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J. Biol. Chem., Vol. 282, Issue 31, 22900-22909, August 3, 2007

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HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels^*

Gina M. Whitaker, Damiano Angoli, Hamed Nazzari, Ryuichi Shigemoto, and Eric A. Accili¹

From the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and the Division of Cerebral Structure, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8787, Japan

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET²), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET² data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.

Received for publication, November 29, 2006 , and in revised form, June 4, 2007.

^* This work was supported by grants from the Heart and Stroke Foundation of British Columbia and the Yukon and the Canadian Institutes for Health Research (to E. A. A.). 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 Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-6900; Fax: 604-822-6048; E-mail: eaaccili{at}interchange.ubc.ca.

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