Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel

Abstract

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 peptides, 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 sugge...

The nonproton ligand of acid-sensing ion channel 3 activates mollusk-specific FaNaC channels via a mechanism independent of the native FMRFamide peptide

“…Here, we demonstrated that GMQ, a previously described nonproton ligand of ASIC3, can activate all four FaNaC orthologues via a mechanism distinct from the native FMRFamide peptide (35), but similar to the one used by GMQ to activate ASIC3 (27). Our data not only indicate a closer evolutionary relationship between ASIC and FaNaC subfamilies, despite the low sequence homology (12-18%), but also a conservation of the "GMQ-activation mode" that was already present in the common ancestor of these two subfamilies.…”

“…For the mutation HaFaNaC ⌬Gln-445-Ala-493 (with an ϳ50-residue truncation at the specific insertion II of the knuckle domain) (Figs. 1B and 8C), its apparent affinity (ϳ80-fold decrease), and the maximal currents of FMRFamide decreased profoundly (35). In contrast, the EC 50 of GMQ and the maximal currents were not changed by a truncation of Gln-445-Ala-493 ( Fig.…”

“…8E). In addition, a covalent modification of I160C decreased the apparent affinity for at least 700-fold and significantly reduced the maximal current of FMRFamide in HaFaNaC (EC 50 ϭ 7.33 Ϯ 0.82 M versus over 5 mM, before versus after the DTNB treatment of HaFaNaC I160C , respectively), and DTT treatment can reverse those effects (35). However, unlike the FMRFamide-evoked activation, DTNB treatment of cells that expressed HaFaNaC I160C or HaFaNaC WT , only a slight potentiation or no apparent difference was detected between the GMQ-induced currents before and after DTNB modifications (Fig.…”

“…8D). Meanwhile, mutations at the residues of other three domains, including I160A, D154A, Y131Q, W359A, and E489A, which significantly reduced (ϳ10-to 40-folds) the apparent affinity of FMRFamide (35) showed little effect on the dose-response curves of GMQ on HaFaNaC (EC 50 ϭ 0.8 Ϯ 0.04, 0.78 Ϯ 0.05, 0.81 Ϯ 0.03, 0.92 Ϯ 0.02, 0.93 Ϯ 0.03, 0.82 Ϯ 0.04 mM, for HaFaNaC WT, I160A, D154A, Y131Q, W359A and E489A, respectively, Ca 2ϩfree/pH 7.4) ( Fig. 8E).…”

An ancient FMRFamide-related peptide-receptor pair induces defense behavior in a brachiopod larva

“…For FMRFamide, there have been three different receptors deorphanized in invertebrates so far. One is an FMRFamide-gated amiloride-sensitive Na + channel (FaNaCh) that has been identified in molluscs [45][46][47]. The two other receptors belong to two different groups of neuropeptide GPCRs.…”