No abstract

Genetic defects leading to epilepsy have been identified in ␥2 GABA A receptor subunit. A ␥2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a ␥2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA A receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant ␥2 constructs and showed that the ␥2(R43Q) mutation prevented surface expression of the subunit, unlike ␥2(K289M) substitution. Several ␥2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with ␣3-and ␤3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the ␥2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant ␥2(R43Q) was retained within intracellular compartments, ␣␤ complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that ␥2(R43Q) was able to associate with ␣3-or ␤3-subunits, although the stoichiometry of the complex with ␣3 was altered. Our data show that ␥2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA A receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the ␥2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous ␥2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

A cDNA encoding a GABA(A) receptor subunit was isolated from rat brain. The predicted protein is 70% identical to the human epsilon-subunit. It was recently reported [Sinkkonen et al. (2000), J. Neurosci., 20, 3588-3595] that the rodent epsilon-subunit mRNA encoded an additional sequence ( approximately 400 residues). We provide evidence that human and rat epsilon-subunit are similar in size. The distribution of cells expressing the GABA(A) epsilon-subunit was examined in the rat brain. In situ hybridization histochemistry revealed that epsilon-subunit mRNA is expressed by neurons located in septal and preoptic areas, as well as in various hypothalamic nuclei, including paraventricular, arcuate, dorsomedial and medial tuberal nuclei. The mRNA was also detected in major neuronal groups with broad-range influence, such as the cholinergic (basal nucleus), dopaminergic (substantia nigra compacta), serotonergic (raphe nuclei), and noradrenergic (locus coeruleus) systems. Immunohistochemistry using an affinity-purified antiserum directed towards the N-terminal sequence unique to the rat epsilon-subunit revealed the presence of epsilon-subunit immunoreactivity over the somatodendritic domain of neurons with a distribution closely matching that of mRNA-expressing cells. Moreover, using in situ hybridization, alpha3, theta and epsilon GABA(A) subunit mRNAs were all detected with an overlapping distribution in neurons of the dorsal raphe and the locus coeruleus. Our results suggest that novel GABA(A) receptors may regulate, neuroendocrine and modulatory systems in the brain.

Ionotropic GABA A receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABA A receptors consist of α1-, β2-, and γ2-subunits that are co-expressed in numerous brain areas. α3-, θ-, And ε-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of ε and θ transcripts before birth, to a θ and α3 expression at birth, and finally to a grouped ε, θ and α3 expression post-partum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), ε and θ appeared to have a greater spatial distribution before the dominance of the α3 subunit transcript around birth. We also revealed expression of α3, θ, and ε in the developing spinal cord and identified neurons that express ε in the post-natal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of α3-, θ-and ε-subunits may be assembled at a regional and developmental level in the brain. Keywordsneuroepithelium; cortex; hindbrain; motoneurons; intermediolateral columnThe physiological actions of GABA, the major inhibitory neurotransmitter in the adult CNS, are mediated by the activation of ionotropic and metabotropic receptors (Sieghart, 2000). Ionotropic GABA A receptors are heteropentameric structures generated by coassembly of α1-6, β1-3, γ1-3, ε, θ and π subunits (Sieghart, 1995;Hevers and Luddens, 1998). Many studies have demonstrated that the subunit composition determines both functional