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The Journal of Neuroscience, September 3, 2003, 23(22):8167-8175

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Disruption of an Intersubunit Interaction Underlies Ca²⁺-Calmodulin Modulation of Cyclic Nucleotide-Gated Channels

Jie Zheng,¹ Michael D. Varnum,² and William N. Zagotta¹

¹Howard Hughes Medical Institute and Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195, and ²Washington State University, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Pullman, Washington 99164

Cyclic nucleotide-gated channels are key molecular elements for olfactory transduction. Olfactory adaptation caused by repeated exposure to an odorant has been proposed to be mediated by the binding of Ca²⁺-calmodulin to the NH₂-terminal domain of the channel, breaking its interaction with the COOH-terminal domain and downregulating the channel. We used a fluorescence resonance energy transfer (FRET) approach to study the structural aspects of this domain-domain interaction under physiological conditions in real time. Fluorescent proteins enhanced cyan fluorescent protein and enhanced yellow fluorescent protein were genetically attached at sites adjacent to the NH₂- and COOH-terminal interacting domains, respectively, allowing direct observation of molecular rearrangements in intact channels. FRET signals caused by the specific interdomain interaction were observed in both intact cells and excised patches. Comparison of the effective FRET efficiencies demonstrated that the interaction occurs specifically between subunits but not within the same subunit. Binding of Ca²⁺-calmodulin caused a reversible decrease in FRET with the same time course as channel downregulation. These results suggest that a separation or reorientation of the interacting domains between subunits by Ca²⁺-calmodulin leads to channel downregulation. The quaternary arrangement presents a structural framework for understanding the molecular mechanism of olfactory adaptation.

Key words: ion channel; Ca²⁺-calmodulin; olfactory adaptation; signal transduction; GFP mutants; FRET; fluorescence

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Received June 11, 2003; revised July 14, 2003; accepted July 15, 2003.

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