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.
Highlights
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
ENaCs open spontaneously, DEGs respond upon mechanical stimulation, acid-sensing ion channels (ASICs) are sensitive to extracellular acidosis, whereas FaNaCs are directly activated by native FMRFamide peptide [1, 2, 23]
This study demonstrated here that the “primitive” member of DEG/ENaC, FaNaCs, including all four orthologues AkFaNaC, LsFaNaC, HaFaNaC, and HtFaNaC, could all be directly activated by GMQ (Figs. 2B and 11D), the nonproton ligand of mammalian ASICs
Summary
We identified GMQ as the first nonproton ligand of ASICs (Fig. 2A), which selectively activates ASIC3 but not ASIC1a, -1b, and -2a [27]. B, the representative current traces illustrating the activation of four FaNaC orthologues in CHO cells with or and FMRFamide (0.1–1 mM) in the absence or presence of extracellular Ca2ϩ. Most of those currents were inhibited by amiloride (100 M) (n ϭ 4 –7). Inhibited the GMQ’s action in HaFaNaC (Fig. 5A), just like what it does on rASIC3 [26, 27], GMQ-evoked currents were potentiated in alkaline solution (pH 8.0 –9.0) (Fig. 5A), which was eliminated by extracellular Ca2ϩo deprivation (Fig. 5B), indicating that GMQ, proton, and Ca2ϩo interacted with each other on gating HaFaNaC via similar but more complicated mechanisms than they do on ASIC3 [27].
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