A pore-facing glycan constrains GABAA receptor subunit stoichiometry and gating behavior

The gating and function of γ-aminobutyric acid type A receptors (GABAARs) depend on their hetero-pentameric subunit composition, yet the rules guiding subunit selection remain unclear. We show that a conserved N-linked glycan in α subunits, uniquely positioned within the extracellular pore, acts as a structural constraint that limits the functional viability of α-containing assemblies. A total of 32 µs of molecular dynamics simulations of native and hypothetical non-canonical assemblies show that adding a third pore-facing glycan or placing two glycans on adjacent subunits disrupts key interactions at the β+/α– interface containing the GABA-binding site. These perturbations propagate allosterically, alter extracellular–transmembrane domain coupling, and promote deep gate closure. Systems with three pore-facing glycans consistently adopt deep-closed conformations, whereas native assemblies with two glycans preserve interfacial integrity and pore hydration. These results identify the pore-facing glycan as a structural constraint on functionally viable GABAAR architecture and gating. Molecular dynamics simulations reveal how additional pore-facing glycans drive GABAA receptors into deep-closed conformations, disrupting gating and constraining structurally and functionally viable subunit stoichiometries.