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J. Biol. Chem., Vol. 280, Issue 45, 37995-38004, November 11, 2005

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Endoplasmic Reticulum Retention and Associated Degradation of a GABA_A Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the 1 Subunit^*

Martin J. Gallagher1, Wangzhen Shen, Luyan Song, and Robert L. Macdonald¶

From the Departments of Neurology, Molecular Physiology and Biophysics, and ^¶Pharmacology, Vanderbilt University, Nashville, Tennessee 37232

A GABA_A receptor 1 subunit epilepsy mutation (1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the 1 subunit. We reported previously that heterologous expression of 1(A322D)22 receptors in mammalian cells resulted in reduced total and surface 1 subunit protein. Here we demonstrate the mechanism of this reduction. Total 1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 ± 6%) or when coexpressed with 2 and 2S subunits (78 ± 6%), indicating an expression reduction prior to subunit oligomerization. In 122S receptors, endoglycosidase H deglycosylated only 26 ± 5% of 1 subunits, consistent with substantial protein maturation, but in 1(A322D)22S receptors, endoglycosidase H deglycosylated 91 ± 4% of 1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the 1 subunit was tagged with yellow (1-YFP) or cyan (1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 ± 4% of 1-YFP22 but only 5 ± 1% 1(A322D)-YFP22 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 ± 4% 1-YFP22S, 86 ± 3% 1(A322D)-YFP). Heterozygous expression of 1-CFP22S and 1(A322D)-YFP22S or 1-YFP22S and 1(A322D)-CFP22S receptors showed that membrane GABA_A receptors contained primarily wild type 1 subunits. These data demonstrate that the A322D mutation reduces 1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane 1 subunits that are almost exclusively wild type subunits.

Received for publication, July 28, 2005

^* This work was supported by United States Public Health Service Grants K08NS44257-01 (to M. J. G.), NS33300 (to R. L. M.), and NS39479 (to R. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Neurology, Vanderbilt University, 6140 Medical Research Bldg. III, 465 21st Ave. South, Nashville, TN 37232-8552. Tel.: 615-322-5979; Fax: 615-322-5517; E-mail: Martin.Gallagher{at}Vanderbilt.edu.

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