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

Abstract

A mutation in the γ2 subunit of the γ-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 α1β2γ 2 and mutant α1β2γ 2R43Q GABARs expressed in HEK 293 cells. The γ2R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing α1β 2γ2 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 α1β 2γ2R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing α1β2γ 2R43Q GABARs confirmed that surface expression of the γ2R43Q subunit was reduced. These data demonstrate that the γ2R43Q 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 γ2R43Q subunit have epilepsy.