Assembly of GABAA receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar single-channel properties

Abstract

Recent experimental evidence has led to the hypothesis that GABAA receptor channel (GABAR) heterogeneity or receptor channel subtypes may occur by differential assembly of a given set of subunits into various configurations. Alternatively, assembly of subunits into mature GABARs may arise from an ordered process to produce a preferred form of the receptor channel, as seen for nicotinic ACh receptors. In the preceding article, we demonstrated that transient expression of GABAR α1, and β1, subunits in mouse L929 fibroblast cells produced two different types of GABARs, when coexpressed with and without the γ2S subunit. Not only did these GABARs differ in their GABA and diazepam pharmacology, but initial single-channel recordings suggested that the two types of GABARs (α1β1, and α1β1γ2S) had different conductance and gating properties. It also appeared that α1β1γ2S GABARs were preferentially formed over α1β1 GABARs, but it was not completely shown if both forms of GABARs were produced when a cell expressed all three subunits. To characterize further the assembly process and determine the preferred form, if it existed, it was necessary to obtain a kinetic "fingerprint" for both α1β1 and α1β1γ2S GABARs. Thus, single-channel patch-clamp recording and kinetic analysis of receptor channel gating were performed. For both α1β1 and α1β1γ2S GABARs, GABA evoked single-channel openings to both a main conductance (15 and 29 pS, respectively) and a subconductance level (10 and 21 pS, respectively) with greater than 90% of the total current through the main conductance level openings. The two GABAR populations were further differentiated by their open and burst properties. On average, α1β1γ2S GABARs opened for almost three times the duration as α1β1 GABARs (6.0 vs 2.3 msec, respectively) and had three openings per burst, α1β1 GABARs opened predominantly as single opening bursts. Using the conductance and gating properties to differentiate the two GABAR populations, we determined that α1β1 GABARs were rarely, if ever, formed upon coexpression of all three subunits, suggesting that α1β1γ2S GABARs were the preferred final form of the receptor channel. Also, the homogeneity of the conductance and gating properties of α1β1γ2S GABARs among the different patches studied implied that a single preferred configuration of GABARs may exist. Thus, the assembly of GABARs from their constituent subunits may occur by an ordered, not random, process akin to nicotinic ACh receptors, suggesting another conserved feature of members of the ligand-gated ion channel supergene family.