Abstracts

We recently reported that the GABA[sub]A[/sub] receptor (GABAR) [alpha]1 subunit mutation ([alpha]1A322D) that is associated with human autosomal dominant juvenile myoclonic epilepsy (ADJME) substantially reduced total and surface [alpha]1 subunit expression in transiently transfected HEK293T cells. The mechanism by which this point mutation reduces [alpha]1 subunit is unknown, but it has been postulated that A322D causes [alpha]1 retention and degradation in the endoplasmic reticulum (ER). Here we utilize confocal microscopy to visualize and quantify the amount of green fluorescent protein- (GFP) tagged wild type and A322D [alpha]1 that colocalizes to the ER. We constructed GFP tagged wild type (GFP[alpha]1) and mutant (GFP[alpha]1A322D) [alpha]1 subunits in which GFP is connected to the N-termini of [alpha]1. We transiently transfected HEK293T cells with wild type human GABAR [beta]2S and [gamma]2S subunits and either GFP[alpha]1 or GFP[alpha]1A322D. Whole cell voltage-clamp GABA-evoked currents were obtained with rapid GABA perfusion to measure the current kinetics of each GABAR type. Western blots cell lysates were probed with a monoclonal antibody directed against GFP, and quantified. GFP fluorescence from the cellular lysates was measured in a fuorometer. We then transfected HEK293T cells with 25 ng pDsRed2-ER (to selectively label the ER), GABAR [beta]2S and [gamma]2S, subunits, and either GFP[alpha]1 or GFP[alpha]1A322D. The cells were imaged via confocal microscopy 48 hours after transfection. Both total GFP fluorescence as well as GFP fluorescence that was colocalized with pDsRed2-ER was quantified for each cell using Metamorph software. Peak whole cell currents from GABARs containing the GFP[alpha]1A322D subunit had 1% of peak currents from GABARs containing the GFP[alpha]1 subunit and lacked fast and intermediate phases of desensitization. Western blots of whole cell lysates demonstrated that GFP[alpha]1A322D expression was 95[plusmn]2% smaller than GFP[alpha]1 expression. Lysates from GABARs containing the GFP[alpha]1A322D subunit had 18% of the GFP fluorescence compared with GFP[alpha]1 subunit-containing GABARs. Confocal microscopy revealed that cells transfected with [beta]2S and [gamma]2S subunit and the GFP[alpha]1 subunit cDNAs had 83[plusmn]3% of the GFP fluorescence colocalized with the ER and cells transfected with [beta]2S and [gamma]2S and GFP[alpha]1A322D subunit cDNAs had 100[plusmn]4% (P[lt]0.05) of the GFP fluorescence colocalized with the ER. GABARs containing the GFP[alpha]1A322D subunit lacked fast and intermediate components of desensitization and had smaller peak currents and [alpha]1 subunit expression levels than GABARs containing the GFP[alpha]1 subunit, differences that were similar to non-GFP tagged wild type and mutant GABARs. GFP[alpha]1A322D subunit-containing GABARs had reduced total GFP fluorescence and a significantly higher percentage of the GFP fluorescence colocalized to the ER compared with GFP[alpha]1 subunit-containing GABARs. These data suggest that the [alpha]1A322D mutation in ADJME causes [alpha]1 subunit retention and degradation in the ER. (Supported by NIH
NS 39479)