Biology subjects

Cowgill, J.

Publications and source records attributed to Cowgill, J..

8 recordsLinked to original sources

bioRxiv2025

Structural basis for activation and potentiation in a human α5β3 GABAA receptor

General anesthetics like etomidate mediate amnesic as well as sedative effects through different populations of gamma amino butyric acid receptors (GABAARs) in the central nervous system. The amnesic effects have largely been attributed to 5-subunit-containing receptors in the hippocampus, indicating a clear role in learning and memory for these receptors. The 5 subunit is thought to primarily coassemble with {beta}3 and, in some cases, {gamma}2 subunits, generating a variety of receptor subtypes with differential functional and pharmacological properties. However, the stoichiometry, structure, and gating mechanisms of these different subpopulations are not well understood. Here we report structures of human 5{beta}3 GABAARs with various modulators, assembled in two stoichiometries. Our cryo-EM structures, combined with electrophysiology in Xenopus oocytes, support a primary assembly of 2:3 :{beta} subunits, though a minority population of 1:4 :{beta} indicates multiple assemblies are possible. Differential glycosylation of the 5 and {beta}3 subunits enabled reconstruction of the heteromeric complex even in the absence of added fiducials. In the resting state of the receptor, Zn2+ binds to histidine residues at the M2-17' position in the {beta}3 subunit, blocking ion passage. In the activated state, GABA binding to the orthosteric site is associated with global rearrangements propagating to unbinding of the 17' Zn2+ atoms and opening of the 9' hydrophobic gate. Unlike the 1{beta}3 receptor where GABA is effectively a partial agonist, saturating GABA binding to 5{beta}3 appears to drive activation of nearly all receptors, resulting in a single desensitized state observed under the cryo-EM conditions. In agreement with this high GABA efficacy, the GABA-bound structure is virtually unaffected by further addition of the anesthetic etomidate at the {beta}- transmembrane domain interface. These structures offer new detailed models for gating and modulation of a GABAAR subtype critical to learning and memory, including prospective templates for structure-based drug discovery.

biochemistry↗

bioRxiv2024

Cryo-EM structures of {rho}1 GABA(A) receptors with antagonist and agonist drugs

The family of {rho}-type GABAA receptors includes potential therapeutic targets in several neurological conditions, and features distinctive pharmacology compared to other subtypes. Here we combine structures, recordings and simulations to characterize the binding and conformational impact of the drugs THIP (a non-opioid analgesic), CGP36742 (phosphinic acid inhibitor) and GABOB (an anticonvulsant) on a human {rho}1 GABAA receptor. We identify a distinctive binding pose of THIP in {rho}1 versus neuronal 4{beta}3{delta} GABAA receptors, offering a rationale for its inverse effects on these subtypes. CGP36742 binding is similar to the canonical {rho}-type inhibitor TPMPA, supporting a shared mechanism of action among phosphinic acid inhibitors. Binding of GABOB is similar to that of GABA, but produces a mixture of primed and desensitized states, likely underlying its weaker agonist activity. Together, these results elucidate interactions of a {rho}-type GABAA receptor with therapeutic drugs, offering mechanistic insights and a prospective basis for further pharmaceutical development.

biophysics↗

bioRxiv2024

Divergent mechanisms of steroid inhibition in the human {rho}1 GABA(A) receptor

{rho}-type {gamma}-aminobutyric acid-A (GABAA) receptors are widely distributed in the retina and brain, and are potential drug targets for the treatment of visual, sleep and cognitive disorders. Endogenous neuroactive steroids including {beta}-estradiol and pregnenolone sulfate negatively modulate the function of {rho}1 GABAA receptors, but their inhibitory mechanisms are not clear. By combining four new cryo-EM structures with electrophysiology and molecular dynamics simulations, we characterize binding sites and negative modulation mechanisms of {beta}-estradiol and pregnenolone sulfate at the human {rho}1 GABAA receptor. {beta}-estradiol binds in a pocket at the interface between extracellular and transmembrane domains, apparently specific to the {rho} subfamily, and disturbs allosteric conformational transitions linking GABA binding to pore opening. In contrast, pregnenolone sulfate binds inside the pore to block ion permeation, with a preference for activated structures. These results illuminate contrasting mechanisms of {rho}1 inhibition by two different neuroactive steroids, with potential implications for subtype-specific gating and pharmacological design.

biophysics↗

bioRxiv2023

Structural Basis for Hyperpolarization-dependent Opening of the Human HCN1 Channel

Hyperpolarization and cyclic-nucleotide (HCN) activated ion channels play a critical role in generating self-propagating action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN channels activate upon membrane hyperpolarization, but the structural mechanisms underlying this gating behavior remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 in Closed, Intermediate, and Open states. Our structures reveal that the inward motion of two gating charges past the charge transfer center (CTC) and concomitant tilting of the S5 helix drives the opening of the central pore. In the intermediate state structure, a single gating charge is positioned below the CTC and the pore appears closed, whereas in the open state structure, both charges move past CTC and the pore is fully open. Remarkably, the downward motion of the voltage sensor is accompanied by progressive unwinding of the inner end of S4 and S5 helices disrupting the tight gating interface that stabilizes the Closed state structure. This "melting" transition at the intracellular gating interface leads to a concerted iris-like displacement of S5 and S6 helices, resulting in pore opening. These findings reveal key structural features that are likely to underlie reversed voltage-dependence of HCN channels.

biochemistry↗

bioRxiv2023

Structure and dynamics of differential ligand binding in the human {rho}-type GABA(A) receptor

The neurotransmitter {gamma}-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABAARs). Pharmacological properties of {rho}-type GABAARs are particularly distinctive, yet the structural basis for their specialization remains unclear. Here we present cryo-EM structures of a lipid-embedded human {rho}1 GABAAR, including a partial intracellular domain, under apo, inhibited, and desensitized conditions. An apparent resting state, determined first in the absence of modulators, was recapitulated with the specific inhibitor (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid and blocker picrotoxin, and provided a rationale for bicuculline insensitivity. Comparative structures, mutant recordings, and molecular simulations with and without GABA further explained the sensitized but slower activation of {rho}1 relative to canonical subtypes. Combining GABA with picrotoxin also captured an apparent uncoupled intermediate state. This work reveals structural mechanisms of gating and modulation with applications to {rho}-specific pharmaceutical design, and to our biophysical understanding of ligand-gated ion channels.

biophysics↗

bioRxiv2022

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state

Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea, 2017). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 second) test pulses. Our study identifies a modified gating current recording protocol to obtain steady state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.

biophysics↗

bioRxiv2022

Interplay between VSD, pore and membrane lipids in electromechanical coupling in HCN channels

Hyperpolarized-activated and Cyclic Nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a "domino effect" during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.

biophysics↗

bioRxiv2018

Bipolar Switching by HCN Voltage Sensor Underlies Hyperpolarization Activation.

Despite sharing a common architecture with archetypal voltage-gated ion channels (VGIC), the hyperpolarization- and cyclic AMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of voltage sensor and pore gates are conserved implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage-sensing S3b-S4 transmembrane segment of the HCN channel has an intrinsic ability to drive pore opening in either direction. Specific contacts at the pore-voltage sensor interface and unique interactions near the pore gate forces the HCN channel into a hERG-like inactivated state, thereby obscuring their opening upon depolarization. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity.

neuroscience↗