Leu85 in the β1-β2 Linker of ASIC1 Slows Activation and Decreases the Apparent Proton Affinity by Stabilizing a Closed Conformation

Acid-sensing ion channels (ASICs) are proton-activated channels expressed in neurons of the central and peripheral nervous systems where they modulate neuronal activity in response to external increases in proton concentration. The size of ASIC1 currents evoked by a given local acidification is determined by the number of channels in the plasma membrane and by the apparent proton affinities for activation and steady-state desensitization of the channel. Thus, the magnitude of the pH drop and the value of the baseline pH both are functionally important. Recent characterization of ASIC1s from an increasing number of species has made evident that proton affinities of these channels vary across vertebrates. We found that in species with high baseline plasma pH, e.g. frog, shark, and fish, ASIC1 has high proton affinity compared with the mammalian channel. The β1-β2 linker in the extracellular domain, specifically by the substitution M85L, determines the interspecies differences in proton affinities and also the time course of ASIC1 macroscopic currents. The mechanism underlying these observations is a delay in channel opening after application of protons, most likely by stabilizing a closed conformation that decreases the apparent affinity to protons and also slows the rise and decay phases of the current. Together, the results suggest evolutionary adaptation of ASIC1 to match the value of the species-specific plasma pH. At the molecular level, adaptation is achieved by substitutions of nonionizable residues rather than by modification of the channel proton sensor.     

Inherent Dynamics of the Acid-Sensing Ion Channel 1 Correlates with the Gating Mechanism

The acid-sensing ion channel 1 (ASIC1) is a key receptor for extracellular protons. Although numerous structural and functional studies have been performed on this channel, the structural dynamics underlying the gating mechanism remains unknown. We used normal mode analysis, mutagenesis, and electrophysiological methods to explore the relationship between the inherent dynamics of ASIC1 and its gating mechanism. Here we show that a series of collective motions among the domains and subdomains of ASIC1 correlate with its acid-sensing function. The normal mode analysis result reveals that the intrinsic rotation of the extracellular domain and the collective motions between the thumb and finger induced by proton binding drive the receptor to experience a deformation from the extracellular domain to the transmembrane domain, triggering the channel pore to undergo “twist-to-open” motions. The movements in the transmembrane domain indicate that the likely position of the channel gate is around Leu440. These motion modes are compatible with a wide body of our complementary mutations and electrophysiological data. This study provides the dynamic fundamentals of ASIC1 gating.     

Single channel properties of rat acid-sensitive ion channel-1alpha, -2a,and -3 expressed in Xenopus oocytes

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1alpha, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na(+) are similar for the three types of channels (23-18 pS) and are not voltage dependent. However, ASIC1alpha exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1alpha and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1alpha, ASIC2a, and ASIC3 are activated by external protons with apparent pH(50) of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1alpha and ASIC3 (2.0 and 4.5 s(-1), respectively) but slow and only partial in ASIC2a (0.045 s(-1)). The response to external Ca(2+) also differs: micro M concentrations of extracellular Ca(2+) are necessary for proton gating of ASIC3 (EC(50) = 0.28 micro M), whereas ASIC1alpha and ASIC2a do not require Ca(2+). In addition, Ca(2+) inhibits ASIC1alpha (K(D) = 9.2 +/- 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca(2+) permeability of ASIC1alpha is very small.     

pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits

The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a+2a and ASIC1b+2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a+3, ASIC1b+3, and ASIC2a+3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a+2a+3 and ASIC1b+2a+3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.     

Molecular dynamics simulations investigate the mechanism of Psalmotoxin 1 regulating gating process of an acid-sensing ion channel 1a at pH 5.5

Acid-sensing ion channel 1a (ASIC1a) is a cation channel activated by protons and causes neuronal death through central nervous system. Psalmotoxin1 (PcTx1) is a gating modifier for ASIC1a. The process of PcTx1 regulating the channel gating from the extracellular domain to the transmembrane domain is unclear. Here we used molecular dynamics (MD) simulations method to investigate how PcTx1 regulates the gating of the ASIC1a. Our results indicated that PcTx1can mainly regulate ASIC1a gating process through hydrogen bonds, which can affect their relative positions of several key domains in ASIC1a, further, a long-range conformational changes path was determined, which is composed of β1, β2, β10, α6, α7, β11, and β12 in ASIC1a.     

Annexin II light chain p11 promotes functional expression of acid-sensing ion channel ASIC1a

Acid-sensing ion channels (ASICs) have been implicated in a wide variety of physiological functions. We have used a rat dorsal root ganglion cDNA library in a yeast two-hybrid assay to identify sensory neuron proteins that interact with ASICs. We found that annexin II light chain p11 physically interacts with the N terminus of ASIC1a, but not other ASIC isoforms. Immunoprecipitation studies confirmed an interaction between p11 and ASIC1 in rat dorsal root ganglion neurons in vivo. Coexpression of p11 and ASIC1a in CHO-K1 cells led to a 2-fold increase in expression of the ion channel at the cell membrane as determined by membrane-associated immunoreactivity and cell-surface biotinylation. Consistent with these findings, peak ASIC1a currents in transfected CHO-K1 cells were up-regulated 2-fold in the presence of p11, whereas ASIC3-mediated currents were unaffected by p11 expression. Neither the pH dependence of activation nor the rates of desensitization were altered by p11, suggesting that its primary role in regulating ASIC1a activity is to enhance cell-surface expression of ASIC1a. These data demonstrate that p11, already known to traffic members of the voltage-gated sodium and potassium channel families as well as transient receptor potential and chloride channels, also plays a selective role in enhancing ASIC1a functional expression.     

Extracellular Chloride Modulates the Desensitization Kinetics of Acid-sensing Ion Channel 1a (ASIC1a)

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. The recent crystallization of ASIC1a identified potential binding sites for Cl⁻ in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of Cl⁻ binding is unknown. We investigated the effect of Cl⁻ substitution on heterologously expressed ASIC1a current and H⁺-gated currents from hippocampal neurons recorded by whole-cell patch clamp. Replacement of extracellular Cl⁻ with the impermeable and inert anion methanesulfonate (MeSO₃⁻) caused ASIC1a currents to desensitize at a faster rate and attenuated tachyphylaxis. However, peak current amplitude, pH sensitivity, and selectivity were unchanged. Other anions, including Br⁻, I⁻, and thiocyanate, also altered the kinetics of desensitization and tachyphylaxis. Mutation of the residues that form the Cl⁻-binding site in ASIC1a abolished the modulatory effects of anions. The results of anion substitution on native ASIC channels in hippocampal neurons mirrored those in heterologously expressed ASIC1a and altered acid-induced neuronal death. Anion modulation of ASICs provides new insight into channel gating and may prove important in pathological brain conditions associated with changes in pH and Cl⁻.     

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.     

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.     

A nonproton ligand sensor in the acid-sensing ion channel

Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H(+))-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular "palm" domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis.     

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.     

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.     

Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel: Implication for simultaneous modulation in ASICs

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.     

Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.     

Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons

Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H(+)-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASIC1a/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H(+)-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H(+)-gated currents and therefore may contribute to normal physiology and pathophysiology.     

Expressing acid-sensing ion channel 3 in the brain alters acid-evoked currents and impairs fear conditioning

Previous studies on mice with a disruption of the gene encoding acid-sensing ion channel 1a (ASIC1a) suggest that ASIC1a is required for normal fear behavior. To investigate the effects of altering the subunit composition of brain ASICs on behavior, we developed transgenic mice expressing ASIC3 via the pan-neuronal synapsin I promoter. These mice express ASIC3 in the brain, where the endogenous ASIC3 protein is not detected. We found that in ASIC3 transgenic mice, ASIC3 co-immunoprecipitated with the endogenous ASIC1a protein and distributed in the same subcellular brain fractions as ASIC1a. In addition, ASIC3 significantly increased the rate of desensitization of acid-evoked currents in cultured cortical neurons. Importantly, ASIC3 reduced Pavlovian fear conditioning to both context and auditory cues. These observations suggest that ASIC3 can heteromultimerize with ASIC1a in the brain and alter the biophysical properties of the endogenous channel complex. Moreover, these data suggest that ASIC subunit composition and channel desensitization may be critical determinants for ASIC-dependent behavior.     

Evolutionarily Conserved Interactions within the Pore Domain of Acid-Sensing Ion Channels

Despite the sequence homology between acid-sensing ion channels (ASICs) and epithelial sodium channel (ENaCs), these channel families display very different functional characteristics. Whereas ASICs are gated by protons and show a relatively low degree of selectivity for sodium over potassium, ENaCs are constitutively active and display a remarkably high degree of sodium selectivity. To decipher if some of the functional diversity originates from differences within the transmembrane helices (M1 and M2) of both channel families, we turned to a combination of computational and functional interrogations, using statistical coupling analysis and mutational studies on mouse ASIC1a. The coupling analysis suggests that the relative position of M1 and M2 in the upper part of the pore domain is likely to remain constant during the ASIC gating cycle, whereas they may undergo relative movements in the lower part. Interestingly, our data suggest that to account for coupled residue pairs being in close structural proximity, both domain-swapped and nondomain-swapped ASIC M2 conformations need to be considered. Such conformational flexibility is consistent with structural work, which suggested that the lower part of M2 can adopt both domain-swapped and nondomain-swapped conformations. Overall, mutations to residues in the middle and lower pore were more likely to affect gating and/or ion selectivity than those in the upper pore. Indeed, disrupting the putative interaction between a highly conserved Trp/Glu residue pair in the lower pore is detrimental to gating and selectivity, although this interaction might occur in both domain-swapped and nonswapped conformations. Finally, our results suggest that the greater number of larger, aromatic side chains in the ENaC M2 helix may contribute to the constitutive activity of these channels at a resting pH. Together, the data highlight differences in the transmembrane domains of these closely related ion channels that may help explain some of their distinct functional properties.     

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.     

Zebrafish acid-sensing ion channel (ASIC) 4, characterization of homo- and heteromeric channels, and identification of regions important for activation by H+

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 zASIC4.1. Surface expression of zASIC4.1 was much increased by formation of heteromeric channels, suggesting that zASIC4.1 contributes to heteromeric ASICs in zebrafish neurons. Robust surface expression of H+-insensitive zASIC4.2 suggests that zASIC4.2 functions as a homomer and is gated by an as yet unknown stimulus, different from H+. Moreover, we identified a small region just distal to the first transmembrane domain that is crucial for the differential H+ response of the two paralogs. This post-TM1 domain may have a general role in gating of members of this gene family.     

The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons.

Members of the BNaC/ASIC family of ion channels have been implicated in mechanotransduction and nociception mediated by dorsal root ganglion (DRG) neurons. These ion channels are also expressed in the CNS. We identified the PDZ domain protein PICK1 as an interactor of BNaC1(ASIC2) in a yeast two-hybrid screen. We show by two-hybrid assays, glutathione S-transferase pull-down assays, and coimmunoprecipitations that the BNaC1-PICK1 interaction is specific, and that coexpression of both proteins leads to their clustering in intracellular compartments. The interaction between BNaC1 and PICK1 requires the PDZ domain of PICK1 and the last four amino acids of BNaC1. BNaC1 is similar to two other BNaC/ASIC family members, BNaC2 (ASIC1) and ASIC4, at its extreme C terminus, and we show that PICK1 also interacts with BNaC2. We found that PICK1, like BNaC1 and BNaC2, is expressed by DRG neurons and, like the BNaC1alpha isoform, is present at their peripheral mechanosensory endings. Both PICK1 and BNaC1alpha are also coexpressed by some pyramidal neurons of the cortex, by pyramidal neurons of the CA3 region of hippocampus, and by cerebellar Purkinje neurons, localizing to their dendrites and cell bodies. Therefore, PICK1 interacts with BNaC/ASIC channels and may regulate their subcellular distribution or function in both peripheral and central neurons.