease that lasted about 100 s. The experiment was done at several PMCA concentrations. Global fitting nonlinear regression analysis of these results led to a model in which PMCA is present in two forms: a closed conformation and an open conformation. Calmodulin reacts with both conformations but reacts faster and with higher affinity for the open conformation. Measurements of the ATPase activity of PMCA under similar conditions revealed that the open form has higher ATPase activity than the closed one. Contrasting with the reaction with the whole pump, TA-calmodulin reacted rapidly (in about 2 s) with a calmodulin-binding peptide made after the sequence of the calmodulin-binding domain of PMCA (C28). Results of TA-calmodulin binding to C28 are explained by a simpler model, in which only an open conformation exists.

Original languageEnglish (US)
Pages (from-to)12115-12124
Number of pages10
JournalBiochemistry
Volume42
Issue number41
DOIs
StatePublished - Oct 21 2003

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Calmodulin
Cell membranes
Protein Isoforms
Chemical activation
Cell Membrane
Pumps
Calcium
Conformations
Fluorescence
Adenosine Triphosphatases
Plasma Membrane Calcium-Transporting ATPases
Baculoviridae
Regression analysis
Regression Analysis
Peptides
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Penheiter, A. R., Bajzer, Ž. ., Filoteo, A. G., Thorogate, R., Török, K., & Caride, A. J. (2003). A Model for the Activation of Plasma Membrane Calcium Pump Isoform 4b by Calmodulin. Biochemistry, 42(41), 12115-12124. https://doi.org/10.1021/bi027098+

A Model for the Activation of Plasma Membrane Calcium Pump Isoform 4b by Calmodulin. / Penheiter, Alan R.; Bajzer, Željko ̌; Filoteo, Adelaida G.; Thorogate, Richard; Török, Katalin; Caride, Ariel J.

In: Biochemistry, Vol. 42, No. 41, 21.10.2003, p. 12115-12124.

Research output: Contribution to journalArticle

Penheiter, AR, Bajzer, Ž, Filoteo, AG, Thorogate, R, Török, K & Caride, AJ 2003, 'A Model for the Activation of Plasma Membrane Calcium Pump Isoform 4b by Calmodulin', Biochemistry, vol. 42, no. 41, pp. 12115-12124. https://doi.org/10.1021/bi027098+
Penheiter AR, Bajzer Ž, Filoteo AG, Thorogate R, Török K, Caride AJ. A Model for the Activation of Plasma Membrane Calcium Pump Isoform 4b by Calmodulin. Biochemistry. 2003 Oct 21;42(41):12115-12124. https://doi.org/10.1021/bi027098+
Penheiter, Alan R. ; Bajzer, Željko ̌ ; Filoteo, Adelaida G. ; Thorogate, Richard ; Török, Katalin ; Caride, Ariel J. / A Model for the Activation of Plasma Membrane Calcium Pump Isoform 4b by Calmodulin. In: Biochemistry. 2003 ; Vol. 42, No. 41. pp. 12115-12124.
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