The Journal of General Physiology
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Published online Aug 28 2006. doi:10.1085/jgp.200609556
The Rockefeller University Press, 0022-1295 $8.00
JGP, Volume 128, Number 3, 337-346
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ARTICLE

 Short-range Molecular Rearrangements in Ion Channels Detected by Tryptophan Quenching of Bimane Fluorescence



Leon D. Islas and William N. Zagotta

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

Correspondence to William N. Zagotta: zagotta{at}u.washington.edu

Ion channels are allosteric membrane proteins that open and close an ion-permeable pore in response to various stimuli. This gating process provides the regulation that underlies electrical signaling events such as action potentials, postsynaptic potentials, and sensory receptor potentials. Recently, the molecular structures of a number of ion channels and channel domains have been solved by x-ray crystallography. These structures have highlighted a gap in our understanding of the relationship between a channel's function and its structure. Here we introduce a new technique to fill this gap by simultaneously measuring the channel function with the inside-out patch-clamp technique and the channel structure with fluorescence spectroscopy. The structure and dynamics of short-range interactions in the channel can be measured by the presence of quenching of a covalently attached bimane fluorophore by a nearby tryptophan residue in the channel. This approach was applied to study the gating rearrangements in the bovine rod cyclic nucleotide-gated ion channel CNGA1 where it was found that C481 moves towards A461 during the opening allosteric transition induced by cyclic nucleotide. The approach offers new hope for elucidating the gating rearrangements in channels of known structure.

Abbreviations used in this paper: CNBD, cyclic nucleotide-binding domain; CNG, cyclic nucleotide-gated; FRET, fluorescence resonance energy transfer; HCN, hyperpolarization-activated cyclic nucleotide-modulated; NEM, N-ethylmaleimide; PCF, patch-clamp fluorometry.


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N. P. Semenova, K. Abarca-Heidemann, E. Loranc, and B. S. Rothberg
Bimane Fluorescence Scanning Suggests Secondary Structure near the S3-S4 Linker of BK Channels
J. Biol. Chem., April 17, 2009; 284(16): 10684 - 10693.
[Abstract] [Full Text] [PDF]


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