The Contact Region between Three Domains of the Extracellular Loop of ASIC1a Is Critical for Channel Function

Self Cite

Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na ؉ channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK a value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/ deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.Acid-sensing ion channels (ASICs) 3 are neuronal nonvoltage-gated Na ϩ channels that are activated by a rapid drop in extracellular pH (1, 2). They are members of the epithelial Na ϩ channel/Degenerin family of ion channel proteins (3). Expression in nociceptive neurons and activation by protons suggest that ASICs may act as pain receptors (4), and evidence for such a role has been provided in several animal pain models (5-7). ASIC1a in the central nervous system plays a role in memory formation and the expression of fear (8, 9). ASIC1a is also an important mediator of cell injury induced by conditions associated with acidosis in the mammalian nervous system (10). Functional ASICs are formed by homo-or heterotrimeric assembly of ASIC subunits 1a, 1b, 2a, 2b, and 3. Each subunit has short intracellular N and C termini and two transmembrane domains that are separated by a large extracellular domain.Extracellular acidification opens ASICs. The ASIC activity is terminated in the continued presence of the acidic stimulus within hundreds of milliseconds to seconds by open channel inactivation, which is also called desensitization (11). Only in some ASIC isoforms and under certain pH conditions can acidification induce a sustained current, which has in most cases a much smaller amplitude than the peak current (12-14). At pH values slightly below the physiological pH, ASICs inactivate without apparent channel opening in a process that is called steady-state inactivation (SSIN) (15). By these two ways ASICs enter the inactivated state, which is a nonconducting, absorbing state. Experimental protocols have been applied to determine the kinetics of the recovery from the inactivated state, showing that channels need exposure for a certain duration ...

Section: Discussionmentioning

confidence: 99%

A Combined Computational and Functional Approach Identifies New Residues Involved in pH-dependent Gating of ASIC1a

Liechti

Bernèche

Bargeton

et al. 2010

Self Cite

“…As the simplest ligand possible, whether H ϩ gates ASICs by titration of a single or multiple polar residues remains controversial (24). In recent years, multiple putative pH sensors have been proposed (4,7,8,24,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43), e.g. the residues located in the cleft between the thumb and finger domains (4); residues around Glu-79 and Glu-423 in the palm domain (4,7,40); a cluster of acidic residues in the post-TM1 and pre-TM2 regions (Glu-63, His-72/ His-73, and Asp-78 of ASIC1a) (24,41).…”

Section: Discussionmentioning

confidence: 99%

Atomic Level Characterization of the Nonproton Ligand-sensing Domain of ASIC3 Channels

Chen

et al. 2011

Acid-sensing ion channels (ASICs) are known to be primarily activated by extracellular protons. Recently, we characterized a novel nonproton ligand (2-guanidine-4-methylquinazoline, GMQ), which activates the ASIC3 channel subtype at neutral pH. Using an interactive computational-experimental approach, here we extend our investigation to delineate the architecture of the GMQ-sensing domain in the ASIC3 channels. We first established a GMQ binding mode and revealed that residues Glu-423, Glu-79, Leu-77, Arg-376, Gln-271, and Gln-269 play key roles in forming the GMQ-sensing domain. We then verified the GMQ binding mode using ab initio calculation and mutagenesis and demonstrated the critical role of the above GMQ-binding residues in the interplay among GMQ, proton, and Ca 2؉ in regulating the function of ASIC3. Additionally, we showed that the same residues involved in coordinating GMQ responses are also critical for activation of the ASIC3 E79C mutant by thiol-reactive compound DTNB. Thus, a range of complementary techniques provide independent evidence for the structural details of the GMQ-sensing domain at atomic level, laying the foundation for further investigations of endogenous nonproton ligands and gating mechanisms of the ASIC3 channels. Acid-sensing ion channels (ASICs)4 are proton-gated cation channels (1-3), opening in response to extracellular pH reduction. To date, at least six ASIC subunits encoded by four distinctive genes (asic1, asic2, asic3, and asic4) have been identified (1, 2): 1a, 1b, 2a, 2b, 3, and 4. The first low pH crystal structure of chicken ASIC1 has been resolved (4, 5), revealing significant insights into many fundamental issues about these trimeric ion channels (6). Recently, we discovered a nonproton ligand, 2-guanidine-4-methylquinazoline (GMQ) (see Fig. 1A), which activates ASIC3 channels under neutral pH conditions (7). Remarkably, GMQ-and acid-induced (pH 5.0) currents exhibit distinct kinetics. The acid-induced currents desensitized rapidly, whereas the GMQ-evoked currents showed no desensitization when GMQ was continuously present. Using electrophysiological analysis of a series of ASIC3 channel mutants, we demonstrated that the carboxyl-carboxylate interaction pair, Glu-79 -Glu-423, is crucial for sustained activation of ASIC3 channels at neutral pH. Furthermore, using cysteine substitution mutants, we showed that covalent modification at E79C by DTNB, an Ellman's reagent used to modify and quantitatively detect sulfhydryl groups in proteins, was able to activate the channel, suggesting the importance of the Glu-79 residue in channel gating (7). Although the key role of Glu-79 and Glu-423 in GMQ-ASIC3 interactions has been elucidated, the makeup of this nonproton ligand-sensing domain remains largely unknown. In this study, taking advantage of computational approaches (8) and the available high resolution threedimensional structure of ASIC channels (4, 5), we aimed to delineate the architecture of the nonproton ligand-sensing domain as well as the GMQ binding mode in ASIC3 ...

“…Kellenberger and co-worker have shown that some residues belonging to the upper palm domain form a contact with the finger and the adjacent ␤-ball in ASIC1a (33). They have demonstrated that this zone undergoes conformational changes during inactivation and that chemical modification of this contact region decreases the probability of channel opening.…”

Section: The Finger/palm/␤-ball Connecting Domain (Domain P) and The mentioning

confidence: 99%

Binding Site and Inhibitory Mechanism of the Mambalgin-2 Pain-relieving Peptide on Acid-sensing Ion Channel 1a

Salinas¹,

Besson²,

Delettre³

et al. 2014

Background: Mambalgin-2 is a snake venom peptide that blocks acid-sensing ion channels (ASICs) to relieve pain. Results: Mambalgin-2 interacts with at least three different regions of ASICs and exerts both stimulatory and inhibitory effects. Conclusion: Binding of mambalgin-2 into the pH sensor traps the channels in the closed conformation. Significance: This might allow development of optimized blockers of ASICs of therapeutic value.