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.