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Glycosylation of {beta}2 subunits regulates GABAA receptor biogenesis and channel gating.

ABSTRACT: ?-aminobutyric acid type A (GABA(A)) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA(A) receptor ?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 ?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 ?2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in ?1?2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of ?2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-?-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient ?1?2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak ?1?2 receptor current amplitudes and altered the gating properties of ?1?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-?-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface ?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 ?1?2 channel, we propose mechanisms for regulation of GABA(A) receptors by glycosylation.

SUBMITTER: Lo WY

PROVIDER: S-EPMC2951209 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Glycosylation of {beta}2 subunits regulates GABAA receptor biogenesis and channel gating.

Lo Wen-Yi WY Lagrange Andre H AH Hernandez Ciria C CC Harrison Rebecca R Dell Anne A Haslam Stuart M SM Sheehan Jonathan H JH Macdonald Robert L RL

The Journal of biological chemistry 20100716 41

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 domai ...[more]

PMID: 20639197

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Glycosylation of {beta}2 subunits regulates GABAA receptor biogenesis and channel gating.

Publications

Glycosylation of {beta}2 subunits regulates GABAA receptor biogenesis and channel gating.

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