The nonproton ligand of acid-sensing ion channel 3 activates mollusk-specific FaNaC channels via a mechanism independent of the native FMRFamide peptide

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide–activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.

family (21); FaNaC has also been deemed to be more closely related to ENaC (Fig. 1A) (15,22). Different DEG/ENaC subfamilies have been known to use different activation mechanisms (1,2,23). For example, ENaCs open spontaneously, DEGs respond upon mechanical stimulation, ASICs are sensitive to extracellular acidosis, whereas FaNaCs are directly activated by native FMRFamide peptide (1,2,23). Recently, there have been multiple breakthroughs in identifying new activation mechanisms of some DEG/ENaCs, for instance, the activation of ENaC by a synthetic compound S3969 (24) or shear stress (25), ASIC3 by a synthetic small molecule GMQ (26,27), and ASIC1a by snake venom MitTx (28). Thus, it is reasonable to speculate that DEG/ENaCs may be activated or modulated by some endogenous agents via allosteric mechanisms similar to that used by the exogenous activators. Moreover, understanding the dual or polymodal mode of DEG/ENaC activation is pivotal for elucidating the functional significance of these channels. In the case of ASICs, although the channels are best known to sense extracellular acidosis (29), some of the physiological functions implicated to them cannot be fully explained by their responses to low pH (27,30). The recent identification of nonproton ligands/toxins should help advance our understanding of ASIC-mediated functions (27,28,31,32) and encourage further exploration of endogenous nonproton ligands for these channels (32)(33)(34). The identification of the synthetic activator of ENaC, S3969, also brings new opportunities for therapeutic development, such as treatment of false aldosterone deficiency (24). Therefore, more in-depth studies into polymodal gating mechanisms of the DEG/ENaC channels have important implications in both basic research and disease intervention.
Here, we demonstrated that GMQ, a previously described nonproton ligand of ASIC3, can activate all four FaNaC orthologues via a mechanism distinct from the native FMRFamide peptide (35), but similar to the one used by GMQ to activate ASIC3 (27). Our data not only indicate a closer evolutionary relationship between ASIC and FaNaC subfamilies, despite the low sequence homology (12-18%), but also a conservation of the "GMQ-activation mode" that was already present in the common ancestor of these two subfamilies. In addition, two distinct activation modes of FaNaCs also reflect that the dual gating may already exist in the ancestor of DEG/ENaC superfamily of ion channels.

GMQ, the nonproton ligand of ASIC3, directly activates FaNaC channels
Previously, we identified GMQ as the first nonproton ligand of ASICs ( Fig. 2A), which selectively activates ASIC3 but not ASIC1a, -1b, and -2a (27). Others, however, argued that the GMQ worked by altering pH dependence and steady-state inactivation of ASIC1a, -1b, -2a, and -3 differentially, and such shifts only allowed ASIC3 activation at the physiological pH 7.4 (36). To look for further evidence that GMQ acts as a direct agonist rather a modulator, we examined its ability to activate other DEG/ENaC subfamily members not known to be activated by extracellular protons. We tested FaNaCs because their native agonist, FMRFamide, has been shown to augment the sustained currents of ASIC3 and ASIC1a (37). Interestingly, GMQ directly activated all four FaNaC orthologues expressed in CHO cells (Fig. 2B), and there is no GMQ current observed in cells without FaNaC expression (Fig. 2B). GMQ-evoked currents in all FaNaCs are sensitive to amiloride (Fig. 2B), which is a common feature of DEG/ENaC superfamily of ion channels (1). GMQ also evoked amiloride-sensitive currents in Xenopus oocytes that were injected with HaFaNaC cRNA (see below), suggesting that the stimulatory effect of GMQ is independent of the host cell types. A, phylogenetic tree generated from genes of ASIC, HyNaC, BASIC, ENaC, FaNaC, DEG, and PPK/RPK subfamilies. The genes of ASIC and FaNaC subfamilies are highlighted with red and purple. B, multiple sequence alignment, made by Clastal W with manual adjustments, from two ASICs and four FaNaCs. The conserved sequence and the key binding regions are indicated with different colors. Purple, conservative residues; cyan, key residues essential for GMQ's action in rASIC3 (Glu-79 (E79) and Glu-423 (E423)); yellow, FaNaC regions identical to key site of GMQ in rASIC3; green, residues essential for FMRFamide-mediated HaFaNaC activation; blue and orange, FaNaC-specific insertions I and II in comparison with other members of DEG/ENaCs.

Nonproton ligand of ASIC3 activates FaNaCs
ASIC3 can be directly activated by deprivation of extracellular Ca 2ϩ (Ca 2ϩ o ) ( Fig. 2A) (27,38,39), acidosis (lower pH than 7.2), or alkalization (pH 8.0) (34). However, neither Ca 2ϩ o deprivation (Fig. 2C), nor high (pH 9.0)/low (pH 5.0) pH (Fig. 2, D-F) activated those four FaNaC orthologues. A small endogenous response to pH 5.0 was observed in all cells (Fig. 2E), and it was not inhibited by amiloride (Fig. 2, E and F). Thus, it is unlikely that GMQ acted on FaNaCs through modulation of any intrinsic activation or inhibition of this DEG/ENaC subfamily by pH or Ca 2ϩ o . Furthermore, GMQ evoked amiloridesensitive currents in Xenopus oocytes with HaFaNaC cRNA injection (Fig. 3, A and B), but caused only a slight inhibition of the spontaneous activity of human ENaC ␣␤␥ expressed (Fig.  3C). Thus, the agonistic effects of GMQ on FaNaCs and ASIC3 are specific, rather than universal, among the DEG/ENaC family of ion channels, which could imply a close evolutionary relationship between FaNaCs and ASICs.

Nonproton ligand of ASIC3 activates FaNaCs
induced by low pH (5.57) (27). Similarly, the pNa ϩ /pK ϩ value of GMQ-evoked HaFaNaC current (6.6) was also markedly lower than that evoked by FMRFamide (9.45) (Fig. 6, A-C). Moreover, neither FMRFamide nor GMQ elicited Cs ϩ or Ca 2ϩ conductance through HaFaNaC (Fig. 6, D and E), similar to GMQ's action on rASIC3 (27). Most importantly, key residues located within the palm domain of HaFaNaC mediated the action of GMQ (see below), further demonstrating that GMQ uses a similar mechanism to activate both ASIC3 (27) and FaNaC channels. The fact that FaNaCs are only identified in invertebrates suggests that this activation mode may evolve earlier before the mammalian subfamilies of DEG/ENaCs.

Differences existed between GMQ-and peptide-mediated FaNaC activations
The above data revealed that the FaNaC currents activated by GMQ exhibited differences in sensitivity to Ca 2ϩ o and selectivity for Na ϩ versus K ϩ as compared with that evoked by FMR-Famide ( To further characterize the GMQ-evoked activation of FaNaC channels, we performed single channel recordings in an outside-out configuration from CHO cells transiently transfected with HaFaNaC. As a control, FMRFamide (0.1-10 M) evoked single channel activities with the main conductance generating ϳ0.9 -1.1 pA of currents at Ϫ80 mV ( Fig. 7, A-D) was similar to previous reports (16,40,41), and there was no single channel activity observed in cells without FaNaC expression (Fig. 7I). Interestingly, the unitary currents of FaNaC induced by 1 mM GMQ were very flickery and only reached ϳ0.4 -0.5 pA at Ϫ80 mV (Fig. 7, E and F). The channels were mostly closed or existed in subconductance states (Fig. 7F). At a higher concentration of GMQ, more fully opened channels were observed (Fig. 7, G and H), but the unitary chord conductance remained to be lower (ϳ4 picosiemens at Ϫ80 mV) than that evoked by FMRFamide (ϳ10 -12 picosiemens), indicating distinct channel gating mechanisms between GMQ and FMRFamide.

FMRFamide and GMQ target distinct regions of FaNaC for channel activation
Given that the key residues essential for the low pH-induced activation of ASIC3 are not involved in the GMQ-mediated channel gating (26,27), it is possible that GMQ and FMRFamide also act on different sites of HaFaNaC. Coapplication of unsaturated GMQ (3 mM) and FMRFamide (1 M) to cells

Nonproton ligand of ASIC3 activates FaNaCs
expressing HaFaNaC (pH 7.4, 2 mM Ca 2ϩ o ) caused a larger current than the sum of the responses from individual applications of GMQ and FMRFamide (Fig. 8, A and B), indicating a likelihood of synergy between the two agonists. For the mutation HaFaNaC ⌬Gln-445-Ala-493 (with an ϳ50-residue truncation at the specific insertion II of the knuckle domain) (Figs. 1B and 8C), its apparent affinity (ϳ80-fold decrease), and the maximal currents of FMRFamide decreased profoundly (35). In contrast, the EC 50 of GMQ and the maximal currents were not changed by a truncation of Gln-445-Ala-493 (Fig. 8D). Meanwhile, mutations at the residues of other three domains, including I160A, D154A, Y131Q, W359A, and E489A, which significantly reduced (ϳ10-to 40-folds) the apparent affinity of FMRFamide (35) showed little effect on the dose-response curves of GMQ on HaFaNaC (EC 50 ϭ 0.8 Ϯ 0.04, 0.78 Ϯ 0.05, 0.81 Ϯ 0.03, 0.92 Ϯ 0.02, 0.93 Ϯ 0.03, 0.82 Ϯ 0.04 mM, for HaFaNaC WT, I160A, D154A, Y131Q, W359A and E489A, respectively, Ca 2ϩfree/pH 7.4) (Fig. 8E). In addition, a covalent modification of I160C decreased the apparent affinity for at least 700-fold and significantly reduced the maximal current of FMRFamide in HaFaNaC (EC 50 ϭ 7.33 Ϯ 0.82 M versus over 5 mM, before versus after the DTNB treatment of HaFaNaC I160C , respectively), and DTT treatment can reverse those effects (35). However, unlike the FMRFamide-evoked activation, DTNB treatment of cells that expressed HaFaNaC I160C or HaFaNaC WT , only a slight potentiation or no apparent difference was detected between the GMQ-induced currents before and after DTNB modifications (Fig. 8, F and G). Collectively, these results demonstrate that the channel regions and critical residues targeted by GMQ and FMRFamide are distinct.
Moreover, although a low concentration of GMQ (100 M) failed to evoke any current in cells that expressed HaFaNaC (Fig. 9A), it significantly potentiated the activation by an unsaturated concentration of FMRFamide (7 M for 2 mM Ca 2ϩ o , 0.1 M for Ca 2ϩ -free) (Fig. 9, A and C), and left-shifted the doseresponse curves of FMRFamide both in the absence (5 mM EGTA) and the presence of Ca 2ϩ o (2 mM) (Fig. 9E). Similar potentiation was produced by a low concentration of FMRF-  o -free) (Fig. 9, G and I), implying that the two agonists opened a common channel pore or ion permeation pathway, despite the key sites for their actions being different.

Residues in the palm domain serve opposite roles in the FaNaC activations by FMRFamide and GMQ
To further understand the mechanism by which GMQ activates the FaNaC subfamily channels, we searched for the channel regions and key amino acid residues involved in the GMQ-mediated HaFaNaC activation. Previously, we demonstrated that residues located at the ␤1and ␤12-sheets of the palm domain, namely Glu-79 and Glu-423, are essential for the GMQ-evoked activation of ASIC3 (Figs. 1B and 10A, shown in cyan) (27), which prompts us to perform mutations on the corresponding residues in HaFaNaC. However, because of the low sequence homology between the HaFaNaC and rASIC3 at this region (Figs. 1B and 10A, shown in yellow), we replaced three to four amino acids of ␤1and ␤12sheets (Fig. 10, A and B) with alanine simultaneously to ensure that the residues corresponding to Glu-79 or Glu-423 in ASIC3 were mutated, yielding two mutants HaFaNaC 99 -102ELSE/AAAA and HaFaNaC 517-519VEY/AAA . Similar to GMQ's action on rASIC3, these two mutations 5 nearly fully abolished GMQ-induced direct activations (Fig. 10C) and allosteric potentiation of HaFaNaC (Fig. 9J), indicating that residues located in the palm domain are also essential for GMQ-mediated HaFaNaC stimulus. Surprisingly, those two mutants led a profound increase in the apparent affinity of FMRFamide in HaFaNaC both in 2 mM Ca 2ϩ o (EC 50 ϭ 7.33 Ϯ 0.82, 0.09 Ϯ 0.01, and 0.78 Ϯ 0.03 M for WT, 99 -102ELSE/AAAA, and 517-519VEY/AAA, respectively) (Fig. 10, D and E) and Ca 2ϩ -free solutions (EC 50 ϭ 110 Ϯ 15, 2.03 Ϯ 0.11, and 21.5 Ϯ 1.97 nM for WT, 99 -102ELSE/ AAAA, and 517-519VEY/AAA, respectively) (Fig. 10, F and G), indicating that the site located in the palm domain of HaFaNaC may be an allosteric site of FMRFamide both in the presence and absence of extracellular Ca 2ϩ , further supporting the idea that GMQ and FMRFamide use distinct mechanisms to gate FaNaC channels.
Echoing the findings from the compound mutations, substitutions of the residues in the ␤1and ␤12-sheets profoundly decreased the EC 50 of FMRFamide for HaFaNaC (EC 50 ϭ 7.33 Ϯ 0.82, 5.01 Ϯ 1.01, 0.2 Ϯ 0.02, 0.52 Ϯ 0.04, 0.14 Ϯ 0.03, 5 Throughout this manuscript, the following designations were used: ELSE/ AAAA, E99A/L100A/S101A/E102A; VEY/AAA, V517A/E518A/Y519A.     (Fig. 11, B and C), which is similar to effects of ASIC3 E79A and ASIC3 E423A on the apparent affinity of GMQ in rASIC3 (27). Because of the extreme difficulty in obtaining the crystal structure of FaNaC-GMQ complex, we cannot conclude that GMQ activates FaNaC by directly interacting with residues in the ␤1and ␤12-sheets. Likewise, it has not been firmly established that GMQ activates ASIC3 by binding to Glu-79 and Glu-423 at the palm domain either, although covalently linking GMQ or TNB to Cys-79 of ASIC3 E79C was sufficient to activate the channel in the absence of acidosis (27). Even so, based on the different ion selectivity, unitary conductance, and the distinct key sites determining their actions, it is reasonable to conclude that GMQ activates FaNaC via a mechanism distinct to that of native FMRFamide peptide.

Discussion
This study demonstrated here that the "primitive" member of DEG/ENaC, FaNaCs, including all four orthologues AkFaNaC, LsFaNaC, HaFaNaC, and HtFaNaC, could all be

Nonproton ligand of ASIC3 activates FaNaCs
directly activated by GMQ (Figs. 2B and 11D), the nonproton ligand of mammalian ASICs. GMQ was the first nonproton ligand ever identified for mammalian ASICs and shown to activate ASIC3 in the absence of acidosis (27). A recent report, however, suggested that GMQ might not directly activate ASIC3, but instead work by altering the pH dependence and steady-state inactivation of ASICs in an isoform-specific manner so that the pH dependence of ASIC3 was shifted to a higher pH range (36). Here, because FaNaC cannot be activated by low pH, high pH or Ca 2ϩ o deprivation (Fig. 2, C-F), the activation of FaNaC by GMQ in the absence of FMRFamide cannot be attributed to a GMQ-induced change in pH or Ca 2ϩ o sensitivity. Thus, we believe that the GMQ, may act as an activator of FaNaCs, rather than a modulator. These results also suggested that GMQ may directly activate ASIC3 channels rather than change its pH sensitivity. This point is supported by the following evidence. First, the residues essential for the low pH-induced channel gating of rASIC3 are not essential for GMQ's action on ASIC3 (27), indicating that distinct channel regions are involved in ASIC3 gating by GMQ and protons. Second, GMQ-evoked inward currents are sustained whereas low-pHinduced currents are transient. If GMQ's activation is merely attributed to shifting the pH sensitivity of ASIC3, the currents should also be transient. Furthermore, it has been demonstrated that the activation of ASIC3 by pH 6.9 and pH 6.0 produced currents with different desensitization rates (2,42). If GMQ were to affect the pH sensitivity of ASIC3, the currents evoked by different concentrations of GMQ should also exhibit distinct desensitization rates. However, both low and high concentrations of GMQ evoke persistent currents (27). Third, the sites of action for GMQ in HaFaNaC and ASIC3 are both located within the lower palm domain, along with the conformational transition pathway of DEG/ENaCs (43)(44)(45). Although the agonistic function of GMQ does not exclude the capability of this compound to potentiate proton-induced activation of ASIC3, just like that GMQ also potentiated the FMRFamideevoked currents of HaFaNaC (Fig. 9), these potentiation effects, occurring normally between agonists that act on separate pathways, should not be taken as evidence that GMQ acted just as a modulator rather than a direct agonist on the channel. As we have demonstrated here, GMQ and FMRFamide are both direct agonists (Figs. 2 and 9) of the FaNaC subfamily and modulators to each other's actions.
Additionally, according to the evolutionary tree of DEG/ ENaC superfamily of ion channels, HyNaC is closely related with ASIC (Fig. 1A) (21), and FaNaC to ENaC (15,22). Unexpectedly, GMQ could activate FaNaC, but not ENaC (Fig. 3C), suggesting a closer relationship between FaNaC and ASICs. The fact that FMRFamide can interact with both ASICs and FaNaC further supports this hypothesis (16,37,46), although FMRFamide only regulates, rather than directly activates ASIC3 (37). As a newly discovered nonproton ligand of ASIC3 (27), GMQ is able to activate DRG neurons and induce pain sensation, indicating that nonproton ligand-mediated activation may be an activation mode with physiological functions (27). Some endogenous ligands of ASIC3 have been identified, such as agmatine, although its potency is weak (27, 33). Recently, lipids have also been demonstrated to activate ASIC3 in neutral environments (32). Therefore, it is possible that there are potent endogenous nonproton ligands of ASICs awaiting identification. FaNaCs are expressed only in invertebrates whereas ASICs are mainly found in mammalian (1,46). These two kinds of subfamilies are distant in the evolution although they share common agonists. Therefore, this activation mode of GMQ maybe already exist in the common ancestor of those two subfamilies of ion channels, rather than appear during the later evolution. However, we could not exclude the possibility that other members of DEG/ENaC belonging to the superfamily can also be activated by other small molecules. For instance, the discovery of the small agonist of ENaC, namely S3969, demonstrates that besides spontaneous activation, ENaC could also be activated by small molecules (24). Furthermore, the key site of ENaC activation for S3969 is also located in the palm domain (24), which is similar to GMQ-induced activation of ASIC3 and FaNaC, suggesting that palm domain is possibly the key site of DEG/ENaC superfamily of ion channels activated by small molecules. However, whether GMQ's actions on FaNaC or ASIC3 are through a direct interaction with the residues of the palm domain or not still require extensive experiments and additional structural determinants of GMQ-ASIC3 and GMQ-FaNaCs, and a similar situation for S3969-mediated ENaC activation. Nevertheless, our observations together with previous reports (23,24) suggest the presence of a distinct gating mode in some members of DEG/ENaC besides activation modes identified previously, for example, the FMRFamide-mediated FaNaC gating, low-pH-evoked ASIC gating, and the spontaneous opening of ENaCs. Some of these gating modes might have disappeared or become unused during evolution of DEG/ ENaCs, like the direct activation by FMRFamide peptides, which is only found in FaNaCs, or they have not been experimentally identified. Others, such as the gating possibly through interaction with the palm domain residues, via the binding of small molecules such as GMQ in the case of ASIC3 (26,27) and FaNaC, and S3969 in the case of ENaC (24) are still preserved (Fig. 11D), indicating that this novel gating mode uncovered by GMQ and S3969 may be essential for the functions of mammalian DEG/ENaC channels.
Additionally, our study unveils a dual gating of FaNaC, induced by FMRFamide and GMQ, (Fig. 11D). Polymodal activation modes exist in other members of DEG/ENaC superfamily. For ENaC, not only the channel is spontaneously open (23), but also it can be activated by proteases (47,48), small molecules, e.g. S3969 stimulation (24), or shearing stress stimulations (25). For ASICs, the activation can be achieved by low pH (29), GMQ (27,31), toxins (28), lipid (32), alkali stimulation (34), and mechanical force (49,50). Therefore, we speculate that DEG/ENaC superfamily is another type of ion channels, similar to transient receptor potential (TRP) channels (51) and K2P channels (52), responding to multiple stimulations. Although it remains to be determined whether the palm domain and the thumb-finger interface located in the extracellular loop represents the only or at least the major areas of gating in the DEG/ENaC superfamily, it is quite clear from the studies on ASIC3 and FaNaCs that these two domains represent distinct gating modes that result in different open states. The polymodal activation of FaNaC indicates that this mode, at Nonproton ligand of ASIC3 activates FaNaCs least dual gating, is a characteristic of DEG/ENaC superfamily of ion channels existed in remote antiquity before the evolution of this superfamily. It should be interesting to explore further these two distinct gating modes among different members of the DEG/ENaC superfamily and with the use of different activation methods, as well as the interplay between them, which will shed more light on the mechanisms of regulation of these channels and help identify new strategies to perturb their function for clinical therapies.

Whole-cell and single-channel recordings
As we described previously (27,35), whole-cell recordings were carried out with CHO cells 24 -48 h post transfection. Patch pipettes (3-5 megaohms) were filled with internal solution, which contains (in mM): 30 NaCl, 120 KCl, 1 MgCl 2 , 10 HEPES, and 5 EGTA, pH 7.4. The standard external solution (SS) contains (in mM): 150 NaCl, 10 glucose, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , and 10 HEPES (pH 6.0 -7.4). HEPES was replaced by equimolar MES when pH Ͻ6.0. CaCl 2 and MgCl 2 were substituted with 5 mM EGTA in the Ca 2ϩ -free solution. During electrophysiological recordings, 80 -90% of the series resistance was compensated. All currents were measured at a holding potential of Ϫ60 mV by Axon Axopatch 200B connected to a DIGIDATA 1440A A/D converter, and analyzed using Clampex 10.2. Single-channel recordings using outside-out configuration were carried out in CHO cells at room temperature (23 Ϯ 2°C) 24 -48 h after transfection. Recording pipettes were pulled for borosilicate glass (World Precision Instruments, Inc.) and fired polished to yield resistance of 13-18 megaohms. The holding potential was set at Ϫ80 mV. The external solution and internal solutions are the same as those of whole-cell recordings. Currents were sampled at 50 kHz with a 2-kHz filter, and a low-pass filtered at 300 Hz, using an AxonPatch 200B ampli-fier in conjunction with pClamp 10 software (Molecular Device).

Calcium imaging
CHO cells expressing HaFaNaC seeded on glass coverslips were incubated with Fluo-4-AM (1 M, Molecular Probes) and Pluronic F127 (0.02% (w/v), Sigma) for 30 min at room temperature (23 Ϯ 2°C) in the dark. After washing the cells three times in standard external solution, the coverslip was mounted to a recording chamber placed on the stage of a Nikon N-SIM microscope. Fluorescent intensities in response to 488 nm laser excitation were monitored and recorded using NIS-Elements Soft in the dark. FMRFamide (100 M) or GMQ (5 mM) were added when desired in the presence of 5 M nimodipine (Aladdin) to block voltage-gated calcium channels. Changes in intracellular calcium concentrations were expressed as F t /F 0 , a ratio of fluorescent intensities at time (t) and time zero (0).

Homology modeling
Homology model of HaFaNaC was created based on the cASIC1 structure (PDB ID: 4NYK) as we described previously (35). The alignment of HaFaNaC (GenBank TM ID: CAA63084) and cASIC1 (GenBank TM ID: AAY28986) were made by Modeler 9.9 (53) and manually adjusted by published alignments (54). The obtained model was checked and validated by Pro-Check (55).

Data analysis
Data were expressed as the mean Ϯ S.E.; statistical comparisons were made using Student's t test (*, p Ͻ 0.05; **, p Ͻ 0.001).

Nonproton ligand of ASIC3 activates FaNaCs
GMQ, I max is the maximum current, k is the slope factor. As we described previously (27,56), the permeability ratios of P Na /P Li and P Na /P K were calculated using the modified Goldman-Hodgkin-Katz equation: P X /P Na ϭ exp (⌬V rev F/RT) because of the equimolar cations in the external and internal solution, where X represents the test cations, ⌬V rev is the change in reversal potential when Na ϩ was replaced by the tested cation, F is the Faraday constant, R is the gas constant, and T is the absolute temperature.