Biology subjects

Lynagh, T.

Publications and source records attributed to Lynagh, T..

7 recordsLinked to original sources

bioRxiv2024

Diarylamidine activation of a brachiopod DEG/ENaC/ASIC channel

Diarylamidines are a group of widely used small molecule drugs. One common use of diarylamidines is their pharmacological inhibition of ligand-gated cation channels, including tetrameric ionotropic glutamate receptors and trimeric degenerin/epithelial sodium channel channel/acid-sensing ion channels (DEG/ENaC/ASICs). Here, we discover a DEG/ENaC/ASIC channel from the brachiopod (lamp shell) Novocrania anomala, at which diarylamidines act as agonists. The channel is closely related to bile acid-gated, pH-gated, and peptide-gated channels but is not activated by such stimuli. We describe activation of the channel by diminazene, DAPI, and pentamidine, examine several biophysical and pharmacological properties, and briefly explore the molecular determinants of channel activity with site-directed mutagenesis. We term this channel the diarylamidine-activated sodium channel (DiaaNaC).

biophysics↗

bioRxiv2023

Constitutive activity of ionotropic glutamate receptors via a hydrophobic plug in the ligand-binding domain

Neurotransmitter ligands electrically excite neurons by activating ionotropic glutamate receptor (iGluR) ion channels. Knowledge of the iGluR amino acid residues that dominate ligand-induced activation would enable the prediction of function from sequence. We therefore explored the molecular determinants of activity in rat NMDA-type iGluRs (NMDA receptors), complex heteromeric iGluRs comprising two glycine-binding GluN1 and two glutamate-binding GluN2 subunits, using amino acid sequence analysis, mutagenesis, and electrophysiology. We find that a broadly conserved aspartate residue controls both ligand potency and channel activity, to the extent that certain substitutions at this position bypass the need for ligand-binding in GluN1 subunits, generating NMDA receptors activated solely by glutamate. Furthermore, we identify a homomeric iGluR from the placozoan Trichoplax adhaerens that has utilized native mutations of this crucial residue to evolve into a leak channel that is inhibited by neurotransmitter binding, pointing to a dominant role of this residue throughout the iGluR superfamily.

biophysics↗

bioRxiv2023

Structural basis for excitatory neuropeptide signaling

Rapid chemo-electric signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain1. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily, which occurs throughout the animal kingdom, is unique in its diversity of gating stimuli, with some DEG/ENaCs gated by conventional ligands such as neuropeptides, and others gated by e.g. pH, mechanical force, or enzymatic activity2-5. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. We have therefore dissected the structural basis for neuropeptide binding and gating in a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus6, using cryo-electron microscopy. High-resolution structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes that mediate channel gating. Complementary mutagenesis experiments confirm the functional roles of particular amino acid residues implicated by the structures. Our results illuminate ligand-induced channel gating in DEG/ENaCs and offer a structural template for the experimental dissection of channel pharmacology and ion conduction in a characteristically metazoan ion channel superfamily.

neuroscience↗

bioRxiv2022

Comparative analysis defines a broader FMRFamide-gated sodium channel family and determinants of neuropeptide sensitivity

FMRFamide and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFamide activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFamide potency in one of its native targets, the excitatory FMRFamide-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of function, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFamide, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching DEG/ENaC superfamily, incorporating mutagenesis and experimental dissection of function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFamide activity at FaNaCs and illuminates evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.

biochemistry↗

bioRxiv2022

Peripheral and central employment of acid-sensing ion channels during early bilaterian evolution

Nervous systems are endowed with rapid chemosensation and intercellular signaling by ligand-gated ion channels (LGICs). While a complex, bilaterally symmetrical nervous system is a major innovation of bilaterian animals, the employment of specific LGICs during early bilaterian evolution is poorly understood. We therefore questioned bilaterian animals employment of acid-sensing ion channels (ASICs), LGICs that mediate fast excitatory responses to decreases in extracellular pH in vertebrate neurons. Our phylogenetic analysis identified an earlier emergence of ASICs from the overarching DEG/ENaC superfamily than previously thought and suggests that ASICs were a bilaterian innovation. Our broad examination of ASIC gene expression and biophysical function in each major bilaterian lineage of Xenacoelomorpha, Protostomia, and Deuterostomia, suggests that the earliest bilaterian ASICs were probably expressed in the periphery, before being incorporated into the brain as it emerged independently in certain deuterostomes and xenacoelomorphs. The loss of certain peripheral cells from Ecdysozoa when they split from other protostomes likely explains their loss of ASICs, and thus the absence of ASICs in model organisms Drosophila and C. elegans. Thus, our use of diverse bilaterians in the investigation of LGIC expression and function offers a unique hypothesis on the employment of LGICs in early bilaterian evolution.

evolutionary biology↗

bioRxiv2021

Differential contributions of M1 and pre-M1 to ion selectivity in ASICs and ENaCs

The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium around 10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). The most obvious explanation for this discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we use conventional and non-canonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that they contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work supports the notion that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. The results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.

biophysics↗

bioRxiv2019

Mechanism and site of action of big dynorphin on ASIC1a

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics. Significance StatementNeuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.

biophysics↗