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J. Biol. Chem., Vol. 283, Issue 21, 14728-14738, May 23, 2008

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C-terminal Movement during Gating in Cyclic Nucleotide-modulated Channels^*

Kimberley B. Craven, Nelson B. Olivier, and William N. Zagotta^¶1

From the Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195, the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and the ^¶Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195

Activation of cyclic nucleotide-modulated channels such as CNG and HCN channels is promoted by ligand-induced conformational changes in their C-terminal regions. The primary intersubunit interface of these C termini includes two salt bridges per subunit, formed between three residues (one positively charged and two negatively charged amino acids) that we term the SB triad. We previously hypothesized that the SB triad is formed in the closed channel and breaks when the channel opens. Here we tested this hypothesis by dynamically manipulating the SB triad in functioning CNGA1 channels. Reversing the charge at positions Arg-431 and Glu-462, two of the SB triad residues, by either mutation or application of charged reagents increased the favorability of channel opening. To determine how a charge reversal mutation in the SB triad structurally affects the channel, we solved the crystal structure of the HCN2 C-terminal region with the equivalent E462R mutation. The backbone structure of this mutant was very similar to that of wild type, but the SB triad was rearranged such that both salt bridges did not always form simultaneously, suggesting a mechanism for the increased ease of opening of the mutant channels. To prevent movement in the SB triad, we tethered two components of the SB triad region together with cysteine-reactive cross-linkers. Preventing normal movement of the SB triad region with short cross-linkers inhibited channel opening, whereas longer cross-linkers did not. These results support our hypothesis that the SB triad forms in the closed channel and indicate that this region expands as the channel opens.

Received for publication, December 22, 2007 , and in revised form, February 25, 2008.

The atomic coordinates and structure factors (code 3BPZ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01 EY010329 (to W. N. Z.) and R01 NS038631 (to E. G.). This work was also supported by the Howard Hughes Medical Institute. 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 Physiology and Biophysics, Howard Hughes Medical Inst., University of Washington, Box 357290, Seattle WA 98195-7290, Tel.: 206-685-3878; Fax: 206-543-0934; E-mail: zagotta{at}u.washington.edu.

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