Biology subjects

Twomey, E. C.

Publications and source records attributed to Twomey, E. C..

11 recordsLinked to original sources

bioRxiv2026

GluDs are ionotropic dopamine receptors tuned by G-proteins

Dopamine is a neurotransmitter essential for cognition, and its dysregulation is associated with neurological diseases1,2. Historically, dopamine has been understood to signal exclusively through metabotropic receptors3. Delta-type ionotropic glutamate receptors (GluDs), which have recently been established as ligand-gated ion channels4,5, are fundamental for synaptic maintenance, are implicated in neurological disorders, and co-localize with dopaminergic machinery. Here, we report that dopamine is a direct agonist of GluDs, eliciting ionotropic activity, as visualized by cryo-electron microscopy (cryo-EM), bilayer recordings, mutagenesis, and patch clamp recordings. Dopamine binds to the GluD ligand binding domain, inducing clamshell closure and channel activation through a distinct molecular interface. GluD channel activity is tightly regulated by G-proteins, which act as molecular switches to tune GluD activity: free G{beta}{gamma} inhibits ligand-gating, while G or inactive G-protein heterotrimers enable dopamine-induced GluD currents. This tuning of GluD activity by G-proteins is uncoupled in a point mutation associated with neurodegeneration. These findings expand mechanisms of neuronal dopaminergic signaling, uncover how G-proteins tune GluD channel activity, and provide a framework for targeting GluDs in neurological diseases.

biophysics↗

bioRxiv2026

Molecular basis for ligand-gating of the human GluD1 receptor

The delta-type ionotropic glutamate receptors (iGluRs) GluD1 and GluD2 are ligand-gated ion channels that are fundamental for regulating both excitatory and inhibitory synapses. Rising evidence points to the role of GluD1 in the development of neurological diseases. However, the ultrastructure of human GluD1 (hGluD1) and the molecular basis for its ligand-gating remain unclear. Here, we define the structure of hGluD1 and resolve its ligand-gating mechanism using cryo-electron microscopy (cryoEM) and single channel bilayer recording. While hGluD1 exhibits a non-swapped architecture, it contains conserved iGluR moieties that enable ligand-gating, such as a ligand-binding domain (LBD) tethered to a transmembrane ion channel. Binding of the neurotransmitter {gamma}-aminobutyric acid (GABA) or D-serine to the LBD enables cation influx through the hGluD1 ion channel. Our findings delineate the molecular architecture and function of hGluD1, provide foundations for understanding patient mutations in hGluD1, and will invigorate therapeutic development against hGluD1.

biophysics↗

bioRxiv2026

Cryo-EM reveals a right-handed double-helix dimer architecture of PCDH15 critical for mechanotransduction

Tip links connect the stereocilia of mechanosensory hair cells in the inner ear and transmit force onto mechanotransduction (MET) channels. Tip links consist of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), which assemble into an extracellular filament approximately 150 nm in length. Rare freeze-etched electron microscopy (EM) images have suggested that tip links could be right-handed double helices in vivo, but direct structural evidence has been lacking. Using cryo-EM we determined the structure of a large part of the extracellular PCDH15 domain. Two PCDH15 molecules form a parallel cis dimer stabilized by several dimerization interfaces, including two strand crossovers and two parallel contacts, yielding a right-handed double helix. Functional studies show that mutations in PCDH15 dimerization-domains impair MET. Our results establish the molecular foundation for how PCDH15 forms a right-handed double helix to enable mechanical sensing.

neuroscience↗

bioRxiv2025

Puromycin-sensitive aminopeptidase acts as an inhibitory auxiliary subunit of volume-regulated anion channels

Volume-regulated anion channels (VRACs) are large-pore channels present in nearly all vertebrate cells, playing key roles in cell volume regulation and autocrine/paracrine signaling. Here, we identify the ubiquitously expressed puromycin-sensitive aminopeptidase (PSA) as a binding partner of the obligatory VRAC subunit SWELL1 (also known as LRRC8A) and report the cryo-electron microscopy structure of the SWELL1-PSA complex. Three PSA molecules associate with a single SWELL1 hexamer, coupling adjacent leucine-rich repeat (LRR) domains into local dimers. Functionally, PSA overexpression suppresses VRAC activation, whereas its deletion results in elevated basal channel activity. Notably, PSAs regulatory role on VRACs is independent of its aminopeptidase activity. Our findings identify PSA as the first auxiliary subunit of VRACs, highlight the role of intracellular LRR domains in allosteric channel gating, and propose a new strategy for modulating VRAC function in diverse physiological contexts, including cGAMP transport and STING signaling.

molecular biology↗

bioRxiv2025

Mechanisms of Ion Permeation in the AMPA Receptor Ion Channel

Excitatory synaptic transmission in the human nervous system is mediated by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), tetrameric ligand-gated ion channels localized in the excitatory post-synaptic membrane. AMPARs are activated by the binding of the neurotransmitter glutamate (Glu), which opens the ion channel and allows the influx of Na+ and Ca2+ ions into the post-synaptic neuron, initiating signal transduction. Despite many efforts, a bona fide ion permeation pathway of both monovalent and divalent cations in AMPARs remains elusive. From analyzing our cryo-electron microscopy (cryo-EM) map of an open calcium-permeable AMPAR (CP-AMPAR) ion channel, we identified potential sites vital to permeation of cations through the channel. To delineate mechanisms of permeation, we studied the channel with all-atom molecular dynamics (MD) simulations. Both Na+ and Ca2+ ions are coordinated by an entry site at the top of the channel prior to entering the selectivity filter. A mutation at the filter (Q607E), implicated in a neurodevelopmental disorder, makes the channel more susceptible to Zn2+ block but also creates a more energetically favorable environment for Na+ and Ca2+ permeation through the ion channel. These findings describe a biophysical basis for ion permeation in CP-AMPARs and how disease mutations alter the channel, which will inform therapeutic design against disease mutations in AMPARs that alter the ion channel.

biophysics↗

bioRxiv2025

Architecture, Activation, and Conformational Plasticity in the GluA4 AMPA Receptor

AMPA-subtype glutamate receptors (AMPARs), composed of subunits GluA1-4, mediate fast, excitatory synaptic transmission in the brain. After glutamate binding, AMPAR ion channels exhibit multiple subconductance states that tune neuronal responses to glutamate. GluA4 is the rarest subunit in the brain but is enriched in interneurons. Rising evidence points to the role of GluA4 AMPARs in the development of neurological diseases, but the structural mechanisms of GluA4 function have remained enigmatic. Here, from bilayer recordings and cryo-electron microscopy (cryo-EM), we report the unique features of GluA4 AMPARs that tune receptor function. We find that GluA4 AMPARs have a canonical "Y" shaped architecture where local dimer pairs are domain-swapped between the amino terminal domain (ATD) and ligand binding domain (LBD), both of which comprise the extracellular domain. All four LBDs are glutamate bound yet open the GluA4 ion channel by asymmetric hinging in all channel helices. We observe that the glutamate-saturated LBD has conformational plasticity, and the different conformations of the LBD tune the ion channel gate below. These data provide a framework for understanding how channel subconductance can occur during conditions of saturating glutamate, outline the unique properties of GluA4, expand our understanding of conformational plasticity in AMPARs, and will inform therapeutic design.

biophysics↗

bioRxiv2024

Temperature Sensitive Glutamate Gating of AMPA-subtype iGluRs

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that mediate the majority of excitatory neurotransmission1. iGluRs are gated by glutamate, where upon glutamate binding, they open their ion channels to enable cation influx into post-synaptic neurons, initiating signal transduction2. The structural mechanism of iGluR gating by glutamate has been extensively studied in the context of positive allosteric modulators (PAMs)3-15. A fundamental question has remained - are the PAM activated states of iGluRs representative of glutamate gating in the absence of PAMs? Here, using the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype iGluR (AMPAR) we show that glutamate gating is unique from gating in the presence of PAMs. We demonstrate that glutamate gating is temperature sensitive, and through temperature-resolved cryo-electron microscopy (cryo-EM), capture all major glutamate gating states. Physiological temperatures augment channel activation and conductance. Activation by glutamate initiates ion channel opening that involves all ion channel helices hinging away from the pores axis in a motif that is conserved across all iGluRs. Desensitization occurs when the local dimer pairs decouple and enables closure of the ion channel below through restoring the channel hinges and refolding the channel gate. Our findings define how glutamate gates iGluRs, provide foundations for therapeutic design, and point to iGluR gating being temperature sensitive.

biophysics↗

bioRxiv2024

Memantine Inhibits Calcium-Permeable AMPA Receptors

Memantine is an US Food and Drug Administration (FDA) approved drug that selectively inhibits NMDA-subtype ionotropic glutamate receptors (NMDARs) for treatment of dementia and Alzheimers. NMDARs enable calcium influx into neurons and are critical for normal brain function. However, increasing evidence shows that calcium influx in neurological diseases is augmented by calcium-permeable AMPA-subtype ionotropic glutamate receptors (AMPARs). Here, we demonstrate that these calcium-permeable AMPARs (CP-AMPARs) are inhibited by memantine. Electrophysiology unveils that memantine inhibition of CP-AMPARs is dependent on their calcium permeability and the presence of their neuronal auxiliary subunit transmembrane AMPAR regulatory proteins (TARPs). Through cryo-electron microscopy we elucidate that memantine blocks CP-AMPAR ion channels in a unique mechanism of action from NMDARs. Furthermore, we demonstrate that memantine reverses a gain of function AMPAR mutation found in a patient with a neurodevelopmental disorder and inhibits CP-AMPARs in nerve injury. Our findings alter the paradigm for the memantine mechanism of action and provide a blueprint for therapeutic approaches targeting CP-AMPARs.

biophysics↗

bioRxiv2023

Allosteric Competition and Inhibition in AMPA Receptors

Excitatory neurotransmission is principally mediated by AMPA-subtype ionotropic glutamate receptors (AMPARs). Dysregulation of AMPARs is the cause of many neurological disorders and how therapeutic candidates such as negative allosteric modulators inhibit AMPARs is unclear. Here, we show that non-competitive inhibition desensitizes AMPARs to activation and prevents positive allosteric modulation. We dissected the noncompetitive inhibition mechanism of action by capturing AMPARs bound to glutamate and the prototypical negative allosteric modulator, GYKI-52466, with cryo-electron microscopy. Noncompetitive inhibition by GYKI-52466, which binds in the transmembrane collar region surrounding the ion channel, negatively modulates AMPARs by decoupling glutamate binding in the ligand binding domain from the ion channel. Furthermore, during allosteric competition between negative and positive modulators, negative allosteric modulation by GKYI-52466 outcompetes positive allosteric modulators to control AMPAR function. Our data provide a new framework for understanding allostery of AMPARs and foundations for rational design of therapeutics targeting AMPARs in neurological diseases.

biophysics↗

bioRxiv2023

Structure of Transmembrane AMPA Receptor Regulatory Protein Subunit γ2

Transmembrane AMPA receptor regulatory proteins (TARPs) are claudin-like proteins that tightly regulate AMPA receptors (AMPARs) and are fundamental for excitatory neurotransmission. We used cryo-electron microscopy (cryo-EM) to reconstruct the 36 kDa TARP subunit {gamma}2 to 2.3 [A] and reveal the structural diversity of TARPs. Our data reveals critical motifs that distinguish TARPs from claudins and define how sequence variations within TARPs differentiate subfamilies and their regulation of AMPARs.

biophysics↗

bioRxiv2023

Time-Resolved Cryo-EM Specimen Preparation with Single Millisecond Precision

Molecular structures can be determined in vitro and in situ with cryo-electron microscopy (cryo-EM). Specimen preparation is a major obstacle in cryo-EM. Typical sample preparation is orders of magnitude slower than biological processes. Time-resolved cryo-EM (TR-cryo-EM) can capture short-lived states. Here, we present Cryo-EM Sample Preparation with light-Activated Molecules (C-SPAM), an open-source, photochemistry-coupled device for TR-cryo-EM with single millisecond resolution, tunable timescales, and broad biological applications.

biophysics↗