Biology subjects

Borghese, C. M.

Publications and source records attributed to Borghese, C. M..

7 recordsLinked to original sources

bioRxiv2026

Single-molecule nanophotonic resolution of binding dynamics from apo to fully liganded for a cyclic nucleotide-gated ion channel in cell-derived vesicles

Many biological macromolecules are activated upon ligand binding at multiple specific binding domains. However, how these domains interact and the transient intermediate conformations that connect binding events to protein activity are typically unknown. Ensemble-averaged measures over stochastic binding events are challenged to resolve the underlying asynchronous dynamics. Single-molecule resolution of these dynamics offers an attractive approach to investigate the ligand-activation process. Optical methods using fluorescently labeled ligands enable observation of individual binding events that report on the energetics of early ligand-bound conformational changes. However, diffraction-limited microscopy limits these methods to low ligand concentrations, often below what is required for physiologically relevant activation. Here, we overcome this limitation using nanophotonic zero-mode waveguides to observe the sequential binding of a fluorescent cyclic nucleotide to each of four subunits in TAX-4 cyclic nucleotide-gated ion channels in cell-derived vesicles. Our observations suggest that binding at one domain positively promotes binding at other domains, and that binding induces an isomerization of the binding domain in individual subunits which we attribute to a sequence of pre-activated intermediate states. This approach provides a broadly applicable tool to dissect the energetic landscape of ligand-binding at macromolecules in native cell membranes.

biophysics↗

bioRxiv2026

An atomic interaction conserved for over 600 million years gates inhibitory neurotransmission

Pentameric ligand-gated ion channels (pLGICs) mediate fast inhibitory neurotransmission critical for neuronal network stability. A tyrosine residue in the M2-M3 linker of inhibitory pLGICs, conserved for over 600 million years, is positioned where it could hydrogen bond (H-bond) to the backbone of the neighboring Cys-loop. Given the pathogenic effects of variants of this tyrosine, we hypothesized that this H-bond stabilizes extracellular-to-transmembrane domain coupling essential for channel gating. To test this hypothesis, we used site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings in Xenopus laevis oocytes to disrupt this hydrogen bond in GABAA and glycine receptors. Loss of this interaction via tyrosine substitutions or backbone amide modifications that ablate the acceptor or donor, respectively, markedly decrease agonist sensitivity and maximal channel activation, with effects localized to specific subunits. Molecular dynamics simulations support a role for this H-bond in channel gating. These findings reveal a critical atomic interaction underlying a shared mechanism for inhibitory receptor gating and provide a mechanistic explanation for disease-associated mutations linked to epilepsy, neurodevelopmental disability, and hyperekplexia.

biophysics↗

bioRxiv2026

The human GABAA receptor α4 subunit variant Ile114Asn linked to epilepsy impairs membrane expression with β3 and δ subunits

GABAA receptors mediate fast inhibitory neurotransmission in the brain, and variants in their subunits are linked to neurological disorders like epilepsy. For many identified variants, it remains to be determined whether the variant is pathogenic. Here, we functionally characterized the 4 subunit variant p.I114N (p.I79N in the mature protein), found in an individual with epilepsy, using patch-clamp electrophysiology in combination with {beta}3 and {delta} subunits in HEK293T cells. Whereas the variant has little to no impact on basal activity or GABA-sensitivity, lower maximal GABA-elicited currents as compared to wild-type channels suggest that it impairs assembly or trafficking to reduce expression of channels in the plasma membrane.

biophysics↗

bioRxiv2026

Introducing a proline in the α1 M2-M3 linker relieves a molecular brake on channel activation in α1β2γ2 GABAA receptors

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) essential for inhibitory synaptic transmission throughout the central nervous system. Despite progress in understanding their three-dimensional structure, the molecular basis for how neurotransmitter binding is transduced to ion channel gating remains poorly understood. Furthermore, relatively little is known about the contributions of distinct subunits to this coupling within typical heteromeric receptors. A highly conserved proline (site 1) in the M2-M3 linker of pLGIC subunits is involved in channel gating - e.g., P273 in the GABAAR {beta}2 subunit. In GABAARs, only the {beta} subunits have an additional proline in the M2-M3 linker (site 2) - e.g., {beta}2(P276) - whereas all other subunits have a non-proline at the homologous site 2 position. Here, we investigate the functional contribution of proline at site 2 in distinct subunits of 1{beta}2{gamma}2 GABAARs. We expressed wild type or mutant 1{beta}2{gamma}2 GABAARs in Xenopus laevis oocytes and used two-electrode voltage clamp electrophysiology to record channel currents in response to GABA and/or other ligands. First, we introduced a proline at site 2 in 1 or {gamma}2 subunits: 1(A280P) and {gamma}2(S291P). Second, we replaced the site 2 proline in the {beta}2 subunit with its homologous non-proline residue from 1 or {gamma}2 subunits: {beta}2(P276A) or {beta}2(P276S). We show that 1(A280P) confers enhanced GABA-sensitivity and spontaneous unliganded channel activity, whereas {gamma}2(S291P) has minor effects on channel activation. In contrast, {beta}2(P276A) or {beta}2(P276S) either had no effect or enhanced GABA-activation, respectively, indicating complex functional dependence on the side chain at site 2 in the {beta}2 subunit. When in combination with other substitutions, the presence or absence of 1(A280P) was consistently correlated with enhanced GABA-sensitivity and spontaneous activity. Thus, introduction of a proline at site 2 in the 1 M2-M3 linker biases the channel towards an activated state and prevents it from remaining closed at rest.

biophysics↗

bioRxiv2026

Open-State Dynamics and Allosteric Modulation of the α1β3γ2 GABAA Receptor Stabilized by L9'T/S Substitutions

GABA type A receptors (GABAARs) mediate inhibitory neurotransmission, and their dysfunction contributes to epilepsy, anxiety, and depression. Although closed and desensitized structures of heteropentameric GABAARs are known, an open-state conformation has been difficult to capture. Here we use in-silico mutagenesis and Gaussian-accelerated MD to stabilize open-like ensembles of the 1{beta}3{gamma}2 receptor via hydrophilic substitutions at the hydrophobic 9' gate (L9'T/L9'S). The mutants expand the pore at 9' and 20' (extracellular pore entry), increase hydration and water flux, and lower Cl- permeation barriers at 9' and -2' (desensitization gate) from [~]19/[~]9 kcal mol-{superscript 1} (WT) to [~]1.3-1.6/[~]3.0-3.4 kcal mol-1, yielding ohmic conductance [~]10-30 pS in computational electrophysiology. Conformationally, the mutants show reduced twist, outward M2 tilts, and C-loop closure, consistent with activation-like signatures. On this open-like background, PAMs (diazepam, ganaxolone) primarily tune the residual -2' constriction, whereas bicuculline (orthosteric antagonist) drives a time-ordered, bottom-to-top closing sequence (-2' first, then 9'/20', then twist) via an asymmetric collapse of the M2 bundle that approaches closed-state landmarks while sampling desensitized-like ECD-TMD coupling. Two-electrode voltage clamp confirms spontaneous activity in L9'T-containing receptors, supporting the mutant-stabilized open-like ensembles. These results provide a coherent atomistic framework for gating and allosteric modulation in 1{beta}3{gamma}2 GABAARs and establish a tractable, ligand-responsive platform for state-selective structure-based design.

biophysics↗

bioRxiv2024

A single main-chain hydrogen bond required to keep GABAA receptors closed

GABAA receptors are the primary inhibitory neurotransmitter receptors throughout the central nervous system. Despite significant progress understanding their three-dimensional structure, a critical gap remains in determining the molecular basis for channel gating. We recently identified M2-M3 linker mutations that suggest linker flexibility has asymmetric subunit-specific correlations with channel opening. Here we use non-canonical amino acids (ncAAs) to investigate the role of main-chain H-hydrogen bonds (H-bonds) that may stabilize the M2-M3 linkers. We show that a single main-chain H-bond within the {beta}2 subunit M2-M3 linker inhibits pore opening and is required to keep the unliganded channel closed. Furthermore, breaking this H-bond during channel opening accounts for approximately one third of the activation energy derived from GABA binding. In contrast, the analogous H-bond in the 1 subunit has no effect on gating. Our observations suggest that channel opening involves state-dependent breakage/disruption of a specific main-chain H-bond within the {beta}2 subunit M2-M3 linker.

biophysics↗

bioRxiv2023

A potential cost of evolving epibatidine resistance in poison frogs

BackgroundSome poison arrow frogs sequester the toxin epibatidine as a defense against predators. We previously identified a single amino acid substitution (S108C) at a highly conserved site in a neuronal nicotinic acetylcholine receptor (nAChR) {beta}2 subunit that prevents epibatidine from binding to this receptor. When placed in a homologous mammalian nAChR this substitution minimized epibatidine binding but also perturbed acetylcholine binding, a clear cost. However, in the nAChRs of poison arrow frogs, this substitution appeared to have no detrimental effect on acetylcholine binding and, thus, appeared cost-free. ResultsThe introduction of S108C into the 4{beta}2 nAChRs of non-dendrobatid frogs also does not affect ACh sensitivity, when these receptors are expressed in Xenopus laevis oocytes. However, 4{beta}2 nAChRs with C108 had a decreased magnitude of neurotransmitter-induced currents in all species tested (Epipedobates anthonyi, non-dendrobatid frogs, as well as human), compared with 4{beta}2 nAChRs with the conserved S108. Immunolabeling of frog or human 4{beta}2 nAChRs in the plasma membrane using radiolabeled antibody against the {beta}2 nAChR subunit shows that C108 significantly decreased the number of cell-surface 4{beta}2 nAChRs, compared with S108. ConclusionsWhile S108C protects these species against sequestered epibatidine, it incurs a potential physiological cost of disrupted 4{beta}2 nAChR function. These results may explain the high conservation of a serine at this site in vertebrates, as well as provide an example of a tradeoff between beneficial and deleterious effects of an evolutionary change. They also provide important clues for future work on assembly and trafficking of this important neurotransmitter receptor.

evolutionary biology↗