Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH

Jasti, J.; Furukawa, Hiroyasu; Gonzales, Eric B.; Gouaux, Eric

doi:10.1038/nature06163

Cited by 969 publications

(1,585 citation statements)

References 52 publications

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“…Detergent-solubilized membranes can also be subjected to fluorescence size-exclusion chromatography (FSEC) to measure the 'monodispersity' of the sample 4 . FSEC was recently used to facilitate the purification of the chicken acid-sensing ion channel which crystallized and diffracted to 1.9-Å resolution 5 . In short, although membrane-integrated expression is no guarantee of function, the GFP-tag, nevertheless, speeds up the empirical process that all membrane proteins are required to go through on the way to obtain stable and homogeneous material for functional and structural work.…”

Section: Introductionmentioning

confidence: 99%

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

et al. 2008

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It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for highresolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 μGFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.

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Section: Introductionmentioning

confidence: 99%

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

et al. 2008

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“…This architecture is the hallmark of all members of the ENaC/Degenerins family regardless of their particular function and mode of activation. Gouaux and his group have solved two atomic structures of chicken ASIC1 both in shut non-conducting conformations 3,4 . These structures correspond most likely to desensitized states of the channel, as the crystallization trials in both instances were conducted at low pH.…”

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confidence: 99%

Outlines of the pore in open and closed conformations describe the gating mechanism of ASIC1

Yang

Canessa

2011

Nat Commun

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The proton-activated sodium channel AsIC1 belongs to the EnaC/Degenerins family of ion channels. Little is known about gating of the pore in any member of this class. Here we outline the shape of the ion pathway of AsIC1 in the open and closed conformations by measuring apparent rates of cysteine modification by thiol-specific reagents in the two transmembrane helices that form the pore (Tm1 and Tm2). Closed channels have a narrowing in the external end of the pore, whereas open channels have a narrowing midway, the length of Tm2 that serves as selectivity filter. Thus, gating of the pore entails straightening the tilt of Tm2 without significant rotation. The findings imply that the external narrowing serves as opening, closing and desensitization gate, and that the selectivity filter of AsIC1 is a transient structure that assembles in the open state and is pulled apart in the closed state.

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“…2). The first crystal structure of chicken ASIC1a (cASIC1a) was solved in 2007 [68,69] , and the pore architecture and ion binding sites were solved in 2009 [68,69] . It is anticipated that a great boost to ASICs research will come from unveiling their crystal structures.…”

Section: Asics Acid-sensing Is Associated With Nociceptionmentioning

confidence: 99%

Proton production, regulation and pathophysiological roles in the mammalian brain

Zeng

2012

Neurosci. Bull.

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Abstract:The recent demonstration of proton signaling in C. elegans muscle contraction suggests a novel mechanism for proton-based intercellular communication and has stimulated enthusiasm for exploring proton signaling in higher organisms. Emerging evidence indicates that protons are produced and regulated in localized space and time. Furthermore, identification of proton regulators and sensors in the brain leads to the speculation that proton production and regulation may be of major importance for both physiological and pathological functions ranging from nociception to learning and memory. Extracellular protons may play a role in signal transmission by not only acting on adjacent cells but also affecting the cell from which they were released. In this review, we summarize the upstream and downstream pathways of proton production and regulation in the mammalian brain, with special emphasis on the proton extruders and sensors that are critical in the homeostatic regulation of pH, and discuss their potential roles in proton signaling under normal and pathophysiological conditions.

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Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH

Cited by 969 publications

References 52 publications

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

Outlines of the pore in open and closed conformations describe the gating mechanism of ASIC1

Proton production, regulation and pathophysiological roles in the mammalian brain

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