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J. Biol. Chem., Vol. 282, Issue 26, 19190-19202, June 29, 2007

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Differential Potency of Dominant Negative Connexin43 Mutants in Oculodentodigital Dysplasia^*

Xiang-Qun Gong, Qing Shao, Stéphanie Langlois, Donglin Bai²¹, and Dale W. Laird²

From the Departments of Physiology and Pharmacology and of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada

Oculodentodigital dysplasia (ODDD) is a congenital autosomal dominant disorder with phenotypic variability, which has been associated with mutations in the GJA1 gene encoding connexin43 (Cx43). Given that Cx43 mutants are thought to be equally co-expressed with wild-type Cx43 in ODDD patients, it is imperative to examine the consequence of these mutants in model systems that reflect this molar ratio. To that end, we used differential fluorescent protein tagging of mutant and wild-type Cx43 to quantitatively monitor the ratio of mutant/wild-type within the same putative gap junction plaques and co-immunoprecipitation to determine if the mutants interact with wild-type Cx43. Together the fluorescence-based assay was combined with patch clamp analysis to assess the dominant negative potency of Cx43 mutants. Our results revealed that the ODDD-linked Cx43 mutants, G21R and G138R, as well as amino terminus green fluorescent protein-tagged Cx43, were able to co-localize with wild-type Cx43 at the gap junction plaque-like structures and to co-immunoprecipitate with wild-type Cx43. All Cx43 mutants demonstrated dominant negative action on gap junctional conductance of wild-type Cx43 but not that of Cx32. More interestingly, these Cx43 mutants demonstrated different potencies in inhibiting the function of wild-type Cx43 with the G21R mutant being two times more potent than the G138R mutant. The potency difference in the dominant negative properties of ODDD-linked Cx43 mutants may have clinical implications for the various symptoms and disease severity observed in ODDD patients.

Received for publication, October 13, 2006 , and in revised form, April 3, 2007.

^* This work was supported by the Canadian Institutes of Health Research (CIHR) and Canada Research Chair program (to D. B. and D. W. L.) and a CIHR Strategic Training Scholarship (to S. L.). 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.

² Both authors contributed equally to this work.

¹ To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada. Tel.: 519-850-2569; Fax: 519-850-2562; E-mail: donglin.bai{at}schulich.uwo.ca.

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