Biology subjects

Goldschen-Ohm, M. P.

Publications and source records attributed to Goldschen-Ohm, M. P..

12 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

Asymmetric roles for M2-M3 linkers of specific GABAA receptor subunits in the intrinsic closed-open equilibrium and its modulation by the benzodiazepine diazepam

GABAA receptors (GABAARs) are neurotransmitter-gated ion channels critical for inhibitory synaptic transmission as well as the molecular target for benzodiazepines (BZDs), one of the most widely prescribed class of psychotropic drugs today. Despite structural insight into the conformations underlying functional channel states, the detailed molecular interactions involved in conformational transitions and the physical basis for their modulation by BZDs are not fully understood. We previously identified that alanine substitution at the central residue in the 1 subunit M2-M3 linker (V279A) enhances the efficiency of linkage between the BZD site and the pore gate. Here, we expand on this work by investigating the effect of alanine substitutions at the analogous positions in the M2-M3 linkers of {beta}2 (I275A) and {gamma}2 (V290A) subunits, which together with 1 comprise typical heteromeric 1{beta}2{gamma}2 synaptic GABAARs. We find that these mutations confer subunit-specific effects on the intrinsic pore closed-open equilibrium and its modulation by the BZD diazepam (DZ). The mutations 1(V279A) or {gamma}2(V290A) bias the channel toward a closed conformation, whereas {beta}2(I275A) biases the channel toward an open conformation to the extent that the channel becomes leaky and opens spontaneously in the absence of agonist (e.g., GABA). In contrast, only 1(V279A) enhances the efficiency of DZ-to-pore linkage, whereas mutations in the other two subunits have no effect. These observations show that the central residue in the M2-M3 linkers of distinct subunits in synaptic 1{beta}2{gamma}2 GABAARs contribute asymmetrically to the intrinsic closed-open equilibrium and its modulation by DZ.

biophysics↗

bioRxiv2022

Zn2+ potentiation of OTOP proton channels identifies structural elements of the gating apparatus

Otopetrin proteins (OTOPs) form proton-selective ion channels that are expressed in diverse cell types where they may mediate detection of acids or regulation of pH. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vesibular system and it forms the receptor for sour taste, while the functions of OTOP2, and OTOP3 are not yet known. Importantly, the gating mechanisms of any of the OTOP channels are not well-understood, and until recently, it was not even known if the channels were gated. Here we show that Zn2+, as well as other transition metals including copper (Cu2+), potently activate murine OTOP3. Zn2+ pre-exposure increases the magnitude of OTOP3 currents to a subsequent acid stimulus by as much as 10-fold. In contrast, OTOP2 currents are insensitive to potentiation by Zn2+. Swapping the extracellular tm 11-12 linker between OTOP3 and OTOP2 was sufficient to eliminate Zn2+ potentiation of OTOP3 and confer Zn2+ potentiation on OTOP2. We also show that H531 within the tm 11-12 linker is essential for potentiation of OTOP3 by Zn2+, likely by forming part of its binding site. Kinetic modeling of the data is consistent with Zn2+ stabilizing the open state of the channel, possibly competing with H+ for activation of the channels. These results establish the tm 11-12 linker as part of the gating apparatus of OTOP channels and a target for drug discovery. Zinc is an essential micronutrient and its regulation of OTOP channels will undoubtedly have important physiological sequelae. Significance StatementA family of proton-activated H+ ion channels was recently identified that includes the sour receptor OTOP1. Here we show that members of the OTOP channel family are differentially sensitive to Zn2+, which strongly activates OTOP3 but not OTOP2. By studying chimeric channels, we identify structural elements in the extracellular linker between transmembrane domains 11-12 that are necessary and sufficient for Zn2+ activation of OTOP channels. In addition to the taste system, OTOP channels play important roles in biomineralization in both vertebrate and invertebrates and are expressed in the digestive tract where their expression is a predictor of cancer prognosis. Our identification of the tm 11-12 linker as part of the gating apparatus makes it a promising target for pharmaceutical discovery.

biophysics↗

bioRxiv2021

smBEVO: A computer vision approach to rapid baseline correction of single-molecule time series

Single-molecule time series inform on the dynamics of molecular mechanisms that are occluded in ensemble-averaged measures. Amplitude-based methods and hidden Markov models (HMMs) frequently used for interpreting these time series require removal of low frequency drift that can be difficult to completely avoid in real world experiments. Current approaches for drift correction primarily involve either tedious manual assignment of the baseline or unsupervised frameworks such as infinite HMMs coupled with baseline nodes that are computationally expensive and unreliable. Here, we develop an image-based method for baseline correction using techniques from computer vision such as lane detection and active contours. The approach is remarkably accurate and efficient, allowing for rapid analysis of single-molecule time series contaminated with nearly any type of slow baseline drift.

bioinformatics↗

bioRxiv2021

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in nonnative lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. We further show that binding of the second ligand in the tetrameric channel is less cooperative than previously estimated from ensemble-averaged binding measures. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

biophysics↗

bioRxiv2021

Unsupervised selection of optimal single-molecule time series idealization criterion

Single-molecule (SM) approaches have provided valuable mechanistic information on many biophysical systems. As technological advances lead to ever-larger datasets, tools for rapid analysis and identification of molecules exhibiting the behavior of interest are increasingly important. In many cases the underlying mechanism is unknown, making unsupervised techniques desirable. The Divisive Segmentation and Clustering (DISC) algorithm is one such unsupervised method that idealizes noisy SM time series much faster than computationally intensive approaches without sacrificing accuracy. However, DISC relies on a user selected objective criterion (OC) to guide its estimation of the ideal time series. Here, we explore how different OCs affect DISCs performance for data typical of SM fluorescence imaging experiments. We find that OCs differing in their penalty for model complexity each optimize DISCs performance for time series with different properties such as signal-to-noise and number of sample points. Using a machine learning approach, we generate a decision boundary that allows unsupervised selection of OC based on the input time series to maximize performance for different types of data. This is particularly relevant for SM fluorescence datasets which often have signal-to-noise near the derived decision boundary and include time series of nonuniform length due to stochastic bleaching. Our approach allows unsupervised per-molecule optimization of DISC, which will substantially assist rapid analysis of high-throughput single-molecule datasets with noisy samples and nonuniform time windows.

bioinformatics↗

bioRxiv2020

A critical residue in the α1M2-M3 linker regulating GABAA receptor pore gating by diazepam

Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs. Their anxiolytic and sedative effects are conferred by modulating the activity of GABAA receptors (GABAARs), which are the primary inhibitory neurotransmitter receptors throughout the central nervous system. However, the physical mechanism by which BZDs exert their effects on the receptor is poorly understood. In particular, BZDs require coapplication with an agonist to effectively open the channel pore, making it difficult to dissect whether the drug has altered either agonist binding or channel gating as these two processes are intimately coupled. To isolate effects on gating we used a spontaneously active gain of function mutant (1L9T{beta}2{gamma}2L) that is directly gated by BZDs alone in the absence of agonist. In the 1L9T background we explored effects of alanine substitutions throughout the 1M2-M3 linker on modulation of the channel pore by the BZD positive modulator diazepam (DZ). The M2-M3 linker is known to be an important element for channel activation. Linker mutations generally impaired unliganded pore opening, indicating that side chain interactions are important for channel gating in the absence of bound agonist. All but one mutation had no effect on the transduction of chemical energy from DZ binding to pore gating. Strikingly, 1V279A doubles DZs energetic contribution to gating, whereas larger side chains at this site do not. In a wild-type background 1V279A enhances DZ-potentiation of currents evoked by saturating GABA, consistent with a direct effect on the pore closed/open equilibrium. Our observations identify an important residue regulating coupling between the BZD site and the pore gate, thereby shedding new light on the molecular mechanism of a frequently prescribed class of psychotropic drugs.

molecular biology↗