Li T et al. (2009), Interaction of the aromatics Tyr-72/Trp-288 in ...

XB-ART-39450

J Biol Chem 2009 Feb 13;2847:4689-94. doi: 10.1074/jbc.M805302200.

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Interaction of the aromatics Tyr-72/Trp-288 in the interface of the extracellular and transmembrane domains is essential for proton gating of acid-sensing ion channels.

Li T , Yang Y , Canessa CM .

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Acid-sensing ion channels are proton-activated ion channels expressed in the nervous system. They belong to the family of ENaC/Degenerins whose members share a conserved structure but are activated by widely diverse stimuli. We show that interaction of two aromatic residues, Tyr-72, located immediately after the first transmembrane segment, and Trp-288, located at the tip of a loop of the extracellular domain directed toward the first transmembrane segment, is essential for proton activation of the acid-sensing ion channels. The subdomain containing Trp-288 is a module tethered to the rest of the extracellular domain by short linkers and intrasubunit interactions between residues in the putative "proton sensor." Mutations in these two areas shift the apparent affinity of protons toward a more acidic range and change the kinetics of activation and desensitization. These results are consisting with displacement of the module relative to the rest of the extracellular domain to allow interaction of Trp-288 with Tyr-72 during gating. We propose that such interaction may provide functional coupling between the extracellular domain and the pore domain.

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Species referenced: Xenopus
Genes referenced: asic1

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	FIGURE 1. Ribbon representation of chicken ASIC1 structure. a, a single subunit is shown for simplicity with subdomains, A to F, indicated in different colors. The arrow points to the putative proton sensor with side chains of charged residues represented as sticks. Amino acids important for ASIC1 gating that were mutated in this study are also shown. The image was obtained with the molecular graphics program Chimera. b, amino acid sequence of subdomain F loop. Residues conserved in all ASIC proteins are in red.
	FIGURE 2. Functional effect of mutations in rat ASIC1. A, whole-cell currents of oocytes expressing wild type or mutant rASIC1 with aromatic residues in positions 72 and 288 stimulated with solution of pH 6.0. Bars above current traces indicate the duration of the test pH. B, family of currents elicited by changes in external pH of ASIC1 WT and mutant Y72R. C, table showing average values for the time constant of desensitization (Ïd) of wild type and mutants Y72R and Y72H obtained at various values of external pH (pHo). D, Western blot analysis of surface-biotinylated and total ASIC1 proteins of wild type and nonfunctional mutants. E, representative current trace of ENaC mutant channels carrying Ala in the corresponding positions 72 and 288 of ASIC1. The dotted line indicates the zero current level, and the bars above the current trace represent application to the bath of the specific blocker amiloride. Membrane holding potential was -60 mV. Bath solution contained 120 mm NaCl, pH 7.4. F, currents of mutant ASIC1-P365A show decreased in the magnitude of the peak current, slow rates of activation and desensitization, and impaired recovery from desensitization. Time and current scales are shown with the bars below each trace.
	FIGURE 3. Double mutant cycle analysis of rASIC1 His mutants in positions 72 and 288. A, thermodynamic mutant cycle where X1, X2, Y1, and Y2 represent change in apparent proton affinity along the arrow. B, family of currents induced by lowering the external pH for mutant channels Y72H and W288H. C, proton activation curves for the channels comprising the thermodynamic mutant cycle shown in A. D, coupling coefficient Î© calculated from the apparent EC50 corresponding to wild type and the single and double His mutants.
	FIGURE 4. Functional effects of amino acid substitutions in putative proton sensor. A, representative current traces of channels with additive substitutions of charged residues in the proton sensor for alanine elicited by pH 6.0. Rates of desensitization Î»d (-s) at pH 6.0 were: WT = 0.45 Â± 0.08; R191A = 0.47 Â± 0.11; D237A/E238A = 0.50 Â± 0.09; E346A/D350A = 2.5 Â± 0.1; D237A/E238A/E346A/D350A = 2.43 Â± 0.1; D237/E238/E346A/D350A/R191A = 2.8 Â± 0.1. B, pH response curves. Lines are fits to the Hill function. n = 6-8 oocytes per data point.
	FIGURE 5. Representation of Arg in positions 72 and 288 in ASIC1. The figure was obtained by substituting Tyr-72 and Trp-288 in the crystal structure of chicken ASIC1 by Arg to illustrate the relative position of the positively charged side chain with respect to the opposite aromatic residue.

References [+] :

Bouzat, Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel. 2004, Pubmed