A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.

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The putative channel-forming MU domains of the nicotinic, y-aminobutyric acid type A, and glycine receptors contain a highly conserved leucine residue. Mutation of this hydrophobic amino acid in the neuronal nicotinic receptor a7 (Leu-247), reconstituted in Xenopus oocytes, modifies the ionic response to acetylcholine and alters desensitization. Furthermore, the Leu -+ Thr (L247T) mutant has two conducting states (46 pS and 80 pS), in contrast with the wild-type (WT) receptor, which has only one (45 pS). We now show that this mutant possesses a rather paradoxical pharmacology: antagonists of the WT receptor such as dihydro-flerythroidin, hexamethonium, or (+)-tubocurarine elicit ionic currents when applied to the L247T a7 mutant and these responses are blocked by a-bungarotoxin. Furthermore, prolonged application of acetylcholine causes desensitization in the WT but leads to a potentiation of the responses to acetylcholine or dihydro-13-erythroidin in the mutant. These data are consistent with a scheme in which mutation of Leu-247 renders a desensitized state in the WT channel a conducting state. They also strengthen the proposal that, in the WT, some competitive antagonists may stabilize desensitized states. Finally, these observations may shed light on properties ofother ion channels, in particular the glutamate receptors, which display multiple conductance levels associated with various pharmacological agents.The structural principles by which the nicotinic acetylcholine receptor (AcChoR) mediates the effect of acetylcholine (AcCho) on ion-channel activation and desensitization have been explored by photolabeling methods and site-directed mutagenesis (for reviews, see refs. 1 and 2). In particular, photolabeling with the channel blockers chlorpromazine (3-6) or triphenylmethylphosphonium (7,8) suggested that MII, the second hydrophobic segment of each subunit, was a possible component of the ion channel. Chlorpromazine was found to label three rings of residues from MII, two polar rings and a leucine ring, spaced one a-helical turn apart (3-6). Sitedirected mutagenesis experiments of charged or polar amino acid residues bordering (9, 10) or located within MII (11-13) confirmed and further extended the photolabeling data, strongly supporting the hypothesis that amino acid side chains from MII line the ion channel. Among the three rings of amino acids labeled by chlorpromazine in Torpedo marmorata AcChoR, the hydrophobic leucine ring is conserved at the same position in almost all subunits of nicotinic, glycine, and y-aminobutyric acid type A receptors (14). By using, as a model system, the neuronal homooligomeric and a-bungarotoxin-sensitive a7 nicotinic receptor (15, 16) expressed in Xenopus oocyte, the function of this leucine ring was investigated by site-directed mutagenesis (17). Replacement of Leu-247 with a polar residue (serine or threonine) altered blocking by QX-222 indicating that this residue faces the lumen of the channel. In addition, these mutations abolished current rectification, reduc...

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood-brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-␤-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-␤-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy cause progressive paralysis, often leading to premature death. Neurotrophic factors have been suggested as therapeutic agents for motor neuron diseases, but their clinical use as injected recombinant protein was limited by toxicity and/or poor bioavailability. We demonstrate here that adenovirus-mediated gene transfer of neurotrophin-3 (NT-3) can produce substantial therapeutic effects in the mouse mutant pmn (progressive motor neuronopathy). After intramuscular injection of the NT-3 adenoviral vector, pmn mice showed a 50% increase in life span, reduced loss of motor axons and improved neuromuscular function as assessed by electromyography. These results were further improved by coinjecting an adenoviral vector coding for ciliary neurotrophic factor. Therefore, adenovirus-mediated gene transfer of neurotrophic factors offers new prospects for the treatment of motor neuron diseases.

The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor y subunit: Implications for the a-helical organization of regions MII and for the structure of the ion channel (channel blockers/ligand-gated ion channels/allosteric proteins) FREDERIC REVAH*, JEAN-LUC GALZI*, JfR6ME GIRAUDAT*, PIERRE-YVES HAUMONTt, FLORENCE 8254), -248 of the a chain (Ser-a248), and leucine -257 of the 8 chain (Leu-,8257) (10)(11)(12), which all belong to the second hydrophobic segment of each subunit, MII. Studies using another photolabile NCB, [3H]triphenylmethylphosphonium, have suggested that this compound labels the same serine residues as [3H]CPZ on the a, f3, and 8 subunits (13,14). Finally, site-directed mutagenesis (15)(16)(17)32) has brought complementary information and additional evidence in favor of the notion that segment MII and neighboring amino acids contribute to the regulation of ion transport through the channel.In the present paper, we demonstrate that on the y subunit of T. marmorata AcChoR, three residues belonging to the MII segment are labeled by [3H]CPZ in a PCP-sensitive manner under equilibrium conditions. The implications of these results in terms of a plausible structural model of the AcChoR ion channel are discussed. MATERIALS AND METHODS 4675The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Three a~tmatic amino acids. Tyr ~'. Trp *° and Tyr '~7 belongin 8 1o three separate domains of the r~7-sttbunit of neuronal nicotinic actty|choline reoel~or were mutated to phenylalanin©, and the electrophysiological response of the resultmg mulant receptors analy~ed in the k'es~pNs oocyte ©~pression system. All mutations signifi~mtly decreased the apparent affinities for a~tyi~olir~ and nicotine, and to a kss~r extent, tho~ for the ~ompetitive antagonists dihydro-~-crythroidine and ~-bungarotoxin. Other properlies investigated, s~h as the voltaire ~dent'y of the itws response as well as its sensitivity to the open channel blocker QX222, were not significantly chanl~d, indicatting that the mutations affected selectively the rex3o~ition of cholin~rgic ligands by the reoeptor protein. The maximal rates for the rapid desensitization proems were slight|y modified, sugil~ting that the contribtttion ofTyr ~z. Trp ~ and Tyr ~ to the binding area might differ in the various conformations of the nicotinic re~-ptor. Other mutations at nearby positions ($94N, WI53F, GISID and ~i82E) did not aff~t the pro;~-tics of tl~ ¢leclrophysiological r~q~msc. rl~ data point to the fun~iona| significan~ of Tyr *~, Trp *a and Tyr ~s' in the binding of cholimsrrlgi~ iigands and ion channel metivati~ of the ni~otin~ receptor, thas supporting a multiple loop model [(1990) J. Biol. Chem. 2455 for the liBand binding area.Neuronal nicotinic acetylcholine receptor; Ac~yicholine bindin 8 site: site-dire-ted mutagenesis

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