(mentions CMT) The GABAA receptor {alpha}1 subunit epilepsy mutation A322D inhib

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Proc Natl Acad Sci U S A. 2007 Aug 1

The GABAA receptor {alpha}1 subunit epilepsy mutation A322D inhibits

transmembrane helix formation and causes proteasomal degradation.

Gallagher MJ, Ding L, Maheshwari A, Macdonald RL.

Departments of Neurology, Molecular Physiology and Biophysics, and

Pharmacology, Vanderbilt University, Nashville, TN 37232.

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.