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J. Biol. Chem., Vol. 279, Issue 5, 3553-3562, January 30, 2004

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Distinct Structural Determinants of Efficacy and Sensitivity in the Ligand-binding Domain of Cyclic Nucleotide-gated Channels^*

Edgar C. Young, Supported by a HHMI Research Associateship. and Natalia Krougliak

From the Center for Neurobiology & Behavior, Columbia University, New York, New York 10032

Cyclic nucleotide-gated (CNG) channels open in response to direct binding of cyclic nucleotide messengers. Every subunit in a tetrameric CNG channel contains a cytoplasmic ligand-binding domain (BD) that includes a -roll (flanked by short helices) and a single C-terminal helix called the C-helix that was previously found to control efficacy (maximal open probability) and selectivity for cGMP versus cAMP. We constructed a series of chimeric CNG channel subunits, each containing a distinct BD sequence (chosen from among six phylogenetically divergent isoforms) fused to an invariant non-BD sequence. We assayed these "BD substitution" chimeras as homomeric CNG channels in Xenopus oo-cytes to compare their functions and found that the most efficient activation by both cAMP and cGMP derived from the BD of the catfish CNGA4 olfactory modulatory subunit (fCNGA4). We then tested the effects of replacing subregions of the bovine CNGA1 BD with corresponding fCNGA4 sequence and hence identified parts of the fCNGA4 BD producing efficient activation. For instance, replacing either the "hinge" that connects the roll and C-helix subdomains or the BD sequence N-terminal to the hinge greatly enhanced cAMP efficacy. Replacing the "loop-8" region (the C-terminal end of the -roll) improved agonist sensitivity for cGMP selectively over cAMP. Our results thus identify multiple BD elements outside the C-helix that control selective ligand interaction and channel gating steps by distinct mechanisms. This suggests that the purine ring of the cyclic nucleotide may interact with both the -roll and the C-helix at different points in the mechanism.

Received for publication, September 24, 2003 , and in revised form, October 27, 2003.

^* This work was supported in part by funds from Steven A. Siegelbaum, Howard Hughes Medical Institute (HHMI). 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.

Deceased July 29, 2003. This paper is dedicated to the memory of Natasha whose grace and resourcefulness were an example to all.

To whom correspondence should be addressed: Center for Neurobiology & Behavior, Columbia University, 722 W. 168th St., Annex 632, New York, NY 10032. Tel.: 212-543-5259; Fax: 212-795-7997; E-mail: ecy4{at}columbia.edu.

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