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J. Biol. Chem., Vol. 282, Issue 35, 25640-25648, August 31, 2007

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The Plasma Membrane Ca²⁺ Pump Isoform 4a Differs from Isoform 4b in the Mechanism of Calmodulin Binding and Activation Kinetics

IMPLICATIONS FOR Ca²⁺ SIGNALING^*

Ariel J. Caride, Adelaida G. Filoteo, John T. Penniston, and Emanuel E. Strehler¹

From the Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905 and the Massachusetts General Hospital, Boston, Massachusetts 02114

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca²⁺. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca²⁺ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., Török, K., and Caride, A. J. (2003) Biochemistry 41, 12115–12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca²⁺ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca²⁺ signal, we then simulated the effect of PMCA4a on the shape of a Ca²⁺ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500–24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.

Received for publication, February 6, 2007 , and in revised form, June 15, 2007.

^* This work was supported by Grants GM28835 and NS51769 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data analysis.

¹ To whom correspondence should be addressed: 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-9372; Fax: 507-284-2384; E-mail: strehler.emanuel{at}mayo.edu.

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