Preferential Coassembly of a4 and d Subunits of the g-Aminobutyric AcidA Receptor in Rat Thalamus

Pharmacological study of rat thalamic g-aminobutyric acidA (GABAA) receptors revealed the presence of two distinct populations, namely, diazepam-sensitive and diazepam-insensitive [H]Ro15–4513 binding sites accounting for 94 6 2% (1339 6 253 fmol/mg protein) and 6 6 2% (90 6 44 fmol/mg protein) of total sites, respectively. Thalamic diazepam-insensitive sites exhibited a pharmacology that was distinct from diazepamsensitive sites but comparable to that of the a4b3g2 subtype of the GABAA receptor stably expressed in L(tk ) cells. Immunoprecipitation experiments with a specific anti-a4-antiserum immunoprecipitated 20 and 7% of total thalamic [H]muscimol and [H]Ro15–4513 sites, respectively. Combinatorial immunoprecipitation using antisera against the a4, g2, and d subunit revealed that a4dand a4g2-containing receptors account for 13 6 2 and 8 6 3% of [H]muscimol sites from thalamus, respectively. It also indicated that all d subunits coexist with an a4 subunit in this brain region. In conclusion, our results show that in rat thalamus both a4bg2 and a4bd subtypes are expressed but a4bd is the major a4-containing GABAA receptor population. g-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Its effects are mediated largely through the GABAA receptors, a family of GABA-gated Cl ion channels (for reviews, see Sieghart, 1995; McKernan and Whiting, 1996), which are pentameric assemblies of the 14 different subunits cloned to date (a1–6, b1–3, g1–3, d, and e). The combination of a and g subunits has been shown to confer specific functional and pharmacological properties, in particular the affinity and efficacy of compounds at the benzodiazepine binding site. These two subunit types also contribute to the affinity and efficacy of GABA and Zn sensitivity of the channel. Dysfunction of GABAergic neurotransmission has been implicated in neurological disorders such as epilepsy. Studies of temporal lobe epilepsy using different animal models have reported up-regulation of various GABAA receptor subunit mRNAs and proteins as well as modification of the pharmacological profile of receptors in rat hippocampus. For example, in electrical kindled rat, Clark and coworkers (1994) found increased levels of a4, b1, and b3 subunit mRNAs in dentate gyrus. Similarly, in kainic acid-induced temporal lobe epilepsy a marked up-regulation of a1, a2, a4, a5, b1, b3, g2, and d subunit proteins has been reported in the molecular layer of the rat dentate gyrus (Schwarzer et al., 1997). A recent study in rat (Brooks-Kayal et al., 1998) investigating GABAergic currents and mRNA expression in single dentate granule cells demonstrated profound changes in subunit expression and GABAA receptor properties after pilocarpine treatment. The most dramatic changes were a 175 and 225% increase in the relative expression of a4 and d subunit mRNAs, respectively, together with an enhanced sensitivity of GABAA receptors to block by Zn . An emerging view from these and other studies (Mahmoudi et al., 1997; Matthews et al., 1998; Smith et al., 1998) is that a4 subunit-containing GABAA receptors are highly plastic and, compared with other subtypes, are rapidly up-regulated in response to changes in neuronal activity. Biochemical and pharmacological reports have shown that in rat brain some a4 receptors bind [H]Ro15–4513 with high affinity (Benke et al., 1997) whereas others do not (Khan et al., 1996), suggesting the existence of a heterogeneous population of a4 subunit-containing GABAA receptors. In the present study, we have used pharmacological analyses and quantitative immunoprecipitation (Sur et al., 1998) to further characterize a4 subunit-containing GABAA receptors. We have focused our attention on subpopulations of a4 subunit-containing receptors present in rat thalamus and hippocampus, brain regions that express high level of a4 subunits and are involved in epilepsy (Wisden et al., 1992;