Insights into the mechanism of pore opening of acid-sensing ion channel 1a

Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains.     

Gating Transitions in the Palm Domain of ASIC1a

Acid-sensing ion channels (ASICs) are trimeric cation-selective proton-gated ion channels expressed in the central and peripheral nervous systems. The pore-forming transmembrane helices in these channels are linked by short loops to the palm domain in the extracellular region. Here, we explore the contribution to proton gating and desensitization of Glu-79 and Glu-416 in the palm domain of ASIC1a. Engineered Cys, Lys, and Gln substitutions at these positions shifted apparent proton affinity toward more acidic values. Double mutant cycle analysis indicated that Glu-79 and Glu-416 cooperatively facilitated pore opening in response to extracellular acidification. Channels bearing Cys at position 79 or 416 were irreversibly modified by thiol-reactive reagents in a state-dependent manner. Glu-79 and Glu-416 are located in β-strands 1 and 12, respectively. The covalent modification by (2-(trimethylammonium)ethyl) methanethiosulfonate bromide of Cys at position 79 impacted conformational changes associated with pore closing during desensitization, whereas the modification of Cys at position 416 affected conformational changes associated with proton gating. These results suggest that β-strands 1 and 12 contribute antagonistically to activation and desensitization of ASIC1a. Site-directed mutagenesis experiments indicated that the lower palm domain contracts in response to extracellular acidification. Taken together, our studies suggest that the lower palm domain mediates conformational movements that drive pore opening and closing events.     

The pre-transmembrane 1 domain of acid-sensing ion channels participates in the ion pore

The acid-sensing ion channel (ASIC) subunits ASIC1, ASIC2, and ASIC3 are members of the amiloride-sensitive Na+ channel/degenerin family of ion channels. They form proton-gated channels that are expressed in the central nervous system and in sensory neurons, where they are thought to play an important role in pain accompanying tissue acidosis. A splice variant of ASIC2, ASIC2b, is not active on its own but modifies the properties of ASIC3. In particular, whereas most members of the amiloride-sensitive Na+ channel/degenerin family are highly selective for Na+ over K+, ASIC3/ASIC2b heteromultimers show a nonselective component. Chimeras of the two splice variants allowed identification of a 9-amino acid region preceding the first transmembrane (TM) domain (pre-TM1) of ASIC2 that is involved in ion permeation and is critical for Na+ selectivity. Three amino acids in this region (Ile-19, Phe-20, and Thr-25) appear to be particularly important, because channels mutated at these residues discriminate poorly between Na+ and K+. In addition, the pH dependences of the activity of the F20S and T25K mutants are changed as compared with that of wild-type ASIC2. A corresponding ASIC3 mutant (T26K) also has modified Na+ selectivity. Our results suggest that the pre-TM1 region of ASICs participates in the ion pore.     

Asn415 in the β11-β12 Linker Decreases Proton-dependent Desensitization of ASIC1

Neurons of the mammalian nervous system express the proton-sensing ion channel ASIC1. Low concentrations of protons in the normal range of extracellular pH, pH 7.4–7.3, shut the pore by a conformational transition referred as steady-state desensitization. Therefore, the potential of local acidification to open ASIC1 relies on proton affinity for desensitization. This property is important physiologically and also can be exploited to develop strategies to increase or decrease the channel response to protons. In a previous study (Li, T., Yang, Y., and Canessa, C. M. (2010) J. Biol. Chem. 285, 22706–22712), we found that Leu-85 in the β1-β2 linker of the extracellular domain decreases the apparent proton affinity for steady-state desensitization and retards openings, slowing down the time course of the macroscopic currents. Here, we show that Asn-415 in the β11-β12 linker works together with the β1-β2 linker to stabilize a closed conformation that delays transition from the closed to the desensitized state. Substitutions of Asn-415 for Cys, Ser, or Gly render ASIC1 responsive to small increases in proton concentrations near the baseline physiological pH.     

Conformational Changes in the Lower Palm Domain of ASIC1a Contribute to Desensitization and RFamide Modulation

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel that contributes to fear and pain as well as neuronal damage following persistent cerebral acidosis. Neuropeptides can affect acid-induced neuronal injury by altering ASIC1a inactivation and/or steady-state desensitization. Yet, exactly how ASIC1a inactivation and desensitization occur or are modulated by peptides is not completely understood. We found that regions of the extracellular palm domain and the β_11-12 linker are important for inactivation and steady-state desensitization of ASIC1a. The single amino acid substitutions L280C and L415C dramatically enhanced the rate of inactivation and altered the pH-dependence of steady-state desensitization. Further, the use of methanethiosulfonate (MTS) reagents suggests that the lower palm region (L280C) undergoes a conformational change when ASIC1a transitions from closed to desensitized. We determined that L280C also displays an altered response to the RFamide peptide, FRRFamide. Further, the presence of FRRFamide limited MTS modification of L280C. Together, these results indicate a potential role of the lower palm domain in peptide modulation and suggest RFamide-related peptides promote conformational changes within this region. These data provide empirical support for the idea that L280, and likely this region of the central vestibule, is intimately involved in channel inactivation and desensitization.     

Protonation controls ASIC1a activity via coordinated movements in multiple domains

Acid-sensing ion channels (ASICs) are neuronal Na⁺-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.     

Asp433 in the closing gate of ASIC1 determines stability of the open state without changing properties of the selectivity filter or Ca2+ block

A constriction formed by the crossing of the second transmembrane domains of ASIC1, residues G432 to G436, forms the narrowest segment of the pore in the crystal structure of chicken ASIC1, presumably in the desensitized state, suggesting that it constitutes the "desensitization gate" and the "selectivity filter." Residues Gly-432 and Asp-433 occlude the pore, preventing the passage of ions from the extracellular side. Here, we examined the role of Asp-433 and Gly-432 in channel kinetics, ion selectivity, conductance, and Ca(2+) block in lamprey ASIC1 that is a channel with little intrinsic desensitization in the pH range of maximal activity, pH 7.0. The results show that the duration of open times depends on residue 433, with Asp supporting the longest openings followed by Glu, Gln, or Asn, whereas other residues keep the channel closed. This is consistent with residue Asp-433 forming the pore's closing gate and the properties of the side chain either stabilizing (hydrophobic amino acids) or destabilizing (Asp) the gate. The data also show residue 432 influencing the duration of openings, but here only Gly and Ala support long openings, whereas all other residues keep channels closed. The negative charge of Asp-433 was not required for block of the open pore by Ca(2+) or for determining ion selectivity and unitary conductance. We conclude that the conserved residue Asp-433 forms the closing gate of the pore and thereby determines the duration of individual openings while desensitization, defined as the permanent closure of all or a fraction of channels by the continual presence of H(+), modulates the on or off position of the closing gate. The latter effect depends on less conserved regions of the channel, such as TM1 and the extracellular domain. The constriction made by Asp-433 and Gly-432 does not select for ions in the open conformation, implying that the closing gate and selectivity filter are separate structural elements in the ion pathway of ASIC1. The results also predict a significantly different conformation of TM2 in the open state that relieves the constriction made by TM2, allowing the passage of ions unimpeded by the side chain of Asp-433.     

Toxin binding reveals two open state structures for one acid-sensing ion channel

Of the three principal conformations of acid-sensing ion channels (ASICs)—closed, open and desensitized—only the atomic structure of the desensitized conformation had been known. Two recent papers report the crystal structure of chicken ASIC1 in complex with the spider toxin psalmotoxin 1, and one of these studies finds that, depending on the pH, channels are in two different open conformations. Compared with the desensitized conformation, toxin binding induces only subtle structural changes in the lower part of the large extracellular domain but a complete rearrangement of the two transmembrane domains (TMDs), suggesting that desensitization gating (the transition from open to desensitized) is mainly associated with conformational rearrangements of the TMDs. Moreover, the study reveals how two different arrangements of the TMDs in the open state give rise to ion pores with different selectivity for monovalent cations.     

The tarantula toxin psalmotoxin 1 inhibits acid-sensing ion channel (ASIC) 1a by increasing its apparent H+ affinity

Acid-sensing ion channels (ASICs) are ion channels activated by extracellular protons. They are involved in higher brain functions and perception of pain, taste, and mechanical stimuli. Homomeric ASIC1a is potently inhibited by the tarantula toxin psalmotoxin 1. The mechanism of this inhibition is unknown. Here we show that psalmotoxin 1 inhibits ASIC1a by a unique mechanism: the toxin increases the apparent affinity for H(+) of ASIC1a. Since ASIC1a is activated by H(+) concentrations that are only slightly larger than the resting H(+) concentration, this increase in H(+) affinity is sufficient to shift ASIC1a channels into the desensitized state. As activation of ASIC1a has recently been linked to neurodegeneration associated with stroke, our results suggest chronic desensitization of ASIC1a by a slight increase of its H(+) affinity as a possible way of therapeutic intervention in stroke.     

A conformation change in the extracellular domain that accompanies desensitization of acid-sensing ion channel (ASIC) 3

Acid-sensing ion channels (ASICs) are thought to trigger some forms of acid-induced pain and taste, and to contribute to stroke-induced neural damage. After activation by low extracellular pH, different ASICs undergo desensitization on time scales from 0.1 to 10 s. Consistent with a substantial conformation change, desensitization slows dramatically when temperature drops (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. PNAS. 98:6459-6463). The nature of this conformation change is unknown, but two studies showed that desensitization rate is altered by mutations on or near the first transmembrane domain (TM1) (Coric, T., P. Zhang, N. Todorovic, and C.M. Canessa. 2003. J. Biol. Chem. 278:45240-45247; Pfister, Y., I. Gautschi, A.-N. Takeda, M. van Bemmelen, S. Kellenberger, and L. Schild. 2006. J. Biol. Chem. 281:11787-11791). Here we show evidence of a specific conformation change associated with desensitization. When mutated from glutamate to cysteine, residue 79, which is some 20 amino acids extracellular to TM1, can be altered by cysteine-modifying reagents when the channel is closed, but not when it is desensitized; thus, desensitization appears to conceal the residue from the extracellular medium. D78 and E79 are a pair of adjacent acidic amino acids that are highly conserved in ASICs yet absent from epithelial Na(+) channels, their acid-insensitive relatives. Despite large effects on desensitization by mutations at positions 78 and 79-including a shift to 10-fold lower proton concentration with the E79A mutant-there are not significant effects on activation.     

The interaction between two extracellular linker regions controls sustained opening of acid-sensing ion channel 1

Activation of acid-sensing ion channels (ASICs) contributes to neuronal death during stroke, to axonal degeneration during neuroinflammation, and to pain during inflammation. Although understanding ASIC gating may help to modulate ASIC activity during these pathologic situations, at present it is poorly understood. The ligand, H(+), probably binds to several sites, among them amino acids within the large extracellular domain. The extracellular domain is linked to the two transmembrane domains by the wrist region that is connected to two anti-parallel β-strands, β1 and β12. Thus, the wrist region together with those β-strands may have a crucial role in transmitting ligand binding to pore opening and closing. Here we show that amino acids in the β1-β2 linker determine constitutive opening of ASIC1b from shark. The most crucial residue within the β1-β2 linker (Asp(110)), when mutated from aspartate to cysteine, can be altered by cysteine-modifying reagents much more readily when channels are closed than when they are desensitized. Finally, engineering of a cysteine at position 110 and at an adjacent position in the β11-β12 linker leads to spontaneous formation of a disulfide bond that traps the channel in the desensitized conformation. Collectively, our results suggest that the β1-β2 and β11-β12 linkers are dynamic during gating and tightly appose to each other during desensitization gating. Hindrance of this tight apposition leads to reopening of the channel. It follows that the β1-β2 and β11-β12 linkers modulate gating movements of ASIC1 and may thus be drug targets to modulate ASIC activity.     

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.     

Independent Contribution of Extracellular Proton Binding Sites to ASIC1a Activation

Acid-sensing ion channels (ASICs) are a group of trimeric cation permeable channels gated by extracellular protons that are mainly expressed in the nervous system. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. In this report, we employed the substituted cysteine accessibility method and site-directed mutagenesis to examine the mechanism of activation of ASIC1a by extracellular protons. We found that the modification of E238C and D345C channels by MTSET reduced proton apparent affinity for activation. Furthermore, the introduction of positively charged residues at position 345 rendered shifted biphasic proton activation curves. Likewise, channels bearing mutations at positions 79 and 416 in the palm domain of the channel showed reduced proton apparent affinity and biphasic proton activation curves. Of significance, the effect of the mutations at positions 79 and 345 on channel activation was additive. E79K-D345K required a change to a pH lower than 2 for maximal activation. In summary, this study provides direct evidence for the presence of two distinct proton coordination sites in the extracellular region of ASIC1a, which jointly facilitate pore opening in response to extracellular acidification.     

Conformational changes in a pore-forming region underlie voltage-dependent “loop gating” of an unapposed connexin hemichannel

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA–biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA–biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent “loop-gating” mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd²⁺ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd²⁺ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.     

Potentiation and Block of ASIC1a by Memantine

Acid-sensing ion channels (ASICs) are modulated by various classes of ligands, including the recently described hydrophobic monoamines, which inhibit and potentiate ASICs in a subunit-specific manner. In particular, memantine inhibits ASIC1a and potentiates ASIC2a homomers. The aim of the present work was to characterize action mechanism of memantine on recombinant ASIC1a expressed in CHO (Chinese hamster ovary) cells. We have demonstrated that effect of memantine on ASIC1a strongly depends on membrane voltage, conditioning pH value and application protocol. When applied simultaneously with activating acidification at hyperpolarized voltages, memantine caused the strongest inhibition. Surprisingly, application of memantine between ASIC1a activations at zero voltage caused significant potentiation. Analysis of the data suggests that memantine produces two separate effects, voltage-dependent open-channel block and shift of steady-state desensitization curve to more acidic values. Putative binding sites are discussed based on the computer docking of memantine to the acidic pocket and the pore region.     

Toxin binding reveals two open state structures for one acid-sensing ion channel

Of the three principal conformations of acid-sensing ion channels (ASICs)—closed, open and desensitized—only the atomic structure of the desensitized conformation had been known. Two recent papers report the crystal structure of chicken ASIC1 in complex with the spider toxin psalmotoxin 1, and one of these studies finds that, depending on the pH, channels are in two different open conformations. Compared with the desensitized conformation, toxin binding induces only subtle structural changes in the lower part of the large extracellular domain but a complete rearrangement of the two transmembrane domains (TMDs), suggesting that desensitization gating (the transition from open to desensitized) is mainly associated with conformational rearrangements of the TMDs. Moreover, the study reveals how two different arrangements of the TMDs in the open state give rise to ion pores with different selectivity for monovalent cations.     

Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.     

Extracellular Subunit Interactions Control Transitions between Functional States of Acid-sensing Ion Channel 1a

Acid-sensing ion channels (ASICs) are neuronal, voltage-independent Na⁺ channels that are transiently activated by extracellular acidification. They are involved in pain sensation, the expression of fear, and in neurodegeneration after ischemic stroke. Our study investigates the role of extracellular subunit interactions in ASIC1a function. We identified two regions involved in critical intersubunit interactions. First, formation of an engineered disulfide bond between the palm and thumb domains leads to partial channel closure. Second, linking Glu-235 of a finger loop to either one of two different residues of the knuckle of a neighboring subunit opens the channel at physiological pH or disrupts its activity. This suggests that one finger-knuckle disulfide bond (E235C/K393C) sets the channel in an open state, whereas the other (E235C/Y389C) switches the channel to a non-conducting state. Voltage-clamp fluorometry experiments indicate that both the finger loop and the knuckle move away from the β-ball residue Trp-233 during acidification and subsequent desensitization. Together, these observations reveal that ASIC1a opening is accompanied by a distance increase between adjacent thumb and palm domains as well as a movement of Glu-235 relative to the knuckle helix. Our study identifies subunit interactions in the extracellular loop and shows that dynamic changes of these interactions are critical for normal ASIC function.     

Impact of Recovery from Desensitization on Acid-sensing Ion Channel-1a (ASIC1a) Current and Response to High Frequency Stimulation

ASIC1a is a neuronal sodium channel activated by external H⁺ ions. To date, all the characterization of ASIC1a has been conducted applying long H⁺ stimuli lasting several seconds. Such experimental protocols weaken and even silence ASIC1a currents to repetitive stimulation. In this work, we examined ASIC1a currents by methods that use rapid application and removal of H⁺. We found that brief H⁺ stimuli, <100 ms, even if applied at high frequency, prevent desensitization thereby generate full and steady peak currents of human ASIC1a. Kinetic analysis of recovery from desensitization of hASIC1a revealed two desensitized states: short- and long-lasting with time constants of τ_Ds ≤0.5 and τ_Dl = 229 s, while in chicken ASIC1a the two desensitized states have similar values τ_D 4.5 s. It is the large difference in stability of the two desensitized states that makes hASIC1a desensitization more pronounced and complex than in cASIC1a. Furthermore, recovery from desensitization was unrelated to cytosolic variations in pH, ATP, PIP₂, or redox state but was dependent on the hydrophobicity of key residues in the first transmembrane segment (TM1). In conclusion, brief H⁺-stimuli maintain steady the magnitude of peak currents thereby the ASIC1a channel is well poised to partake in high frequency signals in the brain.     

Interaction of acid-sensing ion channel (ASIC) 1 with the tarantula toxin psalmotoxin 1 is state dependent

Acid-sensing ion channels (ASICs) are Na(+) channels gated by extracellular H(+). Six ASIC subunits that are expressed in neurons have been characterized. The tarantula toxin psalmotoxin 1 has been reported to potently and specifically inhibit homomeric ASIC1a and has been useful to characterize ASICs in neurons. Recently we have shown that psalmotoxin 1 inhibits ASIC1a by increasing its apparent affinity for H(+). However, the mechanism by which PcTx1 increases the apparent H(+) affinity remained unclear. Here we show that PcTx1 also interacts with ASIC1b, a splice variant of ASIC1a. However, PcTx1 does not inhibit ASIC1b but promotes its opening; under slightly acidic conditions, PcTx1 behaves like an agonist for ASIC1b. Our results are most easily explained by binding of PcTx1 with different affinities to different states (closed, open, and desensitized) of the channel. For ASIC1b, PcTx1 binds most tightly to the open state, promoting opening, whereas for ASIC1a, it binds most tightly to the open and the desensitized state, promoting desensitization.