title: GABAA receptor biogenesis is impaired by the γ2 subunit febrile seizure-associated mutation, GABRG2(R177G).
abstract: A missense mutation in the GABAA receptor γ2L subunit, R177G, was reported in a family with complex febrile seizures (FS). To gain insight into the mechanistic basis for these genetic seizures, we explored how the R177G mutation altered the properties of recombinant α1β2γ2L GABAA receptors expressed in HEK293T cells. Using a combination of electrophysiology, flow cytometry, and immunoblotting, we found that the R177G mutation decreased GABA-evoked whole-cell current amplitudes by decreasing cell surface expression of α1β2γ2L receptors. This loss of receptor surface expression resulted from endoplasmic reticulum (ER) retention of mutant γ2L(R177G) subunits, which unlike wild-type γ2L subunits, were degraded by ER-associated degradation (ERAD). Interestingly, when compared to the condition of homozygous γ2L(R177G) subunit expression, disproportionately low levels of γ2L(R177G) subunits reached the cell surface with heterozygous expression, indicating that wild-type γ2L subunits possessed a competitive advantage over mutant γ2L(R177G) subunits for receptor assembly and/or forward trafficking. Inhibiting protein synthesis with cycloheximide demonstrated that the R177G mutation primarily decreased the stability of an intracellular pool of unassembled γ2L subunits, suggesting that the mutant γ2L(R177G) subunits competed poorly with wild-type γ2L subunits due to impaired subunit folding and/or oligomerization. Molecular modeling confirmed that the R177G mutation could disrupt intrasubunit salt bridges, thereby destabilizing secondary and tertiary structure of γ2L(R177G) subunits. These findings support an emerging body of literature implicating defects in GABAA receptor biogenesis in the pathogenesis of genetic epilepsies (GEs) and FS.
authors.affiliation: Program in Neuroscience, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Todd
authors.affiliation: Program in Neuroscience, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Katharine N
authors.initials: KN
authors.lastname: Gurba
authors.affiliation: Program in Neuroscience, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Emmanuel J
authors.initials: EJ
authors.lastname: Botzolakis
authors.affiliation: Department of Neurology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Aleksandar K
authors.initials: AK
authors.lastname: Stanic
authors.affiliation: Department of Neurology, Vanderbilt University, Nashville, TN 37232, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA; Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA. Electronic address: robert.macdonald@vanderbilt.edu.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Macdonald
mesh_terms: D002462:Cell Membrane / Q000502:physiology; D017124:Conserved Sequence; D003513:Cycloheximide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation / Q000502:physiology; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D020125:Mutation, Missense; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism*; D003294:Seizures, Febrile / Q000235:genetics; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: C546279:GABRG2 protein, human; D011500:Protein Synthesis Inhibitors; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D003513:Cycloheximide
keywords: Epilepsy mutation; GABA(A) receptors; Genetic epilepsy; Impaired assembly and trafficking; Protein degradation; γ2 subunit R177G mutation