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

Abstract

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. 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.

A Na+ leak channel cloned from Trichoplax adhaerens extends extracellular pH and Ca2+ sensing for the DEG/ENaC family close to the base of Metazoa

“…Interestingly, ASIC channels have recently been proposed to exhibit two distinct conductance states (56), a fast-desensitizing conductance triggered by extracellular protons, and a slow-nondesensitizing conductance triggered by nonproton ligands such as the coral snake venom MitTx (57), neuropeptides (58, 59), and the polyamine 2-guanidine-4-methylquinazoline (GMQ) (60). Similarly, GMQ potently activates the mollusc FaNaC channels through a distinct mechanism than their natural ligand FMRF-amide (9), pointing to a conserved and ancient duality in DEG/ENaC channel gating. Considering this, the apparent homoplasy in Na + leak current activity for phylogenetically distant members of the superfamily might instead be attributable to adaptive changes that independently enhanced sustained conductance properties among distinct channel lineages.…”

“…Notably, the Glu-79/Glu-432 residues were also shown to be involved in activation of ASIC3 by the nonproton agonist GMQ, which, like amiloride, generates nondesensitizing currents (60). Furthermore, GMQ was found to activate molluscan neuropeptide-gated FaNaC channels through amino acids within the nonproton ligand-sensing domain, independent of FMRF-amide activation (9), pointing to a deeply-conserved gating mechanism for DEG/ENaC channels that can be acted upon by GMQ and amiloride. Here, we provide a second report of a WT DEG/ENaC channel being activated by amiloride.…”

“…Acid-sensitive ion channels (ASICs), important for extracellular pH sensing, are potently and transiently activated by extracellular acidification/protons, but are dynamically modulated by extracellular Ca 2+ , mechanical stress, proteolytic cleavage, and neuropeptides (1, 6, 7). The peptide-gated FaNaC channels from gastropod molluscs, which are directly activated by the ligand neuropeptide FMRF-amide (8), are also inhibited by protons and Ca 2+ (9). In Caenorhabditis elegans , the mechanosensory degenerin (DEG) and MEC channels are activated by mechanical stimuli (10), whereas ACD-1 acts as a Na + leak channel that is blocked by external protons and Ca 2+ (11).…”

Altered allostery of the left flipper domain underlies the weak ATP response of rat P2X5 receptors

“…They were then subcloned into the pEGFP-N1 and/or pCDNA3.0 (with EE-tag) vectors. All mutants were constructed by the QuikChange mutagenesis kit and were verified by DNA sequencing as we described previously (49,50). The QuikChange mutagenesis kit was also used for inserting a short sequence into the expression construct by carefully designed primers.…”

Discovery and modulation of acid-sensing ion channel modulating venom peptides

“…Interestingly, early studies showed a similar region of FaNaCs to be the recognition site for FMRFa (214). A second distinct binding region in the upper finger domain of FaNaCs has been recently reported for FMRFa activity (215,216). This region is known to be important for FaNaC gating, and is also much closer to the acidic pocket of ASICs, the area that initiates the activation cascade for proton-gating.…”