Endoplasmic Reticulum Retention and Associated Degradation of a GABAA Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the α1 Subunit

Luyan Song,Martin J. Gallagher,Robert L. Macdonald,Wangzhen Shen

doi:10.1074/jbc.m508305200

Abstract

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.

Highlights

We previously demonstrated that the ␣1(A322D) mutation reduces ␣1 subunit expression in a subunit position-dependent manner
The GABAA receptor ␣1(A322D) epilepsy mutation that codes for a simple substitution of a charged for an uncharged amino acid in the M3 transmembrane helix has a novel mechanism by reducing ␣1 subunit expression at a post-translational and pre-oligomerization trafficking step
␣1(A322D) Alters ␣1 Subunit Trafficking after Translation but Before Receptor Assembly—We previously asserted that it was unlikely that the GCC to GAC mutation in the GABRA1 mutant subunit gene reduced ␣1 subunit expression by disrupting transcription or translation

Summary

MATERIALS AND METHODS

Expression of recombinant GABAA Receptors—pcDNA3.1 plasmids containing cDNAs that encode human ␣1, ␤2S, and ␥2S GABAA receptor subunits were a gift from Dr Mathew Jones (University of Wisconsin, Madison, WI). ␣1-YFP and ␣-CFP cDNAs were constructed by first inserting HpaI and SacII restriction sites between the codons encoding amino acids four and five of the mature ␣1 subunit. For the confocal microscopy experiments, cells were transfected in 35-mm dishes using equal amounts (1 ␮g) of ␣1-FP, ␤2S, and ␥2S subunit cDNAs. For the ER colocalization experiments, the cells were transfected with 30 ng of a cyan fluorescent protein-tagged endoplasmic reticulum marker (CFP-ER, BD Biosciences). For experiments in which the cells were transfected with FP-tagged ␣1 subunit, the membranes were incubated with a monoclonal antibody to green fluorescent protein (1:2500) as well as to an antibody to ␤-actin (clone C4, 1 ␮g/ml). Background FP fluorescence for each image was obtained from cells that stained with FM 4-64 but did not express FP. Data Analysis—Values are reported as mean Ϯ S.E. Statistical significance for the endoglycosidase, confocal, and fluorescence spectroscopy experiments were determined using the Student’s unpaired t test and the significance of expression differences in the Western blot experiments and the effect of aspartate substitution on topology prediction scores were determined using the paired t test (GraphPad, San Diego, CA)

RESULTS

Findings

DISCUSSION