Identification of a unique Ca2+-binding site in rat acid-sensing ion channel 3.
ABSTRACT: Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium (Ca2+) as an allosteric modulator and channel blocker. The channel-blocking activity is most apparent in ASIC3, as removing Ca2+ results in channel opening, with the site's location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca2+ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca2+ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-affinity Ca2+ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.
Project description:The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at -60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.
Project description:BACKGROUND AND PURPOSE:There is a major unmet need to develop new therapies for migraine. We have previously demonstrated the therapeutic potential of the acid-sensing ion channel (ASIC) blockade in migraine, via an ASIC1 mechanism. ASIC3 is expressed in the trigeminal ganglion and its response is potentiated by NO that can trigger migraine attacks in patients. Thus we sought to explore the potential therapeutic effect of ASIC3 blockade in migraine. EXPERIMENTAL APPROACH:To investigate this, we utilised validated electrophysiological and behavioural rodent preclinical models. In rats, ASIC3 blockade using APETx2 (50 or 100 ?g·kg-1 , i.v.) was measured by using durovascular and NO-evoked trigeminal nociceptive responses along with cortical spreading depression models. In mice, we sought to determine if periorbital mechanical sensitivity, induced by acute nitroglycerin (10 mg·kg-1 , i.p.), was attenuated by APETx2 (230 ?g·kg-1 , i.p.), as well as latent sensitisation induced by bright light stress in a chronic nitroglycerin model. KEY RESULTS:Here, we show that the ASIC3 blocker APETx2 inhibits durovascular-evoked and NO-induced sensitisation of trigeminal nociceptive responses in rats. In agreement, acute and chronic periorbital mechanosensitivity induced in mice by nitroglycerin and subsequent bright light stress-evoked latent sensitivity as a model of chronic migraine are all reversed by APETx2. CONCLUSION AND IMPLICATIONS:These results support the development of specific ASIC3 or combined ASIC1/3 blockers for migraine-related pain and point to a potential role for ASIC-dependent NO-mediated attack triggering. This has key implications for migraine, given the major unmet need for novel therapeutic targets.
Project description:Acid-sensing ion channels (ASICs) have been implicated in esophageal acid sensing and mechanotransduction. However, insufficient knowledge of ASIC subunit expression profile in esophageal afferent nerves hampers the understanding of their role. This knowledge is essential because ASIC subunits form heteromultimeric channels with distinct functional properties. We hypothesized that the esophageal putative nociceptive C-fiber nerves (transient receptor potential vanilloid 1, TRPV1-positive) express multiple ASIC subunits and that the ASIC expression profile differs between the nodose TRPV1-positive subtype developmentally derived from placodes and the jugular TRPV1-positive subtype derived from neural crest. We performed single cell RT-PCR on the vagal afferent neurons retrogradely labeled from the esophagus. In the guinea pig, nearly all (90%-95%) nodose and jugular esophageal TRPV1-positive neurons expressed ASICs, most often in a combination (65-75%). ASIC1, ASIC2, and ASIC3 were expressed in 65-75%, 55-70%, and 70%, respectively, of both nodose and jugular TRPV1-positive neurons. The ASIC1 splice variants ASIC1a and ASIC1b and the ASIC2 splice variant ASIC2b were similarly expressed in both nodose and jugular TRPV1-positive neurons. However, ASIC2a was found exclusively in the nodose neurons. In contrast to guinea pig, ASIC3 was almost absent from the mouse vagal esophageal TRPV1-positive neurons. However, ASIC3 was similarly expressed in the nonnociceptive TRPV1-negative (tension mechanoreceptors) neurons in both species. We conclude that the majority of esophageal vagal nociceptive neurons express multiple ASIC subunits. The placode-derived nodose neurons selectively express ASIC2a, known to substantially reduce acid sensitivity of ASIC heteromultimers. ASIC3 is expressed in the guinea pig but not in the mouse vagal esophageal TRPV1-positive neurons, indicating species differences in ASIC expression.
Project description:Injury and inflammation cause tissue acidosis, which is a common feature of various painful conditions. Acid-Sensing Ion channels (ASICs) are amongst the main excitatory channels activated by extracellular protons and expressed in the nervous system. Six transcripts of ASIC subunits including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4 are encoded by four genes (Asic1-4) and have been identified in rodents. Most ASIC subunits are present at substantial levels in primary sensory neurons of dorsal root ganglia (DRG) except for ASIC4. However, their expression pattern in DRG neurons remains largely unclear, mainly due to the lack of antibodies with appropriate specificity. In this study, we examined in detail the expression pattern of ASIC1-3 subunits, including splice variants, in different populations of DRG neurons in adult mice using an in situ hybridization technique (RNAscope) with high sensitivity and specificity. We found that in naïve condition, all five subunits examined were expressed in the majority of myelinated, NF200-immunoreactive, DRG neurons (NF200+). However, ASIC subunits showed a very different expression pattern among non-myelinated DRG neuronal subpopulations: ASIC1 and ASIC3 were only expressed in CGRP-immunoreactive neurons (CGRP+), ASIC2a was mostly expressed in the majority of IB4-binding neurons (IB4+), while ASIC2b was expressed in almost all non-myelinated DRG neurons. We also found that at least half of sensory neurons expressed multiple types of ASIC subunits, indicating prevalence of heteromeric channels. In mice with peripheral nerve injury, the expression level of ASIC1a and ASIC1b in L4 DRG and ASIC3 in L5 DRG were altered in CGRP+ neurons, but not in IB4+ neurons. Furthermore, the pattern of change varied among DRGs depending on their segmental level, which pointed to differential regulatory mechanisms between afferent types and anatomical location. The distinct expression pattern of ASIC transcripts in naïve condition, and the differential regulation of ASIC subunits after peripheral nerve injury, suggest that ASIC subunits are involved in separate sensory modalities.
Project description:Acid-sensitive ion channels belonging to the degenerin/epithelial sodium channel (DEG/ENaC) family activate in response to extracellular protons and are considered unique to deuterostomes. However, sensitivity to pH/protons is more widespread, where, for example, human ENaC Na+ leak channels are potentiated and mouse BASIC and Caenorhabditis elegans ACD-1 Na+ leak channels are blocked by extracellular protons. For many DEG/ENaC channels, extracellular Ca2+ ions modulate gating, and in some cases, the binding of protons and Ca2+ is interdependent. Here, we functionally characterize a DEG/ENaC channel from the early-diverging animal Trichoplax adhaerens, TadNaC6, that conducts Na+-selective leak currents in vitro sensitive to blockade by both extracellular protons and Ca2+ We determine that proton block is enhanced in low external Ca2+ concentration, whereas calcium block is enhanced in low external proton concentration, indicative of competitive binding of these two ligands to extracellular sites of the channel protein. TadNaC6 lacks most determinant residues for proton and Ca2+ sensitivity in other DEG/ENaC channels, and a mutation of one conserved residue (S353A) associated with Ca2+ block in rodent BASIC channels instead affected proton sensitivity, all indicative of independent evolution of H+ and Ca2+ sensitivity. Strikingly, TadNaC6 was potently activated by the general DEG/ENaC channel blocker amiloride, a rare feature only reported for the acid-activated channel ASIC3. The sequence and structural divergence of TadNaC6, coupled with its noncanonical functional features, provide unique opportunities for probing the proton, Ca2+, and amiloride regulation of DEG/ENaC channels and insight into the possible core-gating features of ancestral ion channels.
Project description:Extracellular acid can have important effects on cancer cells. Acid-sensing ion channels (ASICs), which emerged as key receptors for extracellular acidic pH, are differently expressed during various diseases and have been implicated in underlying pathogenesis. This study reports that ASIC1 and ASIC3 are mainly expressed on membrane of pancreatic cancer cells and upregulated in pancreatic cancer tissues. ASIC1 and ASIC3 are responsible for an acidity-induced inward current, which is required for elevation of intracellular Ca2+ concentration ([Ca2+]i). Inhibition of ASIC1 and ASIC3 with siRNA or pharmacological inhibitor significantly decreased [Ca2+]i and its downstream RhoA during acidity and, thus, suppressed acidity-induced epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. Meanwhile, downregulating [Ca2+]i with calcium chelating agent BAPTA-AM or knockdown of RhoA with siRNA also significantly repressed acidity-induced EMT of pancreatic cancer cells. Significantly, although without obvious effect on proliferation, knockdown of ASIC1 and ASIC3 in pancreatic cancer cells significantly suppresses liver and lung metastasis in xenograft model. In addition, ASIC1 and ASIC3 are positively correlated with expression of mesenchymal marker vimentin, but inversely correlated with epithelial marker E-cadherin in pancreatic cancer cells. In conclusion, this study indicates that ASICs are master regulator of acidity-induced EMT. In addition, the data demonstrate a functional link between ASICs and [Ca2+]i/RhoA pathway, which contributes to the acidity-induced EMT.
Project description:Stomatin (STOM) is a monotopic integral membrane protein found in all classes of life that has been shown to regulate members of the acid-sensing ion channel (ASIC) family. However, the mechanism by which STOM alters ASIC function is not known. Using chimeric channels, we combined patch-clamp electrophysiology and FRET to search for regions of ASIC3 critical for binding to and regulation by STOM. With this approach, we found that regulation requires two distinct sites on ASIC3: the distal C-terminus and the first transmembrane domain (TM1). The C-terminal site is critical for formation of the STOM-ASIC3 complex, while TM1 is required only for the regulatory effect. We then looked at the mechanism of STOM-dependent regulation of ASIC3 and found that STOM does not alter surface expression of ASIC3 or shift the pH dependence of channel activation. However, a point mutation (Q269G) that prevents channel desensitization also prevents STOM regulation, suggesting that STOM may alter ASIC3 currents by stabilizing the desensitized state of the channel. Based on these findings, we propose a model whereby STOM is anchored to the channel via a site on the distal C-terminus and stabilizes the desensitized state of the channel via an interaction with TM1.
Project description:Acid-sensing ion channels (ASIC) are voltage-insensitive, cationic channels that have recently been identified in vascular smooth muscle (VSM). It is possible that ASIC contribute to vascular reactivity via Na(+) and Ca(2+) conductance; however, their function in VSM is largely unknown. In pulmonary VSM, store-operated Ca(2+) entry (SOCE) plays a significant role in vasoregulatory mechanisms such as hypoxic pulmonary vasoconstriction and receptor-mediated arterial constriction. Therefore, we hypothesized that ASIC contribute to SOCE in pulmonary VSM. We examined SOCE resulting from depletion of intracellular Ca(2+) stores with cyclopiazonic acid in isolated small pulmonary arteries and primary cultured pulmonary arterial smooth muscle cells by measuring 1) changes in VSM [Ca(2+)](i) using fura-2 indicator dye, 2) Mn(2+) quenching of fura-2 fluorescence, and 3) store-operated Ca(2+) and Na(+) currents using conventional whole cell patch-clamp configuration in voltage-clamp mode. The role of ASIC was assessed by the use of the ASIC inhibitors, amiloride, benzamil, and psalmotoxin 1, or siRNA directed towards ASIC1, ASIC2, or ASIC3 isoforms. We found that store-operated VSM [Ca(2+)](i) responses, Mn(2+) influx, and inward cationic currents were attenuated by either pharmacological ASIC inhibition or treatment with ASIC1 siRNA. These data establish a unique role for ASIC1 in mediating SOCE in pulmonary VSM and provide new insight into mechanisms of VSM Ca(2+) entry and pulmonary vasoregulation.
Project description:Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (?fast) that represented voltage-dependent mode, and a slow component (?slow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the ?fast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the ?slow, and produced a 2-3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1.
Project description:Acid-sensing ion channels (ASICs), which belong to the epithelial sodium channel/degenerin family, are activated by extracellular protons and are inhibited by amiloride (AMI), an important pharmacological tool for studying all known members of epithelial sodium channel/degenerin. In this study, we reported that AMI paradoxically opened homomeric ASIC3 and heteromeric ASIC3 plus ASIC1b channels at neutral pH and synergistically enhanced channel activation induced by mild acidosis (pH 7.2 to 6.8). The characteristic profile of AMI stimulation of ASIC3 channels was reminiscent of the channel activation by the newly identified nonproton ligand, 2-guanidine-4-methylquinazoline. Using site-directed mutagenesis, we showed that ASIC3 activation by AMI, but not its inhibitory effect, was dependent on the integrity of the nonproton ligand sensing domain in ASIC3 channels. Moreover, the structure-activity relationship study demonstrated the differential requirement of the 5-amino group in AMI for the stimulation or inhibition effect, strengthening the different interactions within ASIC3 channels that confer the paradoxical actions of AMI. Furthermore, using covalent modification analyses, we provided strong evidence supporting the nonproton ligand sensing domain is required for the stimulation of ASIC3 channels by AMI. Finally, we showed that AMI causes pain-related behaviors in an ASIC3-dependent manner. These data reinforce the idea that ASICs can sense nonproton ligands in addition to protons. The results also indicate caution in the use of AMI for studying ASIC physiology and in the development of AMI-derived ASIC inhibitors for treating pain syndromes.