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J. Biol. Chem., Vol. 282, Issue 48, 35005-35017, November 30, 2007

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Identification of the Calmodulin Binding Domain of Connexin 43^*

Yubin Zhou¹, Wei Yang, Monica M. Lurtz, Yiming Ye^¶, Yun Huang, Hsiau-Wei Lee, Yanyi Chen, Charles F. Louis, and Jenny J. Yang²

From the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, the ^¶Proteomics Laboratory, Biotechnology Core Facility Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, and the Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521

Calmodulin (CaM) has been implicated in mediating the Ca²⁺-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136–158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca²⁺-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its -helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K⁺ is in the range of 0.7–1 µM. Upon binding of the peptide to CaM, the apparent K_d of Ca²⁺ for CaM decreased from 2.9 ± 0.1 to 1.6 ± 0.1 µM, and the Hill coefficient n_H increased from 2.1 ± 0.1 to 3.3 ± 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca²⁺-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136–158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca²⁺-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca²⁺-dependent manner, providing a molecular basis for the well characterized Ca²⁺-dependent inhibition of Cx43-containing gap junctions.

Received for publication, September 14, 2007

^* This work was supported by National Institutes of Health Grants EY-05684 (to C. F. L. and J. Y.) and GM62999 (to J. Y.). 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 Fig. S1.

¹ Fellow of the Molecular Basis of Disease Area of Focus at Georgia State University.

² To whom correspondence should be addressed: Dept. of Chemistry, Georgia State University, University Plaza, Atlanta, GA 30302. Tel.: 404-413-5520; Fax: 404-413-5551; E-mail: chejjy{at}langate.gsu.edu.

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