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Functional roles of aromatic residues in the ligand-binding domain of cyclic nucleotide-gated channels

Li, Jun and Lester, Henry A. (1999) Functional roles of aromatic residues in the ligand-binding domain of cyclic nucleotide-gated channels. Molecular Pharmacology, 55 (5). pp. 873-882. ISSN 0026-895X.

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The ligand-binding domains of cyclic nucleotide-gated (CNG) channels show sequence homology to corresponding region(s) of the Escherichia coli catabolite gene-activator protein (CAP) and to the regulatory subunit of cAMP-dependent or cGMP-dependent protein kinases. The structure of CAP and that of a cAMP-dependent protein kinases regulatory subunit have been solved, prompting efforts to generate structural models for the binding domains in CNG channel. These models explicitly predicted that an aromatic residue in the CNG channel aligning with leucine 61 of CAP forms an interaction with the bound cyclic nucleotide. We tested this hypothesis by site-directed mutagenesis in a rat olfactory channel (rOCNC1) and a bovine rod photoreceptor channel (Brcng). We found that mutations at this site had only weak effects that were not specific to the aromatic or the hydrophobic nature of the substituted residue. This result weakens the hypothesis of a strong or specific interaction at this site. We also separately mutated most of the other aromatic residues in the binding domain to alanine; most of these mutations resulted in channels that either did not function or had only minor changes in sensitivity. However, replacing tyrosine 565 with alanine (Y565A) in rOCNC1 increased agonist sensitivity by approximately 10-fold and resulted in prominent spontaneous activities. Y565 presumably lies between two alpha helices in the binding domain; one of these, the C helix, probably rotates during channel activation. The position of Y565 at the "hinge" between the C helix and another portion of the binding domain, and the consequences of Y565 mutations, strongly suggest that this portion of the binding domain is involved in channel gating processes.

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Lester, Henry A.0000-0002-5470-5255
Additional Information:© 1999 by the American Society for Pharmacology and Experimental Therapeutics. Received June 2, 1998; accepted December 30, 1998. This research was supported by Grant NS11756 from the National Institutes of Health. We thank J. Ho for participating in the early phase of this project. We thank Dr. V. Kumar for the coordinates of the Brcng structural model and Dr. Y. Su for sharing the coordinates of PKA regulatory subunit before publication. We thank Drs. S. Scott and J. Tanaka for exchanging ideas. Dr. William Zagotta provided many stimulating discussions throughout this project.
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Issue or Number:5
Record Number:CaltechAUTHORS:20200626-123655847
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Official Citation:Functional Roles of Aromatic Residues in the Ligand-Binding Domain of Cyclic Nucleotide-Gated Channels Jun Li and Henry A. Lester Molecular Pharmacology May 1, 1999, 55 (5) 873-882
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104081
Deposited By: George Porter
Deposited On:26 Jun 2020 22:51
Last Modified:26 Jun 2020 22:51

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