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.

The neuronal nicotinic alpha 7 (nAChR) and 5-hydroxytryptamine (5HT3) receptors are ligand-gated ion channels with a homologous topological organization and have activation and desensitization reactions in common. Yet these homo-oligomeric receptors differ in the pharmacology of their binding sites for agonists and competitive antagonists, and in their sensitivity to Ca2+ ions. The alpha 7 channel is highly permeable to Ca2+ ions and external Ca2+ ions potentiate, in an allosteric manner, the permeability response to acetylcholine, as shown for other neuronal nAChRs. The 5HT3 channel, in contrast, is not permeable to Ca2+ ions, but blocked by them. To assign these properties to delimited domains of the primary structure, we constructed several recombinant chimaeric alpha 7-5HT3 receptors. We report here that one of the constructs expresses a functional receptor that contains the serotonergic channel still blocked by Ca2+ ions, but is activated by nicotinic ligands and potentiated by external Ca2+ ions.

Introduction by site-directed mutagenesis of three amino acids from the MII segment of glycine or gamma-aminobutyric acid (GABAA) receptors into the MII segment of alpha 7 nicotinic receptor was sufficient to convert a cation-selective channel into an anion-selective channel gated by acetylcholine. A critical mutation was the insertion of an uncharged residue at the amino-terminal end of MII, stressing the importance of protein geometrical constraints on ion selectivity.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smoker's brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4beta2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to act inside the cell and to increase the amount of beta subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4beta2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

The nicotine receptor for the neurotransmitter acetylcholine is an allosteric protein composed of four different subunits assembled in a transmembrane pentamer alpha 2 beta gamma delta. The protein carries two acetylcholine sites at the level of the alpha subunits and contains the ion channel. The complete sequence of the four subunits is known. The membrane-bound protein undergoes conformational transitions that regulate the opening of the ion channel and are affected by various categories of pharmacologically active ligands.

The relative permeability for sodium, potassium, and calcium of chicken a7 neuronal nicotinic receptor was investigated by mutagenesis of the channel domain M2.Mutations in the "intermediate ring" of negatively charged residues, located at the cytoplasmic end of M2 (site 1), reduce calcium permeability without significantly modifying other functional properties (activation and desensitization) of the receptor; a similar change of ion selectivity is also noticed when mutations at site 1 are done in the context of a receptor mutant that conducts ions in a desensitized state. Moreover, mutations of two adjacent rings of leucines at the synaptic end of M2 (site 2) have multiple effects. They abolish calcium permeability, increase the apparent affinity for acetylcholine by 10-to 100-fold, augment Hill numbers (up to 4.6-5.0) of acetylcholine dose-response relationships, slow rates of ionic response onset, and lower the extent of desensitization. Mutations at these two topographically distinct sites within M2 selectively alter calcium transport without affecting the relative permeabilities for sodium and potassium.Influx of calcium (Ca2+) through peripheral and brain nicotinic acetylcholine receptors (nAChRs) has been described in several preparations including muscle (1, 2) (23,27,28). In another study, the simultaneous introduction of three mutations in the M2 segment of the a7 receptor converted its ionic selectivity from cationic to anionic (14). For one of the mutationsThe 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.(E237A), indirect evidence suggested significant alterations of Ca2+ permeability (14).In this study, we further investigate the monovalent vs. divalent cation selectivity of the a7 receptor. We show that a mutation at the cytoplasmic end of M2 (E237A) abolishes Ca2+ permeability without significantly affecting other properties of the pharmacological and physiological responses to ACh. We further identify another site of two adjacent amino acids, close to the extracellular end of M2 (Leu-254 and Leu-255), where mutations reduce Ca2+ permeability and affect other physiological properties of the response such as its apparent affinity for ACh and the rate of currents onset and desensitization.MATERIALS AND METHODS Mutagenesis. Mutants were prepared as described (23,27). Their coding sequence was checked.Electrophysiology. Oocytes were prepared, injected, and recorded as described (29). For voltage-clamp measurements, cells were incubated in OR2 medium (solution B, Table 1) and challenged by ACh application. Data from one oocyte are given in the figures and values (mean ± SEM) determined from 5 to 10 oocytes from more than one donor are given in Table 2.Current-voltage (I-V) curves were obtained by subtracting passive membrane currents from currents evoked in the presence of ACh. ACh was applied at a concentration close to its EC50 value ...

In the alpha7 nicotinic acetylcholine receptors, we analyze the contribution of mutations E237A and V251T, together with the proline insertion P236', in the conversion of the charge selectivity from cationic to anionic. We show that the triple mutant exhibits spontaneous openings displaying anionic selectivity. Furthermore, at position 251, hydrophilic or even negatively charged residues are compatible with an anionic channel. In contrast, the additional proline yields an anionic channel only when inserted between positions 234 and 237; insertion before 234 yields a cationic channel and after 238 alters the receptor surface expression. The coiled 234-238 loop thus directly contributes to the charge selectivity filter of the alpha7 channel.

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...