Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence.
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.
Islas
Leon D
LD
Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
Zagotta
William N
WN
D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D008564:Membrane Potentials; D008958:Models, Molecular; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D014981:Xenopus / Q000235:genetics / Q000378:metabolism
D019086:Bridged Bicyclo Compounds, Heterocyclic; C475741:CNGA1 protein, bovine; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D008301:Maleimides; C514903:bimane-C3-maleimide; C021149:bimanes; D014364:Tryptophan; D006152:Cyclic GMP