Role of the large cytoplasmic loop of the alpha 7 neuronal nicotinic acetylcholine receptor subunit in receptor expression and function

The role of the large intracellular loop of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in the expression of functional channels was studied. For this purpose, systematic deletions and substitutions were made throughout the loop and the ability of the mutated alpha7 subunits to support expression of functional nAChRs at the Xenopus oocyte membrane was tested. Surface nAChR expression was abolished upon removal of sequences at two regions, a 29-amino acid segment close to the N-terminus of the loop (amino acids 297-325) and adjacent to the third transmembrane region and an 11-amino acid segment near the fourth transmembrane region. Some residues (amino acids 317-322) within the 29 amino acids N-terminal segment could be substituted by others but not deleted without loss of expression, suggesting that a certain structure, determined by the number of amino acids rather than by their identity, has to be maintained in this region. The contiguous sequence M323 K324 R325 did not tolerate deletions and substitutions. Removal of the rest of the cytoplasmic loop was not deleterious; even higher expression levels (2-4-fold) were obtained upon large deletions of the loop (Delta399-432 and Delta339-370). High expression levels were observed provided that a minimal sequence of three amino acids (E371, G372, and M373) was present. In addition, some electrophysiological properties of mutant nAChRs were modified. Substitution of the EGM sequence by other protein segments produced a variety of effects, but, in general, insertions were not well tolerated, suggesting the existence of tight structural restrictions in the large cytoplasmic region of the rat alpha7 subunit.     

CHRNB2 is the second acetylcholine receptor subunit associated with autosomal dominant nocturnal frontal lobe epilepsy

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the beta2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes and characterized by an approximately 10-fold increase in acetylcholine sensitivity. CHRNB2 is a new gene for idiopathic epilepsy, the second acetylcholine receptor subunit implicated in ADNFLE.     

Agonist- and competitive antagonist-induced movement of loop 5 on the alpha subunit of the neuronal alpha4beta4 nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that rapidly convert a chemical signal into an electrical signal. Although the structure of the nAChR is quite well described, the coupling between agonist binding and channel gating is still under debate. In this study, we probed local conformational transitions on the neuronal α4β4 nAChR by specifically tethering a conformation-sensitive fluorescent dye on αG98C located on loop 5 (L5), and simultaneously monitoring fluorescence intensity and current after expression in Xenopus oocytes. The potency of acetylcholine (ACh) was significantly higher in the cysteine mutant and further increased upon tetramethylrhodamine-6-maleimide labeling, suggesting a role of L5 in binding or gating. Structural reorganizations of L5 were shown to occur upon activation, as revealed by the fluorescence intensity increase during ACh exposure. Fluorescence changes were also detected at ACh concentrations lower than needed for current activation, suggesting a movement of L5 for a closed, resting or desensitized state. The competitive antagonist dihydro-β-erythroidine also induced a movement of L5 although at concentrations significantly higher than needed for current inhibition. Consequently L5, located inside the lumen of the pentamer, plays a role in both activation and inhibition of the nAChR.     

Nicotine-induced up-regulation and desensitization of alpha4beta2 neuronal nicotinic receptors depend on subunit ratio

Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.     

Study of the interaction of chlorisondamine and chlorisondamine analogues with an epitope of the alpha-2 neuronal acetylcholine nicotinic receptor subunit

Chlorisondamine (CHL), a neuronal nicotinic ganglionic blocker, when injected in the cerebral ventricle of rats chronically blocks the increase in locomotion and rearing by subcutaneous nicotine injection. The blocking of the ion channel(s) prevents nicotine from exerting its rewarding effects on the CNS. When administered intraperitoneally, a dose 400-500 times the intracerebroventricular one is needed to cross the blood-brain barrier and to generate the same level of nicotine antagonism, resulting in severe side-effects, thus making it unlikely to be used as a therapeutical compound. Three CHL analogues, 2-(indolin-1-yl)-N,N,N-trimethylethanaminium iodide, 2-(1,3-dioxoisoindolin-2-yl)- N,N,N-trimethylethanaminium iodide, and 2-(1H-indole-3-carboxamido)- N,N,N-trimethylethanaminium iodide, were synthesized in the hope of circumventing the parent compound's shortcomings. They all share a modified indole ring, lack the four chlorines CHL carries, and have one tertiary amine and one quaternary amine. The CHL analogues form noncovalent complexes with an epitope of the alpha-2 nicotinic receptor subunit, GEREE(p)TEEEEEEEDEN, previously proposed as the possible site of CHL interaction. Complexes were analyzed using matrix-assisted laser desorption/ionization mass spectrometry for comparison with CHL. Overall, all three analogues showed better affinity than CHL for complex formation with both the nonphosphorylated and phosphorylated epitopes.     

NMR structural analysis of alpha-bungarotoxin and its complex with the principal alpha-neurotoxin-binding sequence on the alpha 7 subunit of a neuronal nicotinic acetylcholine receptor.

We report a new, higher resolution NMR structure of alpha-bungarotoxin that defines the structure-determining disulfide core and beta-sheet regions. We further report the NMR structure of the stoichiometric complex formed between alpha-bungarotoxin and a recombinantly expressed 19-mer peptide ((178)IPGKRTESFYECCKEPYPD(196)) derived from the alpha7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in alpha-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the alpha7 19-mer hairpin-like structure. At the contact zone, Ala(7), Ser(9), and Ile(11) in finger I and Arg(36), Lys(38), Val(39), and Val(40) in finger II of alpha-bungarotoxin interface with Phe(186), Tyr(187), Glu(188), and Tyr(194) in the alpha7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the alpha7 receptor, places alpha-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of alpha-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity.     

Interaction of chlorisondamine with the neuronal nicotinic acetylcholine receptor

An epitope was found on the alpha2-nicotinic isoform of the neuronal nicotinic acetylcholine receptor that would likely form salt bridges with quaternary ammonium compounds and a cation-pi interaction with the pi-cloud of an aromatic ring. Chlorisondamine, a nicotinic antagonist, exerts a long-lasting, if not permanent, blockade of the ion channel gated by acetylcholine. Blocking of the ion channel prevents nicotine from exerting its rewarding effect on the CNS. Chlorisondamine contains two quaternary ammonium groups and a tetrachloroisoindoline ring. We propose that chlorisondamine interacts with an epitope on the alpha2 isoform of the rat neuronal nicotinic receptor (residues 388-402, GEREETEEEEEEEDE), where one or both of the quaternary ammonium groups of chlorisondamine form a salt bridge with dither a glutamic acid side chain or a phosphate group, whereas the tetrachlorobenzene portion of the tetrachloroisoindoline ring interacts with the guanidinium group of arginine in a cation-pi association: In this work, a new way of probing the interaction of a receptor epitope (alpha2) with organic molecules (chlorisondamine and hexachlorobenzene) was undertaken using matrix assisted laser desorption/ionization mass spectrometry.     

Chromosomal localization of seven neuronal nicotinic acetylcholine receptor subunit genes in humans.

We have determined the chromosomal location of seven human neuronal nicotinic acetylcholine receptor subunit genes by genomic Southern analysis of hamster/human somatic cell hybrid DNAs. The beta 2 subunit gene was localized to human chromosome 1, the alpha 2 and beta 3 subunit genes were localized to human chromosome 8, the alpha 3, alpha 5, and beta 4 subunit genes were localized to human chromosome 15, and the alpha 4 subunit gene was localized to human chromosome 20. Mapping of the beta 2 subunit gene to chromosome 1 establishes a syntenic group with the amylase gene locus on human chromosome 1 and mouse chromosome 3, while mapping of the alpha 3 subunit gene to chromosome 15 confirms the existence of a syntenic group with the mannose phosphate isomerase gene locus on human chromosome 15 and mouse chromosome 9.     

Barium permeability of neuronal nicotinic receptor alpha 7 expressed in Xenopus oocytes.

The rat alpha 7 neuronal nicotinic acetylcholine receptor was expressed and studied in Xenopus oocytes. The magnitude and reversal potential of instantaneous whole cell currents were examined in solutions containing varying concentrations of either calcium or barium, and in the presence or absence of the intracellular calcium chelator BAPTA. In external barium, application of nicotine elicits an inwardly rectifying response; in calcium the response is larger and has a linear IV relation. Pretreatment of oocytes with BAPTA-AM could not prevent activation of calcium-dependent chloride channels in external Ringer containing calcium. Using an extended GHK equation, the permeability ratio PBa/PNa of the alpha 7 receptor was determined to be about 17. Our results suggest that alpha 7 nicotinic receptors are highly permeable to divalent cations.     

Alpha-selenoconotoxins, a new class of potent alpha7 neuronal nicotinic receptor antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and nonreducible diselenide bonds. Three isoforms of alpha-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one ([Sec(2,8)]ImI or [Sec(3,12)]ImI), or both ([Sec(2,3,8,12)]ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the alpha7 nicotinic acetylcholine receptor, with each selenoanalogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.