Elsevier

Neuropharmacology

Volume 43, Issue 4, September 2002, Pages 467-475
Neuropharmacology

Alternative transcripts of the GABAA receptor ε subunit in human and rat

https://doi.org/10.1016/S0028-3908(02)00153-3Get rights and content

Abstract

In both human and rat tissues, complex patterns of transcripts are derived from the genes that encode the γ-aminobutyric acid (GABA)A receptor ε subunit. An ε subunit transcript (~3.6 kb) is expressed at relatively high levels in regions of the human brain and heart, but is not detected in most other major tissues. The encoded human ε subunit (εh) confers distinctive properties to receptors into which it assembles. A distinct transcript of the gene (6.2 kb) is expressed abundantly in a variety of human tissues. This alternative transcript (ET2) appears to originate from within the ε subunit gene. It is possible that this transcript encodes a truncated subunit (εhS), containing all of the transmembrane and intracellular domains. However, a combination of biochemical and electrophysiological analyses does not support this hypothesis. A distinct transcript of the ε subunit gene, encoding a large extracellular pro/glx domain, is expressed abundantly in rat and mouse brain. Functional analyses also failed to provide evidence for incorporation of this subunit (εrL) into recombinant receptors. However, a shorter rat ε subunit (εr), which lacks the pro/glx domain, conferred εh-like properties to recombinant receptors, providing evidence for a functional rat ε subunit. In common with its human orthologue, incorporation of the εr subunit into recombinant GABAA receptors confers several distinctive properties, including a reduced modulation by the anesthetic propofol and the appearance of spontaneous current.

Introduction

A gene encoding the γ-aminobutyric acid (GABA)A receptor ε subunit was first reported in 1997 (Davies et al., 1997, Garret et al., 1997, Whiting et al., 1997). When expressed in recombinant systems, this subunit has been shown to confer several distinctive properties to the GABAA receptors into which it assembles (e.g. a reduced modulation by anesthetic agents, an absence of outward rectification, spontaneous channel activity, and an altered sensitivity to tracazolate; Davies et al., 1997, Neelands et al., 1999, Thompson et al., 2002). However, its physiological role remains unclear. In the brain, the ε subunit gene is expressed abundantly in only a few discrete regions, such as the hypothalamus and locus ceruleus (Whiting et al., 1997, Sinkkonen et al., 2000). In these locations, the ε subunit may substitute for, or coassemble with, γ subunits (Davies et al., 2001). This could explain the relative insensitivity to benzodiazepines of specific GABAA receptors in these regions (Kasparov et al., 2001). However, it should also be noted that transcripts of the ε subunit gene have been detected throughout the body (Whiting et al., 1997, Akinci and Schofield, 1999, Erlitzki et al., 2000), and the physiological function of these mRNAs (if any) is currently unknown. One of these transcripts (~6 kb) was concluded to be a partially processed product of the primary ε subunit transcript, and is significantly larger than the ε subunit mRNA found in the brain (~3.5 kb; Whiting et al., 1997). Even in the brain, unusual transcripts of uncertain function are expressed abundantly from rodent ε subunit genes (Sinkkonen et al., 2000, Kasparov et al., 2001). Initially, it appeared that all rat ε subunit transcripts encode a large pro/glx domain that is predicted to be located extracellularly, near the N-terminus of the subunit (Sinkkonen et al., 2000). However, Moragues et al. (2000) reported the isolation of an ε subunit cDNA from rat brain that lacks the unusual insertion, and more closely resembles the structure of the human ε subunit mRNA. Notably, current hypotheses of ε subunit function in rodents are based on studies of the human orthologue, and a rat ε subunit cDNA that encodes a functional ε subunit has not yet been described. In this study, we have attempted to address these issues by examining expression patterns and functional properties of several alternative subunit transcripts from human and rat.

Section snippets

Cloning of an alternative transcript, ET2

A transcript of the human ε subunit gene that originates from within intron 6 was isolated from human testes cDNA using the Marathon cDNA amplification system (Clontech). Initially, the 5′- and 3′-ends of several cDNA clones were obtained by anchored PCR, using primers that correspond to nucleotides 1087–1110 (reverse), 215–237 (reverse) or 5667–5692 (forward) of GenBank Accession U92285. The terminal sequences of the amplified cDNAs were then used to design primers for amplification of a

Alternative transcripts of the human ε subunit gene

Since the first report of a gene encoding the human ε (εh) subunit (Davies et al., 1997), sequences for two alternative transcripts of rodent ε subunits, εrL and εr, have been identified (Sinkkonen et al., 2000, Kasparov et al., 2001). In this study we examined whether alternative ε subunit transcripts are also expressed in human tissues. Transcripts of the ε subunit gene are relatively abundant in a variety of human tissues. Among all ligand-gated ion channel subunits, they are among the most

Discussion

Following the first report of an ε subunit gene (Davies et al., 1997), studies of ε subunit transcripts in rat brain have indicated the existence of alternative transcripts (Sinkkonen et al., 2000, Moragues et al., 2000). In this study we identified alternative ε subunit transcripts in human tissue, and examined which of the rodent and human ε subunit isoforms can incorporate into recombinant GABAA receptors in a manner that influences receptor function.

Previously, an abundant, alternative (~6

Acknowledgements

This work was supported by grants from the US National Institutes of Health: NS34702 (EFK) and GM58037 (TGH). We thank Dr M. Garret for providing a partial clone of the rat ε (εr) subunit cDNA.

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