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J. Biol. Chem., Vol. 281, Issue 8, 4691-4698, February 24, 2006
Endogenous and Exogenous Ca2+ Buffers Differentially Modulate Ca2+-dependent Inactivation of CaV2.1 Ca2+ Channels*From the Department of Pharmacology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia 30322 Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Cav2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Cav2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Cav2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mM), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mM). However, when the concentration of BAPTA was reduced to 0.5 mM, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Cav2.1. Cotransfection of Cav2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Cav2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.
Received for publication, November 7, 2005 , and in revised form, December 21, 2005. * This work was supported by Grant NS044922 from the National Institutes of Health (to A. L.), the Whitehall Foundation, and a Predoctoral NRSA Grant F31NS049757 (to L. K.). 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. 1 To whom correspondence should be addressed: Dept. of Pharmacology, Emory University School of Medicine, 5123 Rollins Research Bldg., 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-5991; Fax: 404-727-0365; E-mail: alee{at}pharm.emory.edu.
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