title: A GABAA receptor mutation linked to human epilepsy (gamma2R43Q) impairs cell surface expression of alphabetagamma receptors.
abstract: A mutation in the gamma2 subunit of the gamma-aminobutyric acid (GABA) type A receptor (GABAR), which changes an arginine to a glutamine at position 43 (R43Q), is linked to familial idiopathic epilepsies. We used radioligand binding, immunoblotting, and immunofluorescence techniques to examine the properties of wild-type alpha1beta2gamma2 and mutant alpha1beta2gamma2R43Q GABARs expressed in HEK 293 cells. The gamma2R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing alpha1beta2gamma2R43Q GABARs, the number of binding sites for [3H]flunitrazepam relative to wild-type receptors was decreased 75%. Using surface protein biotinylation, affinity purification, and immunoblotting, we demonstrated that expression of cell surface alpha1beta2gamma2R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing alpha1beta2gamma2R43Q GABARs confirmed that surface expression of the gamma2R43Q subunit was reduced. These data demonstrate that the gamma2R43Q mutation impairs expression of cell surface GABARs. A deficit in surface GABAR expression would reduce synaptic inhibition and result in neuronal hyperexcitability, which could explain why families possessing the gamma2R43Q subunit have epilepsy.
authors.affiliation: Department of Physiology and Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Feyza
authors.initials: F
authors.lastname: Sancar
authors.forename: Cynthia
authors.initials: C
authors.lastname: Czajkowski
mesh_terms: D000495:Allosteric Site; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004827:Epilepsy / Q000235:genetics* / Q000378:metabolism; D005445:Flunitrazepam / Q000494:pharmacology; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics*; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; D011963:Receptors, GABA-A; D005973:Glutamine; D005445:Flunitrazepam; D001120:Arginine