The GABAA receptor alpha1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation

Abstract

A form of autosomal dominant juvenile myoclonic epilepsy is caused by a nonconservative missense mutation, A322D, in the GABAA receptor alpha1 subunit M3 transmembrane helix. We reported previously that the A322D mutation reduced total and surface alpha1(A322D) subunit protein and that residual alpha1(A322D) subunit resided in the endoplasmic reticulum. Here, we demonstrate that the reduction in alpha1(A322D) expression results from rapid endoplasmic reticulum-associated degradation of the alpha1(A322D) subunit through the ubiquitin-proteasome system. We provide direct evidence that the alpha1(A322D) subunit misfolds and show that in at least 33% of alpha1(A322D) subunits, M3 failed to insert into the lipid bilayer. We constructed a series of mutations in the M3 domain and empirically determined the apparent free energy cost (DeltaGapp) of membrane insertion failure, and we show that the DeltaGapp correlated directly with the recently elucidated transmembrane sequence code (DeltaGLep). These data provide a biochemical mechanism for the pathogenesis of this epilepsy mutation and demonstrate that DeltaGLep predicts the efficiency of lipid partitioning of a naturally occurring protein's transmembrane domain expressed in vivo. Finally, we calculated the DeltaDeltaGLep for 277 known transmembrane missense mutations associated with Charcot-Marie-Tooth disease, diabetes insipidus, retinitis pigmentosa, cystic fibrosis, and severe myoclonic epilepsy of infancy and showed that the majority of these mutations also are likely to destabilize transmembrane domain membrane insertion, but that only a minority of the mutations would be predicted to be as destabilizing as the A322D mutation.

Fig. 1.

The A322D mutation reduced total cellular expression of the α1 subunit. (A and B) Total lysate protein (2.5–40 μg) from mock-transfected cells (m) or those cotransfected with β2 and γ2 subunits and either α1 (a) or α1(A322D) (d) subunit (A) or α1-FLAG (a) or α1(A322D)-FLAG (d) subunit (B) were analyzed by immunoblot (n = 4). The blots were probed with a primary antibody directed against the α1 subunit (A) or the FLAG epitope (B). (C) Each point represents the mean normalized IBD ± SEM for cells transfected with α1 (●), α1(A322D) (■), α1-FLAG (○), or α1(A322D)-FLAG (□) subunits. The lines are linear regressions through the linear portions of each plot and the connected data points for the untagged wild-type α1 subunit. (D) The mean apparent ratios of α1(A322D) to α1 (▴) were plotted.

Fig. 2.

The A322D mutation caused rapid degradation of the α1(A322D)-FLAG subunit. Pulse-labeled mock-transfected (m) cells or those transfected with either human α1-FLAG (●, a) or α1(A322D)-FLAG (■, d) subunits were lysed, immunopurified, and fractionated by SDS/PAGE. (A) After a 10-min labeling, cells were chased for the indicated times (n = 5). The amount of α1-FLAG and α1(A322D)-FLAG subunits differed significantly at points labeled with an asterisk. (B) Cells were pulse-labeled for 5, 10, 15, or 20 min before lysis (n ≥ 3).

Fig. 3.

The α1(A322D) subunit was degraded through the ubiquitin–proteasome system. (A) Mock-transfected cells (m) or those transfected with the α1(A322D)-FLAG subunit were incubated for 30 min in the absence (−, ■) or presence (+, □) of 10 μM lactacystin (LAC). The cells were then [³⁵S]methionine pulse-labeled for 10 min and then chased for the indicated time periods (n = 4). Statistical significance was indicated by an asterisk. (B) Mock-transfected cells or cells transfected with α1-FLAG (a) or α1(A322D)-FLAG (d) subunits were incubated in the absence or presence of 10 μM lactasystin (n ≥ 5) and analyzed by immunoblot that was probed with an antibody directed against polyubiquitin (B1) or FLAG (B2). The relative amounts of polyubiquitinated subunits were plotted (B3).

Fig. 4.

The A322D mutation altered α1 subunit transmembrane topology. (A1 and A2) Schematic diagrams demonstrate that if the α1 subunit's M3 domain inserts into the ER membrane, N365 will be cytoplasmic and cannot be glycosylated (A1), but, if M3 fails membrane insertion, N365 will be within the ER lumen and could be glycosylated (A2). Although M4 was shown to be transmembrane, these experiments do not determine the M4 transmembrane status. In all constructs, the N-terminal glycosylation sites N38 and N138 were mutated to glutamines, and amino acids flanking N365 were mutated (K3654D + N366T) to enhance glycosylation. Mock-transfected cells (m) or cells transfected with wild-type (a) or mutant (d) subunits were [³⁵S]-labeled and fractionated by SDS/PAGE. (B) With N365 intact, mutant, but not wild-type, subunit migrated as two distinct bands (n = 7). With N365 mutated to glutamine, both wild-type and mutant subunits migrated as a single band. (C) Mutant subunits were untreated (−) or treated (+) with, peptide N-glycosidase F before SDS/PAGE. (D) Untagged mutant subunits, but not wild-type subunits, migrated as two bands (n = 4); a protein that coimmunopurified with the untagged proteins and migrated with the same molecular mass as the unglycosylated subunit is marked (→).

Fig. 5.

M3 membrane insertion efficiency correlated with its hydrophobicity. We pulse-labeled mock-transfected cells (1) or cells transfected with cDNA encoding α1-FLAG subunits that contained the following M3 substitutions: no substitution (2); A322W (3); A322G (4); A322S (5); A322N (6); A322Q (7); A322N, I329N (8); A322Q, I329Q (9); A322K (10); A322R (11); A322E (12); A322D (13); A332K, I329K (14); and A322D, I329D (15). We listed the mean ΔG_app below the corresponding gel lane. (B) For each mutation, the empirical M3 (ΔΔG_app) relative to alanine was plotted relative to that from the published transmembrane sequence code derived from the Lep protein (ΔΔG_Lep). (C) The M3 ΔG_app was plotted (± SEM, n ≥ 4) relative to the calculated Wimley–White hydrophobicity free-energy change (ΔG_ww). All the data points were plotted with error bars, although for some, the error bars were smaller than the symbol size and thus were obscured.