No abstract

Selective modulators of ␥-aminobutyric acid, type A (GABA A ) receptors containing ␣ 4 subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(؊tk) cells, we stably expressed the native GABA A receptor subunit combinations ␣ 3 ␤ 3 ␥ 2, ␣ 4 ␤ 3 ␥ 2 , and, for the first time, ␣ 4 ␤ 3 ␦ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA A receptor antagonists and, for ␣ 3 ␤ 3 ␥ 2 and ␣ 4 ␤ 3 ␥ 2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with ␣ 4 and ␤ 3 , ␦ subunits, compared with ␥ 2 , conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. ␣ 4 ␤ 3 ␦ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at ␣ 4 ␤ 3 ␦ compared with ␣ 4 ␤ 3 ␥ 2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA A receptor. For GABA A receptors containing ␣ 4 subunits, our experiments reveal that inclusion of ␦ instead of ␥ 2 subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of ␣ 4 ␤ 3 ␦ receptors, consistent with a central role for this subunit combination in premenstrual syndrome. ␥-Aminobutyric acid (GABA)1 is the predominant inhibitory neurotransmitter in the central nervous system, and modulators of type A GABA (GABA A ) receptors are used to treat anxiety, insomnia, muscle spasms, and epilepsy. GABA A receptors are pentameric ligand-gated chloride channels, mediating rapid inhibitory synaptic neurotransmission, and are composed of different combinations of subunits from a family including ␣ 1-6 , ␤ 1-4 , ␥ 1-3 , ␦, ⑀, , and 1-2 (1). They are modulated by a plethora of clinically important drugs including benzodiazepines, barbiturates, steroids, and anesthetics. Subunit stoichiometry has been contentious (2-4), but the evidence is now convincing that receptors composed of ␣, ␤, and ␥ subunits contain two ␣, two ␤, and one ␥ subunit (5). The precise combination of subunits is an important determinant of receptor pharmacology; ␣ subunits govern GABA affinity (6), ␣ and ␥ subunits regulate benzodiazepine site pharmacology (6 -9), and ␤ subunits control loreclezole and etomidate sensitivity (10).␣ 4 subunits comprise only a small percentage of neuronal subunits, concentrated in hippocampus, striatum, cerebral cortex, thalamus, and basal ganglia (11-15). They assemble with ␤ 2/3 and ␥ 2 subunits in most areas of the brain (12), but also with ␤ 2/3 and ␦ subunits in olfactory bulb, dentate gyrus, and thalamus (14 -17). Of the 20 -27% of thalamic GABA A receptors that contain ␣ 4 su...

We have examined the spatial and temporal nature of Ca 2؉ signals activated via the phosphoinositide pathway in oligodendrocytes and the cellular specializations underlying oligodendrocyte Ca 2؉ response characteristics. Cultured cortical oligodendrocytes were incubated with fluo 3 or fura 2, and digital video fluorescence microscopy was used to study the effect of methacholine on [Ca 2؉ ] i . Single peaks, oscillations, and steady-state plateau [Ca 2؉ ] i elevations were evoked by increasing agonist concentration. The peaks and oscillations were found to be Ca 2؉ wave fronts, which propagate via distinct amplification regions in the cell where the kinetics of Ca 2؉ release (amplitude and rate of rise of response) are elevated. Staining with 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolecarbocyanine iodide (JC-1) and 3,3-dihexyloxacarbocyanine iodide revealed that mitochondria are found in groups of three or more in oligodendrocyte processes and that the groups are distributed with considerable distance separating them. Crosscorrelation analysis showed a high degree of correlation between sites where mitochondria are present and peaks in the amplitude and rate of rise of the Ca 2؉ response. Intramitochondrial Ca 2؉ concentration, measured using rhod 2, increased upon treatment with methacholine. Methacholine also evoked a rapid change in mitochondrial membrane potential as measured by the J-aggregate fluorescence of JC-1. Pretreatment with the mitochondrial inhibitors carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 M, 2 min) or antimycin (2 g/ml, 2 min) altered the methacholine-evoked Ca 2؉ response in most cells studied, responses being either markedly potentiated or inhibited. The results of this study demonstrate that stimulation of phosphoinositide-coupled muscarinic acetylcholinoceptors activates propagating Ca 2؉ wave fronts in oligodendrocytes and that the characteristics of these waves are dependent on mitochondrial location and function.Cultured oligodendrocytes express a variety of receptors coupled to the mobilization of Ca 2ϩ from inositol trisphosphate receptor (InsP 3 R) 1 -expressing intracellular stores. These include M 1 muscarinic cholinoceptors (1-3), which are also thought to be expressed on oligodendrocytes in vivo (4). While InsP 3 -evoked Ca 2ϩ responses are known to be present in oligodendrocytes (2, 5), the spatial and temporal nature of these has not been characterized. In a wide variety of cell types, including several other types of glial cells, stimulation of InsP 3 -coupled receptors results in activation of Ca 2ϩ waves and oscillations (6 -10). In astrocytes, Ca 2ϩ waves are initiated in several distinct regions, which then propagate throughout the cell via multiple amplification sites, at which the amplitude of the wave and the rate of rise of the response are markedly elevated compared to surrounding regions (9, 10). 2The molecular specializations underlying wave initiation and propagation sites in glial and other cell types remain poorly defined. Recent reports...

To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK activity in OP cells are influenced by cytoplasmic calcium levels, as shown by their sensitivity to the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The kinetics of CREB phosphorylation in response to the various agonists corresponds to that of MAPK activation. Moreover, CREB phosphorylation and MAPK activation are similarly affected by calcium ions. The MEK inhibitor PD 098059, which selectively prevents activation of the MAPK pathway, strongly reduces induction of CREB phosphorylation by kainate, carbachol, bFGF, and the phorbol ester TPA. We propose that in OPs the MAPK/RSK pathway mediates CREB phosphorylation in response to calcium influx, PKC activation, and growth factor stimulation.