2009-01-13 paper_1

T h e J o u r n a l o f G e n e r a l P h y s i o l o g y J. Gen. Physiol. © The Rocke feller University Press $8.00 Volume 128 Number 3 September 2006 337–346 http://www.jgp.org/cgi/doi/10.1085/jgp.200609556 337 ARTICLE Short-range Molecular Rearrangemen ts 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 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 s uch as action potentials, postsynaptic potentials, and sensory receptor potentials. Recently, the molecular structures o a number o ion channels and channel domains have been solved by x-ray crystallography. These structures have highlighted a gap in our understanding o the relationship between a channel’s unction and its structure. Here we introduce a new technique to fll this gap by simultaneously measuring the channel unction with the inside-out patch-clamp technique and the channel structure with uorescence spectroscopy. The structure and dynamics o short-range interactions in the channel can be measured by the presence o quenching o a c ovalently attached bimane uorophore 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 ound that C481 moves towards A461 during the opening allosteric transition induced by cyclic nucleotide. The approach oers new hope or elu- cidating the gating rearrangements in channels o known structure. INTRODUCTION With the recent unveiling o many high-resolution x-ray crystallographic structures o ion channel proteins, we have entered a new era in ion channel research (Hille, 2001; Swartz, 2004). We are no longer asking simply what does the channel look like but instead are now asking how does the channel structure change or rearrange during gating. Gating is the process by which all ion channels control the opening and closing o their ion-permeable pore. In most cases it is thought to be an allosteric conormational change that is regulated by signals such as changes in transmembrane voltage, the binding o external or internal ligands, or membrane stretch (Sigworth, 1994; Li et al., 1997; Perozo and Rees, 2003). In each case, the regulator dieren- tially aects the energy o the closed and open conor- mations and produces changes in the channel open probability. These changes in open probability are o undamental importance to the physiological unction o ion channels, but their detailed molecular mecha- nisms remain unknown. Unortunately, structural determination o the same channel protein in dierent conormational states has proven difcult. As a result, a number o methods have been developed to iner conormational changes rom more indirect measurements such as gating e- ects o channel mutations, state-dependent changes in cysteine accessibility or disulfde bond ormation, changes in cysteine-linked biotin accessibility, and state dependence o channel modulators and blockers (Holmgren et al., 1997; Liu et al., 1997; Johnson and Zagotta, 2001; Laine et al., 2003; Phillips et al., 2005; Ruta et al., 2005). Recently, site-specifc uorescence labeling o channels has been used to ollow the conormational changes associated with gating in voltage- dependent channels (Mannuzzu et al., 1996; Cha and Bezanilla, 1997; Cha et al., 1999; Zheng and Zagotta, 2000; Posson et al., 2005). The uorescence o a uorophore can report changes in local environment, accessibility to soluble quenchers, or proximity to nearby uorophores by uorescence resonance energy transer (FRET) (Selvin, 1995). This method can be com- bined with whole-cell (or whole-oocyte) recording or excised patch recording, allowing simultaneous moni- toring o channel unction and structure with a rela- tively noninvasive probe. The existing uorescence methods have a number o limitations that have reduced their useulness: (a) the cause o uorescence changes is oten ambiguous, and its time course oten complex, making the molecular interpretation o the results difcult; (b) the uorophores are oten large (e.g., GFP derivatives) or attached by long linkers, making the uorophore a poor reporter o the movement o its attachment point; (c) the uorescent labeling is not always completely specifc to the channel or the cysteine in question; (d) distances reported by standard FRET are too large Correspondence to William N. Zagotta: [email protected] Abbreviations used in this paper: CNBD, cyclic nucleotide-binding domain; CNG, cyclic nucleotide-gated; FRET , uorescence resonance energy transer; HCN, hyperpolarization-activated cyclic nucleotide- modulated; NEM, N -ethylmaleimide ; PCF, patch-clamp uorometry. o n S e p t e b e r 3 0 , 2 0 0 8 w w w . j g p o r g D o w n l o a d e d f r o Published August 28, 2006

2009-01-13 paper_1

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