The Dual Activation And The Channel Gating Evolution Of FMRFamide-gated Sodium Channels

The degenerin/epithelial sodium channels?DEG/ENaC?are superfamily of voltage-insensitive sodium channels with diverse activation mechanisms among different subfamilies.DEG/ENaCs have implicated in many important physiological and pathological functions,such as the feeling,emotion regulation,pain sensation,and cognition.Most DEG/ENaC subfamilies are expressed in mammals,such as acid-sensing ion channels?ASICs?,while some subfamilies are only identified in invertebrates,such as FMRFamide-gated sodium channels?FaNaCs?,suggesting that DEG/ENaC subfamilies diverged early in evolution.ASIC subfamily is sensitive to extracellular acidification.We recently reported that rat ASIC3 could be persistently activated by the nonproton agonist,2-guanidine-4-methylquinazoline?GMQ?,in the absence of acidosis.This GMQ-mediated activation of ASIC3 relies on the residues of the palm domain,one of protonation sites during acidosis,but not all other protonation sites,indicating that the GMQ-mediated activation mechanism is some different with low-pH induced channel gating.A series of questions still remain unclear.Does this new exogenous small molecule-mediated activation only exist in the subfamily of ASIC in whole DEG/ENaC superfamily?Is there identical endogenous activation pattern at neutral pH similar with the GMQ's activation?If this is the case,where is it from?Moreover,does the activation of GMQ act as the modulator or the direct activator?To address these questions,we intended to study the possible activation of GMQ in FaNaCs,an elder subfamily member among the entire DEG/ENaC superfamily,and a member known to not be activated by extracellular protons.Here,we firstly used electrophysiology to confirm that GMQ acts as a direct agonist.Then,we compared mechanisms of FMRFamide-and GMQ-mediated activation by combining experimental methods such as point mutation,homology modeling,single channel recording,chemical modification,calcium imaging and western-blotting.Finally,we discussed the evolution of DEG/ENaCs channel gating,and speculated an ancient dual/multiple gating of DEG/ENaC superfamily.Results show that:1)GMQ could directly activate FaNaCs subfamily ion channels that are only identified in invertebrates,in the absence of acidosis.These GMQ-induced currents reveal several properties in common with other DEG/ENaC superfamily currents,such as Na⁺ion selectivity,and amiloride?a broad-spectrum inhibitor of DEG/ENaCs?,extracellular pH,and Ca²⁺sensitivity,suggesting that GMQ can act as a direct agonist,and the activation of GMQ has been present in the early stage in the evolution of DEG/ENaC superfamily.2)According to the evolutionary tree of DEG/ENaC superfamily,HyNaC?Hydra sodium channel?is closely related with ASIC,while FaNaC to ENaC?epithelial sodium channel?.In addition to ASIC,GMQ can activate FaNaC,but not ENaC,indicates a closer relationship between FaNaC and ASIC.3)The key amino acid residues involved in the GMQ-mediated HaFaNaC activation is located in the palm domain,similar to GMQ's action on rASIC3.Moreover,GMQ-induced FaNaC and ASIC currents have similar current characteristics and concentration level.These results imply that GMQ uses a similar mechanism to activate both ASIC3 and FaNaC channels.4)Differences in ion selectivity,unitary conductance,and effects of substitutions at key residues reveals that GMQ and FMRFamide activate FaNaCs via distinct mechanisms.The presence of two activation mechanisms in the FaNaCs diverge early in the evolution of DEG/ENaCs and suggest that dual gating is an ancient feature in this superfamily.In conclusion,we confirm that the small-molecule,GMQ,acts as an activator of nonproton-activated FaNaCs,rather than a modulator,which enriches the ligand types of DEG/ENaC.Two distinct activation mechanisms of GMQ and FMRFamide in FaNaCs reveal a dual/multiple gating of DEG/ENaC superfamily,similar to transient receptor potential?TRP?channels and two-pore domain K⁺channels?K2P?channels.Notably,the GMQ-gating mode is still preserved in the mammalian ASIC subfamily,whereas FMRFamide-mediated channel gating was lost during evolution.This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs.Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.