The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with a4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering a4* receptors hypersensitive to nicotine. Selective activation of a4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of a4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-binding abilities and (ii) EC 50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the ␣ subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of ␣ tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position ␣149 produces a constitutively active receptor.

No abstract

The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K+conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K+in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of theKir4.1gene (Kir4.1 −/−). Müller cells from Kir4.1 −/− mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes.In situwhole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 −/− Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K+fluxes through Müller cells, was totally absent in retinas from Kir4.1 −/− mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K+channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K+in the mouse retina.

MethodsMutagenesis and preparation of cRNA and Oocytes -Mutant 5-HT 3A receptor subunits were developed using pcDNA3.1 (Invitrogen, Abingdon, U.K.) containing the complete coding sequence for the 5-HT 3A(b) subunit from mouse neuroblastoma N1E-115 cells as previously described 1 . For nonsense suppression the proline codon at 308 was replaced by TAG as previously described 2 . Wild type and mutant receptor subunit coding sequences were then subcloned into pGEMHE. This was linearized with Nhe1 (New England Biolabs) and cRNA synthesised using the T7 mMESSAGE mMACHINE kit (Ambion). Oocytes from Xenopus laevis were prepared and maintained as described previously 2 .Synthesis of tRNA and dCA-amino acids-Unnatural amino acids were chemically synthesised as nitroveratryloxycarbonyl (NVOC) protected cyanomethyl esters and coupled to the dinucleotide dCA, which was then enzymatically ligated to 74-mer THG73 tRNA CUA as detailed previously 3 . Immediately prior to co-injection with mRNA, tRNA-aa was deprotected by photolysis. Typically 5 ng mRNA and 25 ng tRNA-aa were injected into Stage V-VI oocytes in a total volume of 50 nl. For control experiments, mRNA was injected 1) in the absence of tRNA and 2) with the THG73 74-mer tRNA.Experiments were preformed 18-36 h post injection.Characterisation of mutant receptors-5-HT-induced currents were recorded from individual oocytes using two-voltage electrode clamp with either a GeneClamp 500 amplifier or an OpusXpress system (Axon Instruments, Inc., Union City, CA). All experiments were performed at 22-25 ºC. Serotonin (creatinine sulphate complex,

Abstract. Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT 3 , GABA A and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT 3 ) and some are anion selective (e.g. GABA A and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience.In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT 3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them.

Fast synaptic transmission depends on the selective ionic permeability of transmitter-gated ion channels. Here we show changes in the ion selectivity of neuronal P2X transmitter-gated cation channels as a function of time (on the order of seconds) and previous ATP exposure. Heterologously expressed P2X2, P2X2/P2X3 and P2X4 channels as well as native neuronal P2X channels possess various combinations of mono- or biphasic responses and permeability changes, measured by NMDG+ and fluorescent dye. Furthermore, in P2X4 receptors, this ability to alter ion selectivity can be increased or decreased by altering an amino-acid residue thought to line the ion permeation pathway, identifying a region that governs this activity-dependent change.