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

Abstract

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 ...

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

“…This residue lies at the beginning of the ␤6-␤7 loop, which connects the ␤-ball and palm domains (1,16). Electrostatic calculations for the ASIC1 Glu (equivalent to ␣Asp-365) predicted a pK a of 7.4, a prerequisite for proton sensing in the physiological range (17). However, mutating this residue in ASIC1 to Gln had little effect on the pH sensitivity of channel activation (17,18).…”

“…A major challenge in elucidating ENaC gene family function has been the ability to distinguish ligand binding from downstream transduction steps. The crystal structure of ASIC1 suggests several putative proton binding sites, and mutations at select sites affect acid activation of ASIC1 (17)(18)(19). However, one cannot readily distinguish a proton binding site from sites involved in transmitting conformational changes to the channel gate.…”

Deactivation kinetics of acid-sensing ion channel 1a are strongly pH-sensitive

“…Such a model not only accounts for rapid deactivation at pH 8, markedly slower deactivation at pH 7, and the appropriate pH response curve but also harmonizes with existing structural data. Proton activation is thought to proceed from protons binding to pairs of carboxylate residues in the acidic pocket formed by the thumb and finger (53)(54)(55) may also arise from similar pairs in the palm domain (56,57) or elsewhere (60,61). In both regions, carboxylate residues are in close apposition.…”

Gating Transitions in the Palm Domain of ASIC1a*

“…Although mutations introduced at certain positions within the pocket altered apparent proton affinity, these channels remained proton-sensitive, suggesting that additional residues serve as proton-binding sites (12)(13)(14). Recently, Liechti et al (15) combined computational and functional approaches to identify additional acidic residues that contribute to proton gating in the extracellular region of ASIC1a. Overall, these functional studies indicate that residues distributed throughout the extracellular domain contribute to proton gating.…”