Mutations in the palm domain disrupt modulation of acid-sensing ion channel 1a currents by neuropeptides

Abstract

Modulation by neuropeptides enhances several functions of acid-sensing ion channels (ASICs), such as pain sensation and acid-induced neuronal injury. The acid-induced opening of ASICs is transient, because of a rapid desensitization. Neuropeptides containing an Arg-Phe-amide motif affect ASIC desensitization and allow continuous activity of ASICs. In spite of the importance of the sustained ASIC activity during prolonged acidification, the molecular mechanisms of ASIC modulation by neuropeptides is only poorly understood. To identify the FRRFa (Phe-Arg-Arg-Phe-amide) binding site on ASIC1a, we carried out an in silico docking analysis and verified functionally the docking predictions. The docking experiments indicated three possible binding pockets, located (1) in the acidic pocket between the thumb, finger, β-ball and palm domains, (2) in a pocket at the bottom of the thumb domain, and (3) in the central vestibule along with the connected side cavities. Functional measurements of mutant ASIC1a confirmed the importance of residues of the lower palm, which encloses the central vestibule and its side cavities, for the FRRFa effects. The combined docking and functional experiments strongly suggest that FRRFa binds to the central vestibule and its side cavities to change ASIC desensitization.

Figure 1

Docking of FRRFa peptide to the acidic pocket of the desensitized and closed conformation of ASIC1a. The human ASIC1a structural model is based for (a–c) on a desensitized chicken ASIC1 structure, and for (d–g) on the closed chicken ASIC1. (a) General view of the channel, highlighting the location of cavities into which peptides may bind. The central vestibule, which is enclosed by the lower palm domain, is not indicated. Estimated volumes in the desensitized ASIC1a model are ∼1600 ų (acidic pocket), ∼1150 ų (thumb base pocket) and ∼1400 ų (central vestibule, CASTp). (b) Single subunit with structural domains indicated: transmembrane part (red), palm (yellow), β-ball (orange), knuckle (cyan), finger (purple) and thumb (blue). (c) Example of a predicted FRRFa interaction mode with the ASIC1a acidic pocket of the desensitized state, shown in a structural view. (d,e) One of two predicted FRRFa interaction modes with the acidic pocket of closed ASIC1a, shown in a structural view (d) and a schematic 2D representation (e) that illustrates the interactions between peptide and channel (PoseView). The colours in the scheme generated by PoseView were adjusted – the peptide is coloured as in (d and f) and the hydrophobic interactions are presented in purple. The program PoseView does not apply exactly the same stringency in the detection of H-bonds as Chimera, therefore there exist some differences in the H-bond attribution. (f,g) Example of the second predicted FRRFa interaction mode with ASIC1a in the closed conformation, in structural view (f) and schematic 2D representation (g). In (c,d and f) the different domains of ASIC subunits are coloured as in (b) for the subunit on the left, while the second subunit is shown in red. The peptide is shown in green, with light green at the N-terminus and dark green at the C-terminus; the heteroatoms of highlighted residues are colored in blue (nitrogen), red (oxygen) and white (hydrogen). Letters at the end of residue identifiers in (e and g) indicate the ASIC subunit (A, B or C). Residues that were mutated for the functional analysis are presented in ball and stick representation and labelled in bold.

Figure 2

FRRFa modulation of ASIC1a currents is only partially affected by acidic pocket mutations. (a) FRRFa concentration-response curve. Top, representative current traces. Bottom, I_sust/I_peak ratio induced by pH5 as a function of the FRRFa concentration (pre-applied during 45–50 s and present in pH5 solution), n = 7–11. (b) Time course of disappearance of the FRRFa-induced I_sust. FRRFa at 50 μM was applied for 45 s, and current was measured once every minute during washout with FRRFa-free solution. Exponential fit shown as solid line (n = 4, error bars are smaller than the symbols). (c) Time course of appearance of I_sust. Oocytes were exposed for various durations to 50 μM FRRFa in the pH7.4 conditioning solution, before activation of ASIC currents with pH5, including 50 μM FRRFa. Exponential fit shown as solid line, n = 4. (d–f) and (i–k), all Cys mutants had been exposed during 3 min to 1 mM MTSET before the experiment. (d) Bar graph of the I_sust/I_peak ratio induced by pH5. Grey bars, control; red bars, after exposure to 50 μM FRRFa, n = 4–40. *p < 0.05, **p < 0.01, different from the same condition in WT; ^###p < 0.001, ^####p < 0.0001, the FRRFa-induced increase in I_sust/I_peak (see e,f) is different from this ratio in WT. (e) ratio of FRRFa-induced I_sust/I_peak increase by 50 μM FRRFa (n = 4–40). (f) ratio of FRRFa-induced I_sust/I_peak increase by 500 μM FRRFa (n = 5–11). (g) Representative current traces of WT ASIC1a obtained at the indicated pH conditions, without FRRFa (top), with 15 μM FRRFa in the conditioning solution (bottom). (h) pH dependence of steady-state desensitization (SSD). Normalized pH5-induced current amplitude plotted against the conditioning pH (n = 8). The lines represent fits to the Hill equation described in Methods, pH protocol illustrated in inset. FRRFa was administered during the conditioning period (55 s). (i) Midpoint of SSD curve (pHD₅₀) values obtained in the absence of FRRFa (n = 4–12). *p < 0.05; ****p < 0.0001; different from WT. (j) Difference in pHD₅₀ (pHD_50FRRFa-pHD_50ctrl), n = 4–12. (k) nH_FRRFa/nH_ctrl ratio. The pHD₅₀ shifts and nH ratios of mutant channels were not different from those of WT.

Figure 3

Docking results of FRRFa peptide to the thumb base pocket of the desensitized and the closed conformation of ASIC1a. The human ASIC1a structural model is based for (a) on a desensitized ASIC1 structure, and for (b,c) on the closed ASIC1a structure. (a) Predicted FRRFa interaction mode with the desensitized ASIC1a thumb base pocket. (b,c) Predicted FRRFa interaction mode with the closed ASIC1a thumb base pocket in structural view (b) and schematic 2D representation (c). The color code is as described in the legend to Fig. 1. Residues that were mutated for the functional analysis are shown in ball and stick representation in panels a and b and labelled in bold in all three panels.

Figure 4

Thumb base mutations do not prevent FRRFa modulation of ASIC1a currents. (a) Bar graph of the I_sust/I_peak ratio induced by pH5 of WT and thumb base mutants as indicated; grey bars, control; red bars, 50 μM FRRFa, n = 4–22. (b) Ratio of FRRFa-induced increase in I_sust/I_peak in the indicated mutants (n = 4–22). For (a–d) **p < 0.01, ****p < 0.0001, different from the same condition in WT; ^#p < 0.05, ^##p < 0.01, the FRRFa-induced increase in I_sust/I_peak is different from this ratio in WT. (c) Bar graph of the I_sust/I_peak ratio induced by pH5 of WT F302C-MT. (d) Ratio of FRRFa-induced increase in I_sust/I_peak in the indicated mutants. For (c,d) the oocytes had been exposed during 3 min to 1 mM MTSMT (n = 5) prior to the experiment. (e) Representative current traces of ASIC1a-F302A in the absence and presence of 100 μM FRRFa. (f) FRRFa concentration-response curve of the I_sust/I_peak current ratio induced by pH5 in oocytes expressing F302A, n = 8. The WT curve is shown for comparison (n = 7–11). (g) Time course of disappearance of the FRRFa-induced sustained current in the F302A mutant and WT. FRRFa at 50 μM was applied for 45 s, and then washed out. The normalized I_sust/I_peak ratio is plotted as a function of time. Exponential fit shown as solid line, n = 9 (F302A) and n = 4 (WT).

Figure 5

Docking results of FRRFa peptide to the central vestibule of the open and closed conformation of ASIC1a. The structural images are for (a,b) from a human ASIC1a model based on the Mit-toxin-opened ASIC1 structure, and for (c–f) from a model of the closed state. (a) Predicted FRRFa interaction mode with the central vestibule of the open ASIC1a. (b) Predicted FRRFa interaction mode with a side cavity of the central vestibule of the open ASIC1a. Note that the N terminus is located inside the central vestibule. (c–f) Two predicted FRRFa poses in a side cavity of the central vestibule of closed ASIC1a, in structural view (c,e) and schematic 2D representation (PoseView, modified as in Fig. 1, d,f). (e,f) For this pose, the Chimera software detected additional interactions, not detected by PoseView: a hydrogen bond between Arg3 and the Thr370 backbone and a hydrogen bond between Phe1 backbone and the Asn416 side chain. The colour code of the peptide is the same as described in the legend of Fig. 1. The three ASIC subunits are colored in yellow, red, and blue. Residues that were mutated for the functional analysis are are shown in ball and stick representation in panels a, b, c and e and labelled in bold in all panels.

Figure 6

Mutations of the palm change FRRFa modulation of ASIC1a. Mutants containing “-ET” in their name had been exposed during 3 min to 1 mM MTSET prior to the experiment. (a) Representative current traces of the mutant E418C in the absence and presence of 50 μM FRRFa. (b) Bar graph of the I_sust/I_peak ratio induced by pH5 of WT and mutants as indicated (n = 5–41). For (b,g–i) *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001, different from WT. (c) Ratio of 50 μM FRRFa-induced increase in I_sust/I_peak (n = 5–41). (d) Ratio of 500 μM FRRFa-induced increase in I_sust/I_peak (n = 5–11). For (b–d) ^#p < 0.05; ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001; ratio different from the ratio in WT. (e) FRRFa concentration-response curve of the I_sust/I_peak current ratio induced by pH5 in oocytes expressing N416C, n = 8. The WT curve is shown for comparison (n = 7–11). (f) Time course of disappearance of the FRRFa-induced sustained current in the N416C mutant and in WT. 50 μM FRRFa was applied for 45 s, and then washed out. The normalized I_sust/I_peak ratio is plotted as a function of time, and the solid line represents an exponential fit to the data, n = 9 (N416C) and n = 4 (WT). (g) pHD₅₀ values obtained in the absence of FRRFa (n = 5–54). (h) The difference in pHD₅₀ (pHD_50FRRFa-pHD_50ctrl) is plotted in the bar graph, n = 5–54. (i) Plot of the ratio nH_FRRFa/nH_ctrl of the SSD pH dependence.