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J. Biol. Chem., Vol. 278, Issue 44, 43781-43786, October 31, 2003

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Role of Subunit Heteromerization and N-Linked Glycosylation in the Formation of Functional Hyperpolarization-activated Cyclic Nucleotide-gated Channels^*

Barbara Much, Christian Wahl-Schott, Xiangang Zong, Angela Schneider, Ludwig Baumann, Sven Moosmang, Andreas Ludwig, and Martin Biel

From the Department Pharmazie-Pharmakologie für Naturwissenschaften, Ludwig-Maximilians Universität München, Butenandtstrasse 7, 81377 München, Germany and the Institut für Pharmakologie und Toxikologie der Technischen, Universität München, Biedersteinerstrasse 29, 80802 München, Germany

The coassembly of homologous subunits to heteromeric complexes serves as an important mechanism in generating ion channel diversity. Here, we have studied heteromerization in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. Using a combination of fluorescence confocal microscopy, coimmunoprecipitation, and electrophysiology we found that upon coexpression in HEK293 cells almost all dimeric combinations of HCN channel subunits give rise to the formation of stable channel complexes in the plasma membrane. We also identified HCN1/HCN2 heteromers in mouse brain indicating that heteromeric channels exist in vivo. Surprisingly, HCN2 and HCN3 did not coassemble to heteromeric channels. This finding indicates that heteromerization requires specific structural determinants that are not present in all HCN channel combinations. Using N-glycosidase F we show that native as well as recombinant HCN channels are glycosylated resulting in a 10–20-kDa shift in the molecular weight. Tunicamycin, an inhibitor of N-linked glycosylation, blocked surface membrane expression of HCN2. Similarly, a mutant HCN2 channel in which the putative N-glycosylation site in the loop between S5 and the pore helix was replaced by glutamine (HCN2_N380Q) was not inserted into the plasma membrane and did not yield detectable whole-cell currents. These results indicate that N-linked glycosylation is required for cell surface trafficking of HCN channels. Cotransfection of HCN2_N380Q with HCN4, but not with HCN3, rescued cell surface expression of HCN2_N380Q. Immunoprecipitation revealed that this rescue was due to the formation of a HCN2_N380Q/HCN4 heteromeric channel. Taken together our results indicate that subunit heteromerization and glycosylation are important determinants of the formation of native HCN channels.

Received for publication, June 30, 2003 , and in revised form, August 6, 2003.

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft. 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.

To whom correspondence should be addressed: Dept. Pharmazie-Pharmakologie für Naturwissenschaften, Ludwig-Maximilians Universität München, Butenandtstr. 7, 81377 München, Germany. Tel.: 49-89-2180-77327; Fax: 49-89-2180-77326; E-mail: mbiel{at}cup.uni-muenchen.de.

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