title: Glycosylation of {beta}2 subunits regulates GABAA receptor biogenesis and channel gating.
abstract: γ-aminobutyric acid type A (GABA(A)) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA(A) receptor β2 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 α1 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 β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 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 α1β2 channel, we propose mechanisms for regulation of GABA(A) receptors by glycosylation.
authors.affiliation: Department of Neurology, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Wen-Yi
authors.initials: WY
authors.lastname: Lo
authors.forename: Andre H
authors.initials: AH
authors.lastname: Lagrange
authors.forename: Ciria C
authors.initials: CC
authors.lastname: Hernandez
authors.forename: Rebecca
authors.initials: R
authors.lastname: Harrison
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Sheehan
authors.forename: Robert L
authors.initials: RL
authors.lastname: Macdonald
mesh_terms: D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D011963:Receptors, GABA-A; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase