Proton sensitivity of ASIC1 appeared with the rise of fishes by changes of residues in the region that follows TM1 in the ectodomain of the channel

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: Discussionsupporting

confidence: 54%

mentioning

confidence: 99%

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

Liechti

Bernèche

Bargeton

et al. 2010

“…Interestingly, these residues make contact around Glu 178 and Arg 176 of the adjacent subunit, residues that fall within the second region (residues 87-197) that we find necessary and sufficient for high affinity proton sensing. Further support for the importance of these regions comes from previous data indicating that Asp 351 , Gln 358 , and Asn 359 are required in Lamprey ASIC1a to form active ASIC1a channels, suggesting that these residues are critical for proton-dependent gating (37). We find that mutations Q358E and E355K increase the apparent proton sensitivity of ASIC1a.…”

Section: Discussionsupporting

confidence: 49%

“…Experiments using chimeras composed of proton-insensitive and proton-sensitive ASICs suggest that specific residues within the wrist domain of ASICs are required for proton-dependent activation (35)(36)(37). Channels that are unresponsive to protons are produced when these residues are mutated, and because some of these residues can be titrated by protons, it has been suggested that they represent the proton-binding site(s).…”

Section: Discussionmentioning

confidence: 99%

Identification of Protein Domains That Control Proton and Calcium Sensitivity of ASIC1a

Sherwood

Franke

Conneely

et al. 2009

The acid-sensing ion channels (ASICs) open in response to extracellular acidic pH, and individual subunits display differential sensitivity to protons and calcium. ASIC1a acts as a high affinity proton sensor, whereas ASIC2a requires substantially greater proton concentrations to activate. Using chimeras composed of ASIC1a and ASIC2a, we determined that two regions of the extracellular domain (residues 87-197 and 323-431) specify the high affinity proton response of ASIC1a. These two regions appear to undergo intersubunit interactions within the multimeric channel to specify proton sensitivity. Single amino acid mutations revealed that amino acids around Asp 357 play a prominent role in determining the pH dose response of ASIC1a. Within the same region, mutation F352L abolished PcTx1 modulation of ASIC1a. Surprisingly, we determined that another area of the extracellular domain was required for calcium-dependent regulation of ASIC1a activation, and this region functioned independently of high affinity proton sensing. These results indicate that specific regions play overlapping roles in pH-dependent gating and PcTx1-dependent modulation of ASIC1a activity, whereas a distinct region determines the calcium dependence of ASIC1a activation.The acid-sensing ion channels (ASICs) 3 are proton-gated ion channels expressed in neurons throughout the central and peripheral nervous system (1-3). ASICs are activated by extracellular acidosis, and protons act as ligands triggering channel opening (4). Disruption of the accn2 gene (which encodes ASIC1) dramatically reduces proton-gated currents in central neurons and alters a variety of behaviors, including fear, learning, and memory (5, 6). ASIC1 also contributes to neuronal damage and death during the prolonged acidosis accompanying cerebral ischemia (7). Specifically, mice with disruptions in the accn2 gene display 60% smaller lesion size compared with normal mice in models of stroke (8). Application of PcTx1, a venom peptide that prevents ASIC1a activation, is similarly neuroprotective, even when administered hours after injury (8, 9). Thus, ASIC1a represents a novel pharmacological target for the prevention of neuronal death following stroke.Mammals have four genes encoding ASICs (accn1 to -4) that encode at least six different ASIC subunits (1-3, 10). Like all members of the DEG/ENaC family, individual ASIC subunits have two transmembrane regions separated by a large cysteinerich extracellular region. Three ASIC subunits associate to form homomeric or heteromeric channels with distinct biophysical characteristics (11)(12)(13)(14). Specifically, ASIC1a homomeric channels activate at pH values much closer to neutral pH compared with ASIC2a homomeric channels. The high affinity proton sensitivity of ASIC1a plays a prominent role in acidosisinduced neuronal death, and modulators that alter the pH dose response of ASIC1a affect neuronal sensitivity to prolonged acidosis (8, 9, 15). For example, the neuroprotective venom peptide PcTx1 increases the proton sensitivity of the ...

“…Therefore, our results provide only circumstantial evidence that the post-TM1 domain has a general role in H ϩ gating of ASICs. However, a different study also found that the post-TM1 domain is crucial to distinguish H ϩ -sensitive from H ϩ -insensitive ASICs (32). Moreover, there is much more circumstantial evidence from other studies that reinforce the idea that the post-TM1 domain plays an important role in the gating of ion channels from the degenerin/epithelial sodium channel gene family.…”

Section: Discussionmentioning

confidence: 48%

Zebrafish Acid-sensing Ion Channel (ASIC) 4, Characterization of Homo- and Heteromeric Channels, and Identification of Regions Important for Activation by H+

Chen

Polleichtner

Kadurin

et al. 2007

There are four genes for acid-sensing ion channels (ASICs) in the genome of mammalian species. Whereas ASIC1 to ASIC3 form functional H ؉ -gated Na ؉ channels, ASIC4 is not gated by H ؉ , and its function is unknown. Zebrafish has two ASIC4 paralogs: zASIC4.1 and zASIC4.2. Whereas zASIC4.1 is gated by extracellular H ؉ , zASIC4.2 is not. This differential response to H ؉ makes zASIC4 paralogs a good model to study the properties of this ion channel. In this study, we found that surface expression of homomeric zASIC4.2 is higher than that of zASIC4. . Upon a drop in extracellular pH, ASICs rapidly open, depolarizing the cell, and during sustained acidification they desensitize. The genome of mammals, such as mice and humans, contains four asic genes that code for at least six distinct ASIC subunits (1). ASIC subunits contain two transmembrane domains, a large extracellular loop and relatively short intracellular N and C termini (2). Functional ASICs are homo-or hetero-oligomeric assemblies of individual subunits (3-9).The ASIC1a subunit is broadly expressed throughout the central and peripheral nervous system and contributes to synaptic transmission (10, 11). ASIC1b, a splice variant of ASIC1a, is specifically expressed in the peripheral nervous system and might contribute to the perception of pain (12, 13). ASIC2a is broadly expressed in the brain and contributes to synaptic transmission by the formation of heteromeric channels with ASIC1a (3,6,8). ASIC2b, a splice variant of ASIC2a, does not generate functional channels on its own but forms heteromeric channels with other ASIC subunits, notably ASIC3 (14). ASIC3, like ASIC1b, is specifically expressed in the peripheral nervous system (15), and there is evidence for a role in the perception and processing of painful stimuli (16 -19). ASIC4 has the most restricted expression of all ASIC subunits. In humans, ASIC4 mRNA is strongly expressed only in the pituitary gland; expression in other parts of the brain is faint (20). Like ASIC2b, ASIC4 does not form a functional homomeric channel (20, 21), and unlike ASIC2b, it does apparently also not form functional heteromeric channels with other ASIC subunits (20). These properties distinguish ASIC4 from other ASIC subunits, and, whereas possible functions are emerging for all other ASIC subunits, the function of ASIC4 is still unknown.Recently, we have reported the cloning of a family of ASICs from the zebrafish (22). Like their mammalian homologs, zASICs are broadly expressed in the zebrafish central nervous system (22). We identified six ASIC subunits, zASIC1.1, 1.2, 1.3, 2, 4.1, and 4.2, that are encoded by six different genes (22). zASIC1.2 and 1.3 are paralogs of zASIC1.1, and zASIC4.2 is a paralog of zASIC4.1, respectively. The existence of paralogs in zebrafish can be at least partly explained by the fish-specific genome duplication in ray-finned fish that happened ϳ350 million years ago (23). The amino acid sequences of zASIC4.1 and 4.2 are 68% identical; within the ectodomains identity is even 83%. Unlike rat AS...