We present herein the cloning of the human nicotinic acetylcholine receptor ␣9-ortholog and the identification of a new ␣-like subunit (␣10) that shares 58% identity with ␣9. Whereas ␣10 fails to produce functional receptors alone, it promoted robust acetylcholine-evoked currents when coinjected with ␣9. The presence of ␣10 modifies the physiological and pharmacological properties of the ␣9 receptor indicating that the two subunits coassemble in a single functional receptor. Fusing the N-terminal domain of ␣9 with the rest of the ␣10-cDNA yielded a functional ␣9:␣10-chimera that displays the acetylcholine binding properties of ␣9 and ionic pore characteristics of ␣10-containing receptors. In addition, ␣9-and ␣10-subunit mRNAs show limited similar tissue distribution patterns and are expressed in cochlea, pituitary gland, and keratinocytes. These data suggest that, in vivo, ␣9-containing receptors coassemble with ␣10-subunit.

In this paper, we report on the pharmacological and functional profile of SSR180711 (1,4-Diazabicyclo[3.2.2]nonane-4-carboxylic acid, 4-bromophenyl ester), a new selective a7 acetylcholine nicotinic receptor (n-AChRs) partial agonist. SSR180711 displays high affinity for rat and human a7 n-AChRs (K i of 2274 and 1471 nM, respectively). Ex vivo 3 [H]a-bungarotoxin binding experiments demonstrate that SSR180711 rapidly penetrates into the brain (ID 50 ¼ 8 mg/kg p.o.). In functional studies performed with human a7 n-AChRs expressed in Xenopus oocytes or GH4C1 cells, the compound shows partial agonist effects (intrinsic activity ¼ 51 and 36%, EC 50 ¼ 4.4 and 0.9 mM, respectively). In rat cultured hippocampal neurons, SSR180711 induced large GABA-mediated inhibitory postsynaptic currents and small a-bungarotoxin sensitive currents through the activation of presynaptic and somato-dendritic a7 n-AChRs, respectively. In mouse hippocampal slices, the compound increased the amplitude of both glutamatergic (EPSCs) and GABAergic (IPSCs) postsynaptic currents evoked in CA1 pyramidal cells. In rat and mouse hippocampal slices, a concentration of 0.3 mM of SSR180711 increased long-term potentiation (LTP) in the CA1 field. Null mutation of the a7 n-AChR gene totally abolished SSR180711-induced modulation of EPSCs, IPSCs and LTP in mice. Intravenous administration of SSR180711 strongly increased the firing rate of single ventral pallidum neurons, extracellularly recorded in anesthetized rats. In microdialysis experiments, administration of the compound (3-10 mg/kg i.p.) dosedependently increased extracellular acetylcholine (ACh) levels in the hippocampus and prefrontal cortex of freely moving rats. Together, these results demonstrate that SSR180711 is a selective and partial agonist at human, rat and mouse a7 n-AChRs, increasing glutamatergic neurotransmission, ACh release and LTP in the hippocampus.

The molecular composition of the nicotinic acetylcholine receptors (nAChRs) located on dopaminergic neurons and modulating their activity is unclear. Using the reverse transcriptase-polymerase chain reaction we have analyzed the mRNA for nAChR subunits expressed in the substantia nigra (SN) and ventral tegmental area (VTA) following unilateral 6-hydroxydopamine lesion of the dopaminergic system. In contrast to the unlesioned hemisphere, no signal was found in the lesioned hemisphere for alpha3, alpha5, alpha6 and beta4 subunits in the SN nor for alpha2, alpha3, alpha5, alpha6, alpha7 and beta4 subunits in the VTA, indicating the expression of these subunits in dopaminergic neurons. mRNA for alpha4, beta2 and beta3 subunits (and alpha7 in the SN) were still detected after lesion, suggesting that they are expressed in GABAergic neurons and interneurons of these brain areas. These results demonstrate the selective localisation of a number of nAChR subunit mRNA within dopaminergic neurons, strongly suggesting that a heterogenous population of nAChRs play a role in modulating dopaminergic neuronal activity.

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Abstract:Nicotinic acetyicholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetyicholine receptor a subunits from a major insect pest, the peach-potato aphid Myzus persicae. These two subunits, termed Mpal and Mpa2, are respective structural homologues of the Drosophila Da2/Schistocerca gregaria aLl a-subunit pair and the Drosophila ALS a subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine-or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick /32 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mpa2 and none with Mpal. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor a subunits from different species can exhibit distinctive physiological and pharmacological characteristics.