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Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Washington Medical School, Seattle, WA 98195

Address correspondence to William N. Zagotta, Dept. of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290. Fax: (206) 543-0934; email: zagotta{at}u.washington.edu

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca²⁺ ions. Rod CNG channels are directly inhibited by Ca²⁺-calmodulin (Ca²⁺/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca²⁺/CaM-dependent modulation. The molecular basis for Ca²⁺/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca²⁺/CaM to a site in the NH₂-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH₂-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca²⁺/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca²⁺/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH₂- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca²⁺/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH₂- and COOH-terminal regions underlies Ca²⁺/CaM-dependent inhibition.

Key Words: Ca²⁺/calmodulin • cyclic nucleotide-gated channels • patch-clamp • fluorescence • FRET

Abbreviations used in this paper: CaM, calmodulin; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CNBD, cyclic nucleotide-binding domain; CNG, cyclic nucleotide-gated; eCFP, enhanced cyan fluorescent protein; eYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; PCF, patch-clamp fluorometry.

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