Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel
Journal of Biological Chemistry
FMRFamide (Phe-Met-Arg-Phe-NH 2 )-activated sodium channel (FaNaC) is an amiloride-sensitive sodium channel activated by endogenous tetrapeptide in invertebrates, and belongs to the epithelial sodium channel/degenerin (ENaC/DEG) superfamily. The ENaC/DEG superfamily differs markedly in its means of activation, such as spontaneously opening or gating by mechanical stimuli or tissue acidosis. Recently, it has been observed that a number of ENaC/DEG channels can be activated by small molecules or
... eptides, indicating that the ligand-gating may be an important feature of this superfamily. The peptide ligand control of the channel gating might be an ancient ligand-gating feature in this superfamily. Therefore, studying the peptide recognition of FaNaC channels would advance our understanding of the ligand-gating properties of this superfamily of ion channels. Here we demonstrate that Tyr-131, Asn-134, Asp-154, and Ile-160, located in the putative upper finger domain of Helix aspersa FaNaC (HaFaNaC) channels, are key residues for peptide recognition of this ion channel. Two HaFaNaC specificinsertion motifs among the ENaC/DEG superfamily, residing at the putative ␣4-␣5 linker of the upper thumb domain and the ␣6-␣7 linker of the upper knuckle domain, are also essential for the peptide recognition of HaFaNaC channels. Chemical modifications and double mutant cycle analysis further indicated that those two specific inserts and key residues in the upper finger domain together participate in peptide recognition of HaFaNaC channels. This ligand recognition site is distinct from that of acid-sensing ion channels (ASICs) by a longer distance between the recognition site and the channel gate, carrying useful information about the ligand gating and the evolution of the trimeric ENaC/DEG superfamily of ion channels. The epithelial sodium channel/Degenerin (ENaC/DEG) 4 superfamily, cloned in the early 1990s, is nonvoltage-gated, amiloride-sensitive, and sodium-selective ion channels, including ASIC, ENaC, DEG, FaNaC, bile acid-sensitive ion channel (BASIC), and pickpocket (PPK) channels, etc. (1, 2) . ENaC/DEG ion channels are implicated in many physiological and pathological functions such as synaptic plasticity, learning and memory, emotion regulation, neurodegenerative diseases, epileptic seizures, pain sensation, mechano-sensation, blood pressure regulation, and cystic fibrosis (1-3), which makes them potential drug targets for those disorders. The members of the ENaC/ DEG superfamily differ markedly by means of activation. For example, DEG channels are mechano-sensitive; ENaC channels open spontaneously; ASIC channels are capable of sensing tissue acidosis, while FaNaC is activated by RFamide peptides, etc. (4). Recently, great advances have been made in the exploration of the activation mechanism of this superfamily. For example, it is now known that ENaC, ASIC3, BASIC, and ASIC1a channels can be activated by the small molecules S3969 (5), GMQ (6), bile acid (7) , and peptide toxin (8, 9) respectively. These new findings suggest an important small molecule or peptide ligand-gating property of this superfamily of ion channels, in addition to the subtype-specific gating mechanisms such as spontaneously opening or gating by mechanical stimuli and tissue acidosis, can play an important role in ENaC/DEG channel activation. However, the structural components underlying the ligand gating of ENaC/DEG remain largely unclear except for ASIC channels, mainly due to the high resolution structure of chicken ASIC1 (cASIC1) that was determined recently. However, extensively studying gating properties of ASIC channels remains difficult because of multiple proton-binding sites located in different domains of ASIC channels (10 -13). . B, representative current traces of ⌬445-493 induced by different concentrations of FMRFamide. C, pooled data showing ratio of 7 M and 100 M FMRFamide triggered currents of the mutants in HaFaNaC specific-insertion II. Data are presented as means Ϯ S.E. (n ϭ 4). ## , p Ͻ 0.01; ** , p Ͻ 0.01 versus WT, Student's t test. D, representative images showing Western blots of total and cell surface expressions of HaFaNaC WT channels and ⌬445-493 mutants. E, dose-response relationship of HaFaNaC WT and mutants in HaFaNaC specific-insertion II. Data points are means Ϯ S.E. (n ϭ 4 -8) and fit with Hill equation. Downloaded from FIGURE 7. Hypothetical activation models for DEG, ASIC and FaNaC. A, DEG model showing ectodomain sensing the mechanical stimulus. B, ASIC model showing proton and GMQ binding sites, respectively. C, putative FMRFamide binding sites in HaFaNaC, * indicates residues from a neighboring subunit.