Abstracts

GABAA receptors (GABAAR) are pentameric ion channels usually formed from combinations of the six [alpha], three [beta], three [gamma] and/or one [delta] subunit isoforms. Although many combinations are possible, relatively few are actually found in the central nervous system. Each combination has a specific pattern of expression that varies among brain regions, cell types and even synaptic versus extrasynaptic locations. The expression of these isoforms is altered in certain pathological states and may play a role in the pathogenesis of diseases, such as epilepsy.
The subunit composition of individual GABAARs determines much of their pharmacologic properties. However, because GABAARs rapidly activate and desensitize ([lt]10-100ms) , less is known about the role of subunit composition in determining receptor kinetics. We characterized the current kinetics of GABAARs containing [alpha]1, 3, 4 or 5 with [beta]3 and [gamma]2L using an ultrafast GABA perfusion. 4 [mu]g each of cDNA for [beta]3 and [gamma]2L and one of the [alpha] subunits, with 2 [mu]g of pHook were transfected into HEK 293T cells with a calcium phosphate co-precipitation technique. The next day, transfected cells were selected using a magnetic immuno-bead approach. Whole cell voltage clamp was performed the next day, and kinetic properties were determined using lifted cells exposed to 1 mM GABA using an ultrafast drug application system (exchange times [asymp] 500 [mu]s). As shown in table 1, [alpha]1 and [alpha]4 GABAARs activate and desensitize quickly. In contrast, [alpha]3 and [alpha]5 GABAAR have very little fast desensitization. Furthermore, [alpha]3 containing GABARs are unique in that their activation is an order of magnitude more slowly than the others.[table1] These studies demonstrate that the [alpha] subunit subtype is critical in determining kinetics GABAAR currents. Further studies will explore the response of these receptors to different types of GABA application, including paired pulse, repetitive stimulation and determining the concentration dependence of these receptors. (Supported by R01 NS33300-10)