The Conorfamide RPRFa Stabilizes the Open Conformation of Acid-Sensing Ion Channel 3 via the Nonproton Ligand–Sensing Domain

Abstract

Acid-sensing ion channel 3 (ASIC3) is a proton-gated Na channel with important roles in pain. ASIC3 quickly desensitizes in less than a second, limiting its capacity to sense sustained acidosis during pain. RFamide neuropeptides are modulators of ASIC3 that slow its desensitization and induce a variable sustained current. The molecular mechanism of slowed desensitization and the RFamide binding site on ASIC3 are unknown. RPRFamide, a RFamide from the venom of a cone snail, has a comparatively high affinity for ASIC3 and strongly slows its desensitization. Here we show that covalent binding of a UV-sensitive RPRFamide variant to ASIC3 prevents desensitization, suggesting that RPRFamide has to unbind from ASIC3 before it can desensitize. Moreover, we show by in silico docking to a homology model of ASIC3 that a cavity in the lower palm domain, which is also known as the nonproton ligand-sensing domain, is a potential binding site of RPRFamide. Finally, using extensive mutagenesis of residues lining the nonproton ligand-sensing domain, we confirm that this domain is essential for RPRFamide modulation of ASIC3. As comparative analysis of ASIC crystal structures in the open and in the desensitized conformation suggests that the lower palm domain contracts during desensitization, our results collectively suggest that RPRFamide, and probably also other RFamide neuropeptides, bind to the nonproton ligand-sensing domain to stabilize the open conformation of ASIC3.

Mechanism and site of action of big dynorphin on ASIC1a

“…Together, this argues for an extended interaction surface on ASIC1a, likely extending from the peripheral thumb domain around the α5 helix and into the acidic pocket. While the location of the BigDyn binding site is distinct from that suggested for RFamide neuropeptides on ASICs (41,42), it does overlap with the binding site for PcTx1 (10,25,26,31). This is consistent with the finding that RFamides do not functionally compete with BigDyn or PcTx1 (10,43).…”

Mutations in the palm domain disrupt modulation of acid-sensing ion channel 1a currents by neuropeptides

“…This indicated that FRRFa interferes with conformational changes in the palm, or may impair the association of MTSET with L280C. A very recent study that applied docking and mutagenesis, suggests the central vestibule as binding site of the related peptide RPRFa on ASIC3 37 .…”

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

“…Ion channels have proven highly suited to ncAA incorporation, as evidenced by the success in introducing photocrosslinking, photoswitchable or fluorescent ncAAs into numerous members of this large and diverse protein family (Klippenstein et al ., 2018; Paoletti et al ., 2019; Braun et al ., 2020). Among the ncAA subclasses, photocrosslinkers have proven particularly versatile, as they allow for the trapping of ion channels in certain conformational states (Klippenstein et al ., 2014; Zhu et al ., 2014; Poulsen et al ., 2019; Rook et al ., 2020b), capturing of protein-protein interactions (Martin et al ., 2016; Murray et al ., 2016; Tian & Ye, 2016; Westhoff et al ., 2017) and covalent linking of receptor-ligand complexes to delineate ligand binding sites (Coin et al ., 2013; Rannversson et al ., 2016; Reiners et al ., 2018; Borg et al ., 2020; Bottke et al ., 2020).…”