Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating

Abstract

Acid-sensing ion channels (ASICs) are proton-activated Na⁺ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

Keywords: acid-sensing ion channel; conformational changes; kinetic model; pH sensing; voltage clamp fluorometry.

Fig. 1.

Combined mutation of AcP residues preserves almost normal ASIC1a function. (A) Structural image, showing a human ASIC1a model based on the chicken ASIC1 structure (8). (Left) Trimer structure. Individual domains of one subunit are colored and labeled. (Right) Close-up view of the AcP formed by the thumb, the finger, and the β-ball. The residues that were mutated are indicated. (B) Mutant composition. Each number in the right column represents a neutralization mutation, Glu to Gln, Asp to Asn, and His to Asn. The new mutations from one construct to another are marked in red. (C) Representative current traces of WT and the mutant AcP11. The pH protocol is schematically indicated on the left. (D) The pH dependence of activation and SSD of WT and AcP11 (n = 4–6). Normalized current amplitudes are plotted as a function of stimulation pH for activation (filled symbols) and as a function of the conditioning pH for SSD (open symbols). The solid lines represent fits to the Hill equation (SI Materials and Methods). The kinetic scheme of ASIC functional states is shown, emphasizing, with a green arrow, the activation, and, with a blue arrow, the SSD transition. (E) The pH dependence of activation, plotting pH of half-maximal activation (pH50) values as green bars (left axis) and the Hill coefficient (nH) in gray (right axis). The conditioning pH in these experiments was 7.6 to 8.0, depending on the mutant, to ensure stable recordings without occurrence of SSD. For each mutant, the numbers in red indicate the residues mutated in addition to the mutations already present in the preceding mutant; n = 4–125. (F) The pH dependence of SSD, showing pH of half-maximal SSD, pHDes50 values as blue bars, and nH values of SSD in gray; n = 5–56. (G) Experimental current traces (black) of an activation curve of ASIC1a WT and corresponding traces generated by the 32-state model (red; SI Materials and Methods). (H) Activation and SSD curves of WT, AcP11, and AcP14 generated experimentally (filled symbols) or by the 32-state model (open symbols). (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; different from WT.)

Fig. S1.

ASIC modeling and properties of AcP mutants. (A) Desensitization kinetics of AcP mutants. The current decay phase of ASIC currents was fitted to a single exponential; n = 3–19. The red numbers in the labels indicate the residues mutated in addition to the mutations already present in the preceding mutant (with a lower number of mutations; generally on the left). (B) Sustained current/peak current amplitude (I_sust/I_peak) ratio of AcP mutants determined at pH 5, 4.5, and 4, as indicated; n = 3–18. (C and D) Illustration of the effect of a neutralization mutation on the pH50 and Hill coefficient in (C) a four-state model with two protonation sites. The neutralization mutation is modeled as one of the sites being always protonated and corresponds to a two-state kinetic model. Results for the two-state models (corresponding to the mutant) were obtained from Eq. S9 and are shown as black circles, to which a Hill function was fitted (black line) yielding a pH50 = 7.0 and Hill coefficient n = 1. WT channels correspond to a four-state model (Eqs. S2–S5) with one of the protonation transitions (transition from C_a to ${\text{C}}_{\text{a}}^{\text{b}}$) having the exact same parameters as in the two-state model (black circles). If both protonations are essential for the observed transition, the state of interest is $C_a^b$; therefore we plot $P\left(C_a^b\right)$ obtained from Eq. S5. In this case, WT channels (red triangles) have lower pH50 and higher (or equal) Hill coefficient than the mutant (black circles), both for positive (red upward triangles) and negative (red downward triangles) cooperativity between the two protonation sites. As before, pH50 and Hill coefficients were obtained from the fit of a Hill function (red lines). Finally, if protonation a is accessory to the transition of interest (NE, not essential), the states of interest correspond to $C_a^b$ and $C^b$; therefore we plot $P\left(C^b\right)+P\left(C_a^b\right)$ (blue triangles) obtained from Eqs. S4 and S5. In this case, the WT channel can have a pH50 that is either larger (downward blue triangles) or smaller (upward blue triangles) than the mutant channel. Note that, as shown analytically in Eq. S13, the Hill coefficient of the WT channel is smaller (respectively larger) than that of the mutant for negative (respectively positive) cooperativity. (E) ${\left[H_{50}\right]}_{4state}^{accessory}/{\left[H_{50}\right]}_{2state}$ (i.e., the ratio of the [H₅₀] of the four-state model where protonation of site a is accessory vs. the [H₅₀] of the mutant in which site a has been mutated to a residue mimicking protonation) is shown as a function of $\left[H_{50}^a\right]/[H_{50}^b]$ and α (Eq. S12). Red regions indicate ${\left[H_{50}\right]}_{4state}^{accessory}>{\left[H_{50}\right]}_{2state}$ (pH₅₀ is increased by the mutation) and blue indicates ${\left[H_{50}\right]}_{4state}^{accessory}<{\left[H_{50}\right]}_{2state}$ (pH₅₀ is decreased by the mutation). (F) Model used for fitting experimental traces of WT, AcP11, and AcP14 activation and SSD, as described in SI Materials and Methods. The model is composed of 32 states corresponding to three sets of protonation sites (o = activation, d = desensitization, and a = AcP) and four conformations [C = closed, O = open, D = closed-desensitized, and (OD) = open-desensitized].

Fig. 2.

Combined mutations of palm residues accelerate desensitization and induce a sustained current. (A) Structural image of the palm (yellow) and β-ball (orange) domains of one ASIC1a subunit, showing the acidic residues investigated here, with residues of the “palm core” (see Results) highlighted in bold. (B) Mutant composition. The new mutations from one construct to another (compared with the construct with lower number of mutations) are marked in red. B, β-ball; PaC, palm core. (C) Representative current traces of different palm mutants. The vertical bar corresponds to (in microamperes) 6 (WT), 3 (PaC3), 4 (Pa4b), 1 (PaC4), 1.25 (PaC5), 0.5 (PaC6), and 0.24 (PaC6.2B). (D) Representative current recordings of WT, PaC4, and PaC6 at the indicated temperatures. The vertical bar corresponds to (in microamperes) 4.6 (WT), 3.2 (PaC4), and 1 (PaC6). In C and D, the conditioning pH was 7.4. (E) The pH dependence of activation, pH50 values (colored bars), and nH of activation (gray bars); n = 5–128. The pH50 values of transient currents are shown in purple, and those of sustained currents are shown in orange. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; different from WT.)

Fig. S2.

Properties of palm mutants. (A) I_sust/I_peak ratio of combined palm mutants; n = 3–18. (B) The I_peak/I_sust ratio is plotted for WT, PaC4, and PaC6 at the indicated temperatures. Data were recorded at pH 4 from a conditioning pH of 7.4 (n = 7–10). (C) I_sust/I_peak ratio of single palm mutants; n = 4–28. Mutations to Cys of E79 and E421 have been shown to induce an I_sust/I_peak ratio of 5 ± 1% and 1 ± 1%, respectively (21). For bar graphs of I_sust/I_peak, note that, for some mutants, the I_sust/I_peak ratio was not measured at all three pH values. (D−G) Current−voltage relationship of (D) ASIC1a WT-I_peak, (E) PaC4-I_peak, (F) PaC4-I_sust, and (G) PaC6-I_sust in the presence of either Na⁺ (red), K⁺ (blue), or Cs⁺ (green) in the extracellular solution. The holding potential was −60 mV, and 90-ms voltage ramps from −100 mV to +80 mV were applied. The pH 5-induced ramp current was calculated as the difference between the ramp current obtained during the acidification (during the peak or the sustained phase) and the ramp current measured during the conditioning period at pH 7.4. For each cell, the pH 5-induced currents with extracellular Na⁺-, K⁺-, and Cs⁺-containing solution were normalized to the amplitude measured with Na⁺ at −80 mV. Dotted lines represent SEM of independent experiments (n = 3–11). (H) The pH 5.5-induced current as a function of amiloride concentration, normalized to the control condition. IC₅₀ values obtained from the fits were 125 ± 43 μM (WT-I_peak) and 65 ± 13 μM (PaC4-I_peak); n = 6. (I) Effect of 1 mM amiloride on WT and mutant currents as indicated. The I_amiloride/I_control ratio obtained at pH 5.5 is shown; n = 6–8. (J) Structural image showing residues pointing to the wrist that were mutated. Note that D78 is oriented toward H73 of a neighboring subunit. (K) Representative current traces of palm mutants in which residues H73, D78, and/or E421 were mutated in the background of various PaC mutants. H73 was mutated to Ala or Lys, because mutation to Asn resulted in very small currents. The vertical bar corresponds to the following current amplitude, in microamperes: 0.6 (PaC4+78), 0.5 (PaC4+73A+78), 0.45 (PaC4+73K+78), 5 (PaC5+78), 2 (PaC6+78), 0.6 (PaC6.2B+78), 0.2 (PaC6.2B+421), and 1 (PaC6.2B+78+421). The color of the label refers, as the color of bars in L−N, to the PaC mutant on which the mutant is based. (L) I_sust/I_peak of the indicated mutants; n = 6–28. (M and N) The pH50 and nH of activation of palm−wrist mutants; n = 6–129. The color indicates the palm mutant on which the mutant is based; the pattern refers to the added mutations. Statistically significant differences from WT or the corresponding palm mutant on which palm−wrist mutants are based (M and N; i.e., PaC4+78 is compared with PaC4_tr, etc.) are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. For A, C, and L, all values close to 1 and higher were different from the WT I_sust/I_peak; for other values as indicated with the asterisks.

Fig. S3.

VCF experiments of the AcP. (A) (Left) Close-up view of the AcP showing the residues of pairs that gave rise to fluorescence signals. (Right) Distances (in angstroms) between the Cα atoms of the corresponding residues, measured in homology models of the open [4NTW (8)] and desensitized [4NYK (6)] structures. (B) Current and ΔF kinetics at various stimulation pH conditions. Comparison of current activation and ΔF kinetics (of the first ΔF component) of selected mutants measured at different pH; n = 3–12. ΔF and current signals were correlated (Table S2) in all conditions except pH5.5 for D347C/T236W^first and pH6.3 for E355C^first. (C) Representative current (black) and fluorescence (red) traces of triple mutants in response to extracellular acidification to pH 6. They represent the double mutants used in the VCF part of the study, in which in addition the mutation W233V was introduced to verify that this nearby Trp residue had not influenced the ΔF signals. The conditioning pH for these experiments was 7.4. The traces are representative of 4 to 9 oocytes. Note that all these mutants gave consistent ΔF and current signals, except for D351C/F257W/W233V, which produced small signals that were not present in all oocytes tested. (D and E) Possible errors in the measurement of the kinetics of ΔF signals containing two components, and correction of the rise time values. (D) Representative ΔF traces of the D347C/T236W mutant obtained under different pH changes, to illustrate the overlap of the negative ΔF component over the positive component at more acidic pH conditions. (E) ΔF amplitude ratio of the first ΔF component at the indicated pH and its amplitude at pH 7 (where the signal was maximal); n = 6–21. These ratios were used to correct the rise time values as indicated in SI Materials and Methods.

Fig. 3.

Fluorescence changes in the AcP associated with channel opening and desensitization. (A) Close-up view of the AcP, showing the residues that were mutated to Cys (to dock fluorophores) and/or to the quenching residue Trp. (B) Structure of the fluorophore AlexaFluor488. (C) Representative current and ΔF traces at pH 7 and 6 of the mutant D237C/D347W, highlighting, in green and blue, the parts of the pH 6 traces used for kinetic analysis. Note that the ΔF trace has two components, as highlighted with the arrows. (D) Schematic view of the oocyte recording chamber used for measurements of current and ΔF kinetics (SI Materials and Methods). (E) Scatter graph comparing the rise time of the channel opening (black) and the ΔF onset (red) in response to acidification from the conditioning pH 7.4 to the stimulation pH 6 in paired experiments; n = 6–12. For mutants containing two ΔF components, the two are distinguished as first and second. Correlation between the ΔF and current signal is indicated by labeling in bold turquoise (see criteria in Table S2). (F) Representative current and ΔF traces showing (Left) a mutant in which the start of the ΔF signal precedes that of the current (D347C/T236W) and (Right) one in which the two signals start at the same time (D347C/E238W). The vertical dashed line indicates the beginning of the ΔF. (G) Scatter dot plot of the difference in the ΔF and current delay of appearance (delay_ΔF − delay_I) measured at pH 6; n = 5–11. (H) Scatter graph comparing the current decay time (black) and the fluorescence rise time (red) in response to acidification from pH 7.4 to pH 6 in paired experiments; n = 6–12. Bold turquoise labels indicate correlation between ΔF onset and current decay kinetics (Table S2). (Inset) In blue, the parts of current and ΔF traces whose kinetics were compared. (I) The pH of half-maximal amplitude (pH50) for current (black columns) and fluorescence activation (red), and the pH of current SSD (green); n = 4–16.

Fig. 4.

Predicted conformational changes in the AcP. (A) Representative, paired current (black) and fluorescence (red) traces of double mutants in response to extracellular acidification to pH 6. Black arrows point to fast ΔF components. (B) View of the AcP with the mutated residues, indicating, by dashed black lines, the different Cys/Trp pairs. For each double mutant, the nature of the ΔF signal is indicated by “+” or “−“ signs (“+” denotes increase in ΔF); for mutants with composite ΔF signal, the left sign represents the first component. (C) Scheme indicating the deduced conformational changes in the AcP during opening and steps preceding and correlated with desensitization, as discussed in the Results. The gray cylinders represent residues mutated for the VCF experiments. The dotted outlines of the finger loop and α5 thumb helix represent their hypothesized position in the closed state. Hypothesized conformational changes are illustrated by arrows, the green arrows standing for conformational changes occurring during activation, and the orange arrows standing for subsequent steps occurring before and during desensitization. (D) Representative current and ΔF traces of the mutants AcP13/N237C/N347C, AcP13/N237C, and PaC5/E355C. Note that not only the N237C/N347W pair, but also the single mutation N237C, induced measurable ΔF signals, indicating that, in the changed environment of the AcP13 mutant, the N237C ΔF did not depend on the presence of a nearby Trp residue and does not reflect a change in distance between the fluorophore and the Trp residue. The PaC5/E355C mutant showed, in approximately half of the experiments, a rapid transient and a sustained component and, in the other half, only a sustained current.

Fig. S4.

Combined neutralization and VCF mutations. The figure shows pH and kinetic parameters of the combined mutants AcP13/N237C/N347W and PaC5/E355C (red and black symbols and bars) and compares them to these mutants in the WT background, D237C/D347W and E355C (gray and faintly colored symbols and bars). (A and B) The pH50 and nH of current activation (black and gray) and ΔF (red tones) of the Cys and Cys/Trp mutants combined with the mutant AcP13 or PaC5, compared with values obtained with the Cys and Cys/Trp mutants alone (n= 4–7). (C) Current and ΔF rise time. (D) Current decay time and ΔF rise time. Orange symbols in C and D represent the first ΔF component. (E) Delay_ΔF − Delay_I of the same mutants (n = 5–12). *P < 0.05; **,P < 0.01; ***P < 0.001, and ****P < 0.0001 for the comparison between AcP13/N237C/N347W and D237C/D347W, and between PaC5/E355C and E355C; for mutants with two ΔF components, only the kinetics of the second ΔF components were compared.