A GABAA receptor mutation linked to human epilepsy (γ2R43Q) impairs cell surface expression of αβγ receptors

A mutation in the gamma(2) 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 alpha(1)beta(2)gamma(2) and mutant alpha(1)beta(2)gamma(2)R43Q GABARs expressed in HEK 293 cells. The gamma(2)R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing alpha(1)beta(2)gamma(2)R43Q GABARs, the number of binding sites for [H-3]flunitrazepam relative to wild-type receptors was decreased 75%. Using surface protein biotinylation, affinity purification, and immunoblotting, we demonstrated that expression of cell surface alpha(1)beta(2)gamma(2)R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing alpha(1)beta(2)gamma(2)R43Q GABARs confirmed that surface expression of the gamma(2)R43Q subunit was reduced. These data demonstrate that the gamma(2)R43Q 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 gamma(2)R43Q subunit have epilepsy.