Abstract
FMRFamide (Phe-Met-Arg-Phe-NH2)-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 ofHelix aspersaFaNaC (HaFaNaC) channels, are key residues for peptide recognition of this ion channel. Two HaFaNaC specific-insertion motifs among the ENaC/DEG superfamily, residing at the putative α4-α5 linker of the upper thumb domain and the α6-α7 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.
Highlights
APRIL 1, 2016 VOLUME 291 NUMBER 14 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
Residues in the Putative Upper Finger Domain Determine the Peptide Recognition of Helix aspersa FaNaC (HaFaNaC) Channels—Previous studies suggested that residues located near the first transmembrane domain (TM) and the extracellular domain, numbered 89 –211, are responsible for different EC50 values of FMRFamide between HaFaNaC and HtFaNaC, the detailed sites remain unclear [24, 25]
Assuming that residues 89 –211 contribute to the different FMRFamide affinity between HaFaNaC and HtFaNaC [24, 25], we constructed a series of chimeras by substitution of the sequences of HaFaNaC with corresponding sequences on the HtFaNaC, resulting in 13 chimeras, namely chimera 1 (Ch1, Gln-110 –Ile-116, HaFaNaC numbering, hereinafter), chimera 2 (Ch2, Leu-124 – Thr-126), chimera 3 (Ch3, Arg-128 –Tyr-131), chimera 4 (Ch4, Phe-132–Asn-134), chimera 5 (Ch5, Ser-136 –Asn-138), chimera 6 (Ch6, Leu-139 –Thr141), chimera 7 (Ch7, Arg-144 – Arg-150), chimera 8 (Ch8, D154QK), chimera 9 (Ch9, Asn158 –Ile-160), chimera (Ch10, Gln-169 –Leu-174), chimera (Ch11, Asn-177–Met-183), chimera (Ch12, Leu-191– Val-194) and chimera (Ch13, Ser-198 –Phe-200) (Fig. 2A)
Summary
APRIL 1, 2016 VOLUME 291 NUMBER 14 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. It is 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. Analyzing the structure of cASIC1 (Fig. 1B) suggests that insertion I is a ϳ20 amino acid sequence between ␣4 and
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