Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

gamma-Aminobutyric acid type A (GABA(A)) receptors are hetero-pentameric glycoproteins. Based on consensus sequences, the GABA(A) receptor beta 2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the alpha 1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted beta 2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in alpha 1 beta 2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of beta 2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-beta-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient alpha 1 beta 2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak alpha 1 beta 2 receptor current amplitudes and altered the gating properties of alpha 1 beta 2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-beta-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface beta 2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of alpha 1 beta 2 channel, we propose mechanisms for regulation of GABA(A) receptors by glycosylation.