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

Area of Science:

• Neuroscience
• Molecular Biology
• Computational Chemistry

Background:

• GABA type A receptors (GABAARs) are crucial for inhibitory neurotransmission.
• Dysfunction of GABAARs is implicated in epilepsy, anxiety, and depression.
• Capturing the open-state conformation of heteropentameric GABAARs has been challenging.

Purpose of the Study:

• To stabilize and characterize open-like ensembles of the α1β3γ2 GABAAR.
• To investigate the effects of mutations on receptor pore properties and ion permeation.
• To elucidate the mechanisms of allosteric modulation by PAMs and antagonists.

Main Methods:

• In-silico mutagenesis and Gaussian-accelerated molecular dynamics (MD).
• Computational electrophysiology to determine ion conductance.
• Two-electrode voltage clamp experiments to validate functional changes.

Main Results:

• Hydrophilic substitutions (L9'T/L9'S) stabilized open-like GABAAR ensembles.
• Mutants exhibited expanded pore, increased hydration, and significantly reduced Cl⁻ permeation barriers.
• Allosteric modulators (PAMs, bicuculline) demonstrated distinct binding and gating mechanisms on the stabilized open-like state.
• L9'T receptors showed spontaneous activity, validating the computational model.

Conclusions:

• The study provides a stable, open-like GABAAR model for atomistic investigations.
• This platform facilitates understanding of GABAAR gating and allosteric modulation.
• The findings support structure-based drug design for neurological conditions targeting GABAARs.