A Pore-Facing Glycan Determines GABAA Receptor Subunit Stoichiometry and Gating Behavior

The assembly and gating of γ-aminobutyric acid type A receptors (GABA_ARs) are tightly regulated by their hetero-pentameric subunit composition, yet the molecular determinants governing the pentameric form remain elusive. Here, we demonstrate that a conserved N-linked glycan on α subunits, uniquely positioned within the central pore of the extracellular domain, acts as a structural gatekeeper limiting α subunit incorporation. Using a total of 28 μs of molecular dynamics simulations across native and putative GABA_ARs assemblies, we show that introducing a third pore-facing glycan or positioning two glycans on adjacent subunits disrupts key interfacial salt bridges and hydrogen bonds, particularly at the β+/α- interface that hosts the GABA binding site. These disruptions propagate allosterically, reduce internal loop flexibility, and alter extracellular-to-transmembrane domain coupling, ultimately leading to deep closure of the activation and desensitization gates in the transmembrane domain. Systems containing three glycans consistently shift toward dehydrated, non-conductive conformations. In contrast, native form with two pore-facing glycans preserved native interfacial networks and pore radius. Our findings provide a mechanistic insight for the long-observed α-limiting assembly pattern and identify glycan-mediated steric hindrance as a critical factor of receptor gating. These insights bridge evolutionary conservation, N-glycosylation, and dynamic structure-function relationships, highlighting pore-facing glycosylation as a key determinant of GABA_ARs architecture and function.