Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain.

Abstract

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...

Desensitization of neuronal nicotinic receptors

“…Although conventional models (Fig. 2) have been used to explain desensitization of ␣7 receptors (Bertrand et al, 1992), a novel multistate model in which desensitization depends on the fractional agonist occupation (five states ϭ five ligand binding sites; Palma et al, 1996) has been proposed to explain the unusual behavior of ␣BTX-sensitive receptors in hypothalamic cells . The model predicts that high concentrations of ligand will cause complete desensitization (more fractional occupation of nonconducting states); conversely, low concentrations (less occupancy) will allow prolonged channel opening.…”

“…Moreover, because of the high affinity of desensitized states for agonists, the mutant ␣7 receptor is now sensitive to both ACh and nicotine in the concentration range that would cause desensitization of wildtype nAChRs (Revah et al, 1991). Interestingly, the desensitized conducting state of the mutant channel can be activated by some competitive antagonists, implying that antagonists, like agonists, can bind to desensitized states with high affinity (Bertrand et al, 1992;Palma et al, 1996;see Sabey et al, 1999 for ␣4␤2 receptors). Note that such binding to desensitized states of nAChRs has potential implications for up-regulation of receptor function during chronic exposure to antagonists Peng et al, 1994).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…Mutations at this position in α7 alter receptor desensitization, rectification, agonist potency, and antagonist effects [218][219][220][221][222]. Similarly complex effects have been reported for M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 mutations at other positions within the M2 domain of α7 (for example, the 6' position [223,224]).…”

Water‐mediated conformational transitions in nicotinic receptor M2 helix bundles: a molecular dynamics study

“…Chicken ~7 acetylcholine receptors form homopentameric assemblies [22,23]. Simulated annealing via restrained molecular dynamics (SA/ MD) was used to generate ensembles of pentameric M2ct7 helix bundies.…”

“…The numbers below the sequence correspond to the channel-lining sidechains shown in bold type and define the numbering scheme used throughout the paper, in which the glutamate residue of the cytoplasmic intermediate ring [23] of M2~7 is numbered 12 The N-termini and C-termini were blocked with an acetyl group and an amide group, respectively, in order to mimic the effect of preceding and succeeding peptide bonds.…”