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J. Biol. Chem., Vol. 283, Issue 21, 14461-14468, May 23, 2008

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Ca²⁺-stimulated Basal Adenylyl Cyclase Activity Localization in Membrane Lipid Microdomains of Cardiac Sinoatrial Nodal Pacemaker Cells^*

Antoine Younes¹, Alexey E. Lyashkov¹, David Graham¹, Anna Sheydina¹, Maria V. Volkova¹, Megan Mitsak, Tatiana M. Vinogradova, Yevgeniya O. Lukyanenko, Yue Li, Abdul M. Ruknudin, Kenneth R. Boheler, Jennifer van Eyk, and Edward G. Lakatta²

From the Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224 and the Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21224

Spontaneous, rhythmic subsarcolemmal local Ca²⁺ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca²⁺ releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca²⁺ and cAMP/PKA regulatory components of automaticity are coupled via Ca²⁺ activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca²⁺-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca²⁺ over the entire physiological Ca²⁺ range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca²⁺ cycling proteins drive sarcoplasmic reticulum Ca²⁺ releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

Received for publication, September 10, 2007 , and in revised form, March 5, 2008.

^* This work was supported, in whole or in part, by the National Institutes of Health Intramural Research Program, NIA. 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 Figs. S1–S3 and Tables S1 and S2.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Gerontology Research Center, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8202; Fax: 410-558-8150; E-mail: lakattae{at}mail.nih.gov.

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