Mechanism and site of action of big dynorphin on ASIC1a

Abstract

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is up-regulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs, and noncanonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is primarily mediated through electrostatic interactions of basic amino acids in the BigDyn N terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the noncanonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn, and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.

Keywords: acid-sensing ion channel; ligand–receptor interaction; neuropeptide; noncanonical amino acids; voltage-clamp fluorometry.

Fig. 1.

Mechanism of ASIC1a modulation by BigDyn. (A) Amino acid sequences of BigDyn, DynA, and DynB. (B and C) Representative current traces (B) and averaged data (C) obtained by pH 5.6 application (black bar in B) at mASIC1a WT-expressing Xenopus laevis oocytes after preincubation in pH 7.1 (gray bar) with or without 1 µM of the indicated peptide or peptide combination (green bar); in B, control currents (after pH 7.4 conditioning) are shown in gray for comparison. Asterisks indicate significant difference to control condition (P < 0.0001); n = 5–68). (D) Concentration-response curves for activation (Act.) and steady-state desensitization (SSD) of WT ASIC1a in the presence and absence of 0.1 µM BigDyn (n = 4–15). (E) Structure of the ECD and transmembrane domain (TMD) of cASIC1 (PDB: 4NTW) with individual subunits color coded and Inset showing the location of Lys105. (F) Representative current (black) and fluorescence (red) traces obtained by application of pH 5.5 (black bars) at mASIC1a labeled with Alexa Fluor 488 at position 105, the indicated peptide (PcTx1, 0.3 µM: blue bar; BigDyn, 1 µM: green bar; DynA, 10 µM: dark green bar; and DynB, 10 µM: light green bar) or pH 9.0 (purple bar). (G) Averaged change in fluorescence obtained by application of PcTx1, BigDyn, DynB, or pH 9.0, as shown in F (normalized to that obtained by application of pH 5.5) (n = 5–15). Error bars in C, D, and G represent 95CI.

Fig. 2.

Positive charges near the BigDyn N terminus are essential for ASIC1a modulation. (A) Sequence of BigDyn with basic side chains highlighted in green. (B and C) Representative current traces (B) and averaged data (C) obtained by pH 5.6 application (black bar in B) at ASIC1a WT-expressing Xenopus laevis oocytes after preincubation in pH 7.1 (gray bar) with or without 1 µM of the indicated BigDyn analog (green bar); in B, control currents after pH 7.6 conditioning are shown in gray for comparison. (D) Concentration-response curves for WT ASIC1a modulation by BigDyn (black) and Arg9Ala (green). Error bars in C and D represent 95CI. In C, n.s. indicates no statistically significant difference to control condition. n = 7–48 in C, and n = 7–9 in D.

Fig. 3.

Double charge-neutralizing mutations around the ASIC1a acidic pocket reduce BigDyn modulation. (A) Structure of cASIC1 (PDB: 4NTW) with individual subunits color coded and Inset showing the location of acidic side chains mutated to Gln or Asn. (B and C) Representative current traces (B) and averaged data (C) obtained by pH 5.6 application (black bar in B) after preincubation in SSD-inducing pH (gray bar; see SI Appendix, Fig. S4 and Table S6 for details) with or without (control) 1 µM BigDyn (green bar) at Xenopus laevis oocytes expressing the indicated ASIC1a construct; in B, control currents after pH 7.6 conditioning are shown in gray for comparison (n = 4–45). n.s. indicates no statistically significant difference to control condition. (D) Concentration-response curves for BigDyn-mediated modulation of WT (black) and Glu238Gln/Asp345Asn mutant (green) ASIC1a (n= 7–9). (E) Representative current (black) and fluorescence (red) traces from channels labeled with Alexa Fluor 488 at position 105 obtained by application of pH 5.5 (black bar), 7.0 (gray bar) or 1 µM BigDyn (green bar) at mASIC1a Lys105Cys (Left) and Lys105Cys/Glu238Gln/Asp345Asn (Right). (F) Averaged change in fluorescence at pH 7.0 with and without 1 µM BigDyn as obtained in E, normalized to that obtained by application of pH 5.5; n = 7–22, unpaired t test **P < 0.01; ****P < 0.0001. Error bars in C, D, and F represent 95CI.

Fig. 4.

Photocrosslinking confirms the BigDyn interaction site at the acidic pocket. (A) Structure of cASIC1 (PDB: 4NTW) with individual subunits color coded and Insets highlighting side chains replaced by 4-Azido-l-phenylalanine (AzF) in the acidic pocket (Left Inset) and lower extracellular domain (Right Insets). (B) Structure of 4-Azido-l-phenylalanine (AzF) and schematic workflow for cross-linking to BigDyn. HEK 293T cells expressing AzF-containing ASIC1a variants are incubated with 3 µM BigDyn and exposed to UV light for 15 min to form covalent ASIC1a–BigDyn complexes. The complex is purified via a C-terminal 1D4 tag on ASIC1a and visualized via Western blotting. (C) Western blot of purified hASIC1a WT and variants carrying AzF in the extracellular domain detected using the specified antibodies (AB). BigDyn is detected only in UV-exposed samples containing AzF in the acidic pocket, but absent in control samples not exposed to UV, those carrying AzF in the lower extracellular domain (Right Insets in A), or WT. See also SI Appendix, Fig. S7.