Nonproton ligand sensing domain is required for paradoxical stimulation of acid-sensing ion channel 3 (ASIC3) channels by amiloride

Abstract

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.

FIGURE 1.

Effects of AMI on ASIC3 channels. A, chemical structure of AMI and AMI-activated ASIC3 currents. AMI dose-dependently evoked inward currents in ASIC3-transfected (right panel) but not in nontransfected CHO cells (data not shown). Although the acid (pH 5.0)-induced current desensitized rapidly (left panel), little desensitization was observed for the AMI-evoked currents (above right panel). Similar results were obtained in six other experiments. Concentration-response relationship of AMI is shown below right panel. Each point is the mean ± S.E. of seven measurements normalized to the pH 5.0-induced peak currents, and the solid line was the fit to the Hill equation. The EC₅₀ and n values of AMI are 0.56 ± 0.02 mm and 1.76 ± 0.07, respectively. B, effects of AMI on the activation of ASIC3 channels by acid (pH 5.0). AMI dose-dependently inhibited the pH 5.0-induced currents (i.e. both peak and sustained components) of ASIC3 channels as shown in the representative example (upper panel). Data points are means ± S.E. of four to eight measurements normalized to the currents induced by pH 5.0 in the absence of AMI, and the solid lines were the fit to the Hill equation giving an IC₅₀ value of 7.2 ± 1.4 μm (n = 0.97 ± 0.22) for the sustained (gray) and 412.2 ± 2.0 μm (n = 0.86 ± 0.11) for the peak component (black), respectively. C, subunit selectivity of AMI action at the neutral pH. Shown is the summary of AMI (1 mm)-induced currents in CHO cells transfected with one or two different ASIC subunits. AMI activated ASIC3 homomeric channels and heteromeric ASIC3 + ASIC1b channels but not ASIC1a, ASIC1b, or ASIC2a homomeric channels, or heteromeric ASIC3 + 1a, ASIC3 + 2a, or ASIC3 + 2b channels at the neutral pH. Data are means ± S.E. n = 3–5. n.d., no detectable response to AMI. **, p < 0.001 versus base-line level.

FIGURE 2.

AMI sensitizes ASIC3 channels under mild acidosis. AMI at low micromolar concentrations (1–300 μm) sensitizes the response to mild acidosis (A, pH 7.2; B, pH 7.0; and C, pH 6.8) but not to a more extreme acidosis (D, pH 6.5). Upper panels show representative current traces at −60 mV, and lower panels are means ± S.E. of four to seven measurements normalized to the currents induced by acidosis alone (control; dashed line). *, p < 0.05; **, p < 0.001 versus controls.

FIGURE 3.

Activation of ASIC3 channels by AMI is determined by residues (Glu-79 and Glu-423) of the nonproton ligand sensing domain. A, representative current traces illustrating the effects of E79A and E423A mutations on 1 and 3 mm AMI-induced currents. B, pooled data from experiments as in A and similar results from other mutations of Glu-79 or Glu-423. Data points are means ± S.E. of four to seven measurements normalized to the pH 5.0-induced peak currents. Note: for most mutations, the maximal pH 5.0-induced currents were generated following pH 9.0 pretreatment to restore the steady-state desensitization of acid-dependent currents at pH 7.4. *, p < 0.05 versus wild-type (WT). The dashed line represents the current amplitude induced by AMI (3 mm) from WT ASIC3 channels. C, ratio of current amplitude induced by 1 mm AMI to that by 3 mm AMI. The data were generated from B and are means ± S.E. *, p < 0.05 versus WT (dashed line).

FIGURE 4.

Constant efficacy of AMI inhibition on acid (pH 5.0)-induced currents from ASIC3 mutants at Glu-79 or Glu-423. A, representative current traces showing AMI inhibition of the pH 5.0-induced currents from WT, E79A, and E423A mutant channels. B, pooled data from experiments in A. The inhibition ratio of AMI was quantitatively calculated as follows: ((1 − I_{AMI + pH 5.0}/I_{pH 5.0})·100) where I_{AMI + pH 5.0} and I_{pH 5.0} are the pH 5.0-induced currents measured in the presence or absence of AMI. Data points are means ± S.E. of four to seven measurements. There is no significant difference in the degree of inhibition by either 100 or 300 μm AMI among WT, E79A, and E423A groups. The WT data, taken from Fig. 1B, are regraphed again for comparison.

FIGURE 5.

Structure-activity relationship of AMI derivatives. A, schematic demonstration for structure-activity relationship of AMI. B and C, representative current traces showing the channel activation under neutral pH (B) or inhibition of pH 5.0-activated currents (C) from ASIC3 expressing CHO cells induced by compounds as indicated. Every compound was used at the concentration of 1 mm. Different colored traces represent the currents induced in the presence of different compounds, in which black is for AMI; red is for methyl-3,5-diamino-6-chloropyrazine-2-carboxylate; and blue is for 5-(N-methyl-N-isobutyl)amiloride, respectively. The stimulatory and inhibitory results from 5-(N,N-dimethyl)- or 5-(N-methyl-N-isobutyl)amiloride (data not shown) were similar to that of 5-(N-ethyl-N-isopropyl) amiloride (blue). D, pooled data from experiments in C. The inhibition ratio was defined similarly as shown in Fig. 4B. Data points are means ± S.E. of four to seven measurements. **, p < 0.001 versus zero.

FIGURE 6.

Effects of DTDP on AMI stimulation of ASIC3^E79C channels. A, structure of DTDP. B, illustration of TDP covalently linked to E79C via a mechanism of Ellman's reaction. C, typical recording showing the effect of DTDP (0.5 mm, pH 7.4) on the AMI stimulation of ASIC3^E79C channels. AMI at 1 and 3 mm evoked inward currents from CHO cells expressing ASIC3^E79C. Following DTDP treatment, a successive administration of AMI failed to induce ASIC3^E79C channel activation because of the formation of E79C-S-S-TDP complexes preventing AMI binding. DTT (4 mm, pH 7.4, and 5 min) restores channel activation by AMI presumably by breaking the disulfide bond in the E79C-S-S-TDP complexes, rendering the channel responsive to subsequent AMI application. D, cotreatment of DTDP (0.5 mm, pH 7.4) and DTT (4 mm, pH 7.4) preserves channel activation by AMI. After washout of DTDP/DTT, DTDP treatment again abolished AMI activation of ASIC3^E79C channel because of steric effects. E and F, statistical analysis of effects of DTDP on the sustained currents induced by AMI. Shown in E and F are pooled data from experiments in C and D, respectively. The dashed lines in C and D represent the base-line level. Data points are means ± S.E. of six measurements of sustained currents normalized to that induced by AMI (3 mm) before DTDP treatment. **, p < 0.001, demonstrating the significant difference in the sustained currents induced by both 1 and 3 mm AMI between the two different groups as indicated. N.S., not significant.

FIGURE 7.

Effects of AMI on covalently activated ASIC3 channels. A, typical recording showing the effect of AMI (1 mm) on 0.5 mm DTNB (pH 7.4)-induced ASIC3^E79C activation as a result of a covalent modification (26). Notably, a successive coadministration of AMI (1 mm) and DTNB (0.5 mm at pH 7.4) slowed down the development of DTNB currents (see below) because of steric competition between AMI and DTNB. B, illustration of 5-thio-2-nitrobenzoic acid (TNB) covalently linked to E79C via a mechanism of Ellman's reaction (lower panel) and a typical recording showing the effect of AMI (1 mm) on DTNB-induced ASIC3^E79C activation (upper panel) with a different drug application sequence from A. C, pooled data from the combination of experiments in A and B. The rate (pA/ms) is defined as the maximal current (pA) divided by the duration (ms) of covalent modification treatment as indicated. For AMI-treated, the maximal current during AMI washout (as indicated by SS) is measured to minimize the contribution of AMI-induced inhibition. Different colored points and lines represent paired measurements for individual cells. D, sequence alignment between rat ASIC2a (rASIC2a) and rat ASIC3 subunits. The blue open box indicates the conserved Deg site (Gly-430 versus Gly-438 in ASIC2a and ASIC3, respectively). E, illustration of 2-aminoethyl (EA) group covalently linked to G438C via a mechanism of Ellman's reaction (lower panel) and a typical recording showing the effect of AMI on 0.2 mm MTSEA (pH 7.4)-induced ASIC3^G438C activation (upper panel). F, pooled data from the experiments in E. G, typical recording showing the attenuated effect of AMI on MTSEA-induced ASIC3^G438C-E423A activation. Similar results were obtained in five other experiments.

FIGURE 8.

AMI cause pain-related behaviors through activation of ASIC3 channels. A, pain-related behaviors as determined by the time spent for following saline or AMI (300 μm or 1 mm or 3 mm) injection (10 μl) in asic3^+/+ and asic3^−/− mice. Data are means ± S.E. n = 6–9. **, p < 0.001 versus saline; ##, p < 0.001, asic3^+/+ versus asic3^−/−. B, pain-related behaviors as determined by the time spent for following saline or AMI (3 mm) injection (10 μl) in asic1^+/+ and asic1^−/− mice. Data are means ± S.E. n = 10. *, p < 0.05, asic1^+/+ versus asic1^−/−.