Neuronally Restricted RNA Splicing Regulates the Expression of a Novel GABA_AReceptor Subunit Conferring Atypical Functional Properties

Abstract

We report the isolation and characterization of a cDNA encoding a novel member of the GABA receptor gene family, ⑀. This polypeptide is 506 amino acids in length and exhibits its greatest amino acid sequence identity with the GABA A receptor ␥3 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth ␥ subunit. The ⑀ subunit coassembles with GABA A receptor ␣ and ␤ subunits in Xenopus laevis oocytes and transfected mammalian cells to form functional GABA-gated channels. ␣1␤1⑀ GABA A receptors, like ␣1␤1␥2s receptors, are modulated by pentobarbital and the steroid 5␣-pregnan-3␣-ol-20-one but, unlike ␣1␤1␥2s receptors, are insensitive to flunitrazepam. Additionally, ␣1␤1⑀ receptors exhibit rapid desensitization kinetics, as compared with ␣1␤1 or ␣1␤1␥2s. Northern analysis demonstrates widespread expression of a large ⑀ subunit transcript in a variety of nonneuronal tissues and expression of a smaller transcript in brain and spinal cord. Sequence analysis demonstrated that the large transcript contained an unspliced intron, whereas the small transcript represents the mature mRNA, suggesting regulation of expression of the ⑀ subunit via neuronally restricted RNA splicing. In situ hybridization and immunocytochemistry reveal a pattern of expression in the brain restricted primarily to the hypothalamus, suggesting a role in neuroendocrine regulation, and also to subfields of the hippocampus, suggesting a role in the modulation of long term potentiation and memory.

Studies of ethanol actions on recombinant δ-containing γ-aminobutyric acid type A receptors yield contradictory results

“…Several papers were published concerning the GABA A -R ε subunit: one describing receptors α1β3ε and α2β1ε expressed in HEK293 cells as insensitive to pentobarbital (Davies et al, 1997), and two others describing receptors α1β1ε expressed in Xenopus oocytes showing modulation by pentobarbital, as well as direct effects by this barbiturate (Thompson et al, 1998;Whiting et al, 1997). This controversy would be eventually resolved (Thompson et al, 2002): using α1β1ε expressed in Xenopus oocytes, it was shown that the differences between the ε subunits were not due to amino acid sequences nor to untranslated regions, but to levels of expression determined by the vector in which the DNA encoding the ε subunit was inserted.…”

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

“…The ε subunit shares the greatest sequence homology with the γ subunits, and so it is generally thought to take the place of the single γ subunit in the pentameric receptor. However studies examining the sensitivity of recombinant ε-containing receptors to the positive neurosteroids have provided varying results in which the extent of enhancement by the positive neurosteroids of current through recombinant GABA A receptors appears to be inversely proportional to the level of ε subunit expression (Davies et al, 1997;Whiting et al, 1997;Thompson et al, 1998). A recent study by Thompson et al (2002) suggest that the discordant findings with respect to steroid sensitivity of these ε-containing receptors can be reconciled if it is assumed that ε can substitute for not only for the γ, but also for non-γ subunits within the pentamer such that receptors with alternative stoichiometries are produced as ε subunit expression increases.…”

“…Thus, it is tempting to postulate that allopregnanolone modulation of these extrasynaptic, rather than synaptic, conductances may be the critical factor in mediating the changes in oxytocin neuronal firing that occur during late pregnancy and parturition. Delta-containing receptors are known to give rise to most tonic, extrasynaptic conductances in other brain regions, however, given that δ subunit expression in the hypothalamus/basal forebrain is debatable (Fritschy and Mohler, 1995;Pirker et al, 2000) and expression of ε in this brain region is marked (Whiting et al, 1997;Sinkkonen et al, 2000;McIntyre et al, 2002), most notably in the GnRH neurons (Moragues et al, 2003), an intriguing possibility is that ε-containing receptors may play a comparable role in regulating tonic GABAergic conductances and allosteric modulation to both the endogenous neurosteroids and the AAS.…”

Analysis of the Set of GABAA Receptor Genes in the Human Genome

“…We have confirmed a total of 9 ⑀ subunit exons in the genome, all nine being coding exons. Four expressed ⑀ variants can be found, the first being the full-length functional transcript (33,(53)(54)(55). In variant 2, exons 1-3 are spliced out, with protein translation then starting at an alternative Met.…”