Ribozyme-mediated reduction of the GABA_A receptor α1 subunit

Abstract

As an approach to understanding the role of the α1 subunit of the GABA_A receptor, ribozymes were designed to reduce expression of this subunit protein by hydrolysis of α1 subunit message and antisense inactivation. The ribozyme cleavage sites were selected through homology comparison of all known murine GABA_A receptor subunits at the amino acid and nucleotide sequence level. Two ribozymes were designed and synthesized: one against the extracellular domain and the other against the cytoplasmic domain. These ribozymes were cloned in a mammalian expression plasmid, pZeoSV2 (+). Cleavage of both extracellular and cytoplasmic domain transcripts by the respective ribozymes was observed when each ribozyme was tested against in vitro transcribed mRNA. The stable cell line, 122, expressing recombinant human GABA_A α1, β2 and γ2S subunits of receptor was stably transfected with the cytoplasmic domain ribozyme (cy) alone and with both the cytoplasmic (cy) and extracellular domain (ex) ribozyme expression plasmids. Northern analysis showed a 55–60% reduction of α1 mRNA in clones of cells transfected with either the single ribozyme (Cy) or with both ribozymes (EC). The α1 protein level was reduced 75% in a stable Cy clone and more than 90% in a stable EC clone when compared with α1 expression in 122 cells and the vector transfected (Zeo) cells. Electrophysiological analysis revealed that the GABA_A receptor properties were very similar in 122 cells and in stable clones in which the subunit protein expression had been greatly reduced. No significant difference was detected in the potentiation of the receptor response by either bretazenil or zolpidem. These data demonstrate the efficacy of the ribozyme approach in dramatically reducing GABA_A subunit protein levels in transfected cells and identify those elements that will be important to the application of similar ribozymes to knock-down transmitter receptor subunit proteins under inducible promoters in transgenic mice.

Introduction

Molecular cloning of the GABA_A receptor subunit genes has revealed four major classes of subunits namely α, β, γ and δ. Each class has several subtypes: α (1–6), β (1–3)) and γ (1–3) [32] (reviewed in Refs. [22], [25], [31], [41]). Homology within a class of subunits is 70–80% and between classes is 20–40%. These subunits are highly conserved in phylogeny. For example, the homology among rat, mouse and human α1 subunits is around 95%. Each subunit consists of an extracellular domain, four transmembrane domains and a cytoplasmic loop region. Variations among subunits and subtypes are mainly seen in the cytoplasmic loop region with the least variation in the transmembrane domains.

Recombinant GABA_A receptor expression in mammalian cell lines [12], [19], [29], [30], [39] and Xenopus oocytes [23], [24], [33], [34] has demonstrated that functional receptors are heteropentameric and consist of ternary subunit combinations of α, β, and γ/δ. The specificity and affinity of the GABA_A receptor complex for GABA as well as potentiation of GABA response by clinically important compounds like benzodiazepines, barbiturates and steroids, is conferred by the subunit subtype. For example, α1 containing receptor complexes have high affinity for benzodiazepines. Zhu et al. [44] have shown that treatment of primary cerebellar granule cells, which express only α1 and α6 subunits, with antisense oligonucleotide against α6 subunit enhanced flunitrazepam-induced potentiation of GABA currents. Treatment of these cells with antisense oligonucleotides against the γ2 subunit increased GABA current but decreased potentiation by flunitrazepam.

Analysis of the roles of the individual subunits within GABA_A receptors has been obtained through subunit expression analysis in neurons [27], transfection in cell lines, through targeted disruption/gene deletions in mice [7], [11] and through application of antisense oligonucleotides to receptor expressing neurons or brain tissue slices [3] or injection into rats [35]. An alternative and less widely applied method involves the expression of subunit-selective ribozymes to cells in culture and in transgenic mice. Ribozymes are RNA enzymes, which cleave mRNA with sequence specificity. They can act both as antisense oligonucleotides to disrupt mRNA translation as well as endoribonuclease activities that hydrolyze RNA [14], [38]. Hammerhead and hairpin ribozymes are made up of two parts: (i) a catalytic core formed by 13 conserved nucleotides in association with a stem and loop structure in the shape of hammerhead or hairpin; (ii) two variable sequences located on each side of the catalytic core conferring specificity to the ribozyme by base pairing with mRNA containing the complementary sequence [1], [17], [38]. The cleavage sites within RNA targets are the sequences GUC, CUC, UUC and AUC [14], [38]. Ribozymes have been reported to reduce expression up to 90% when expressed in cell lines. They have been evaluated for gene therapy in AIDS [36], cancer [8], [9], [18], [21] and sickle cell anemia [20] but have not been reported in the analysis of the role of specific subunits in receptor function. Expression of multiribozymes [21] is reported to be more effective than a single ribozyme.

Transfection of cells with vectors from which ribozymes are transcribed under the control of an active promoter element provides the opportunity to selectively knock-down the levels of specific mRNA and decrease expression of the protein specified by that mRNA. We have used this method to examine the role of the α1 subunit in GABA_A receptor function by selectively reducing the mRNA and protein of the α1 receptor subunit in a cell line that expresses the human α1, β2 and γ2S subunits.