Human Muscle Nicotinic Acetylcholine Receptor Research Articles

High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

Potential roles of recombinant acetylcholine receptor α subunit 1–211 in immunoadsorbent and DNA immunization

The open reading frame of the α-subunit (amino acids 1–211) of human muscle nicotinic acetylcholine receptor (hAChR α211) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR α211 (recombinant hAChR α211) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K–hAChR α211 with a yield of 25 mg/L. rhAChR α211 was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChR α211 was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA–hAChR α211. Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patient's sera and establish an animal model of myasthenia gravis by recombinant hAChR α211 immunization.

The role of the amino acid residue at α1:189 in the binding of neuromuscular blocking agents to mouse and human muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites. A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [(125)I]alpha-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant alpha1 subunits. The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC(50) results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC(50) values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at alpha1:189 and proline at alpha1:194 to serine. However the alpha1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs. The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The alpha1:Phe189Ser substitution, but not alpha1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC(50) values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary 'reinforcement'.

Contrasting actions of philanthotoxin-343 and philanthotoxin-(12) on human muscle nicotinic acetylcholine receptors.

Whole-cell recordings and outside-out patch recordings from TE671 cells were made to investigate antagonism of human muscle nicotinic acetylcholine receptors (nAChR) by the philanthotoxins, PhTX-343 and PhTX-(12). When coapplied with acetylcholine (ACh), PhTX-343 caused activation-dependent, noncompetitive inhibition (IC50 = 17 microM at -100 mV) of whole-cell currents that was strongly voltage-dependent. However, preapplication of PhTX-343 unveiled a voltage-independent antagonism that also required receptor activation, which is suggestive of desensitization enhancement. In single-channel studies, 10 microM PhTX-343 significantly reduced the mean open time of channel openings evoked by 1 microM ACh from 4.42 +/- 0.44 to 1.58 +/- 0.10 ms with a minor increase (1.26-fold) in mean closed time. These data indicate that PhTX-343 predominantly blocks the open channel gated by ACh. In contrast, PhTX-(12) caused potent (IC50 = 0.77 microM at-100 mV), activation-dependent, noncompetitive inhibition of ACh-induced whole-cell currents that was only weakly voltage-dependent and suggestive of desensitization enhancement. It caused only a small decrease (7.5%) in the mean open time of channel openings induced by 1 microM ACh, whereas the mean closed time was significantly increased from 200 +/- 45 ms to 586 +/- 145 ms. The different voltage-dependencies of the two modes of action of these philanthotoxins suggest two binding sites, one deep in the nAChR pore, the other near the extracellular entrance to the pore.

Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris: ROLE OF GLYCOSYLATION IN α-BUNGAROTOXIN BINDING

The N-terminal extracellular domain (amino acids 1-210; halpha-(1-210)) of the alpha subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. halpha-(1-210) bound (125)I-alpha-bungarotoxin with a high affinity (K(d) = 5.1 +/- 2.4 nm), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K(i) approximately 7.5 mm). Interestingly, (125)I-alpha-bungarotoxin binding was markedly impaired by in vitro deglycosylation of halpha-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to halpha-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR alpha subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies.

Inhibition of ligand-gated cation-selective channels by tamoxifen

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT 3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC 50 values of 1.2±0.03 μM ( n H=0.84±0.02) and 1.2±0.1 μM ( n H=1.1±0.1), respectively. Tamoxifen could also block native 5-HT 3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC 50=0.81±0.15 μM and n H of 1.3±0.3. The characteristics of block by tamoxifen at the 5-HT 3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.

Myasthenic nicotinic receptor mutant interpreted in terms of the allosteric model

An extended Monod—Wymctn—Changeux allosteric-type model is applied to human muscle nicotinic acetylcholine receptors expressed in HEK cells, for both the normal form and the high-affinity human myasthénic mutant, ε T264P. The model is based on a concerted transition between the basal (resting) B state and the active (open-channel) A state, with the equilibrium in the absence of ligand determined by the allosteric constant, L o = [B 0]/[A 0]. For wild-type receptors the model with L 0 = 910 8 provides a satisfactory representation of published patch-clamp recordings that yields a distribution of open-channel dwell times with a single peak at 0.7 ms. For the εT264P mutant, the model with L 0 = 100 accounts for the trimodal distribution reported for open-channel dwell times, with peaks at 0.15, 3.8 and 60 ms that correspond to non-, mono- and bi-liganded receptors, respectively. Possible applications of the allosteric model to other myasthénic mutants are considered.

Characterisation, crystallisation and preliminary X-ray diffraction analysis of a Fab fragment of a rat monoclonal antibody with very high affinity for the human muscle acetylcholine receptor

The Fab fragment of a rat monoclonal antibody (no. 192) with very high affinity for the main immunogenic region of the human muscle nicotinic acetylcholine receptor (AChR) has been purified, characterised and crystallised using vapour diffusion techniques. Its Kd for human AChR was determined to be 5×10 −11 M. Its cross-reactivity pattern suggests that residue α23 of the AChR strongly affects its epitope. Crystals suitable for X-ray analysis, obtained by micro- and macroseeding techniques, belong to the orthorhombic space group C222 1 and they diffract to 2.8 A resolution using synchrotron radiation. The unit cell dimensions are α = 83.4 Å, b =110.0 A ̊ and c = 212.2 A ̊ and there are two Fab molecules per asymmetric unit.

Functional and non-functional isoforms of the human muscle acetylcholine receptor.

1. The properties of a recently identified isoform of the human muscle nicotinic acetylcholine receptor (AChR) alpha subunit (alpha +), which in muscle is expressed at similar levels to the alpha subunit, were investigated by both electrophysiological and biochemical approaches following expression in Xenopus laevis oocytes. The single-channel properties of adult (alpha 2 beta delta epsilon) and fetal (alpha 2 beta delta gamma) forms of the human AChR were also investigated. 2. The mean burst duration of adult channels (4.1 +/- 0.3 ms, mean +/- S.E.M., n = 5) is half that of fetal channels (7.9 +/- 0.6 ms, n = 4), while the single-channel conductance is larger (62.2 +/- 0.8 and 37.9 +/- 1.6 pS for adult and fetal channels, respectively), comparable to the developmental changes in single-channel properties observed for other mammalian species. 3. In contrast to the alpha isoform, the alpha + subunit does not bind 125I-labelled alpha-bungarotoxin or monoclonal antibodies directed against the AChR 'main immunogenic region' (MIR), illustrating why the alpha + subunit was first detected through screening of cDNA libraries. 4. By using site-directed mutagenesis to produce subunits that conferred different single-channel conductances on the AChR, we demonstrate that the alpha + isoform is not integrated into functional AChRs. 5. The mutagenesis experiments also revealed that the two alpha subunits within an AChR pentamer are not equivalent within the pore lining region.

Single channel properties of human muscle nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Single channel properties of human muscle nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Five Subunit Genes of the Human Muscle Nicotinic Acetylcholine Receptor Are Mapped to Two Linkage Groups on Chromosomes 2 and 17

RFLPs were detected in the five subunit genes of the human muscle nicotinic acetylcholine receptor (nAChR) using genomic DNA or cDNA probes from the homologous mouse loci. The RFLPs at the alpha-, beta-, gamma-, delta-, and epsilon-subunit gene loci were analyzed for genetic linkage in 16 families (n = 188). Significant evidence was obtained for close linkage of the β- and ϵ-nAChR genes and much greater genetic distance between the α-nAChR gene and the γ/δ-nAChR gene complex. The linkage analysis program CRI-MAP was used to map the positions of the β- and ϵ-nAChR genes relative to seven markers on chromosome 17. The results indicate the β- and ϵ-nAChR genes are separated by about 5 cM and located in the region of chromosome 17p occupied by D17S1, D17S31, TP53, and D17S513. The statistical evidence was confirmed by hybridization of the β- and ϵ-nAChR probes to a panel of human-hamster somatic cell hybrids. The α-, γ-, and δ-nAChR genes were placed on a map of 13 chromosome 2 markers. The linkage analysis placed the nAChR genes at two sites on chromosome 2q about equidistant from the marker CRYGP1, with the α-nAChR gene about 27 cM proximal and the γ/δ-nAChR gene complex about 31 cM distal to CRYGP1.

The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon.

Analysis of acetylcholine receptor clones isolated from a human leg muscle cDNA library, revealed that the alpha-subunit existed as two isoforms. A novel exon, coding for 25 amino acids, was located in the human genomic DNA sequence; its insertion into the alpha-subunit gives the new isoform of 462 amino acids. In addition, mRNAs for the two isoforms were found in equal proportions in poly(A)+ RNA obtained from three further sources including partially denervated and innervated human muscle and the rhabdomyosarcoma cell line TE671. Both protein isoforms can be expressed in E. coli. No evidence of a sequence related to that of the new exon was found in cDNA derived from poly(A)+ RNA isolated from fetal calf or embryonic chick muscle or Torpedo marmorata electric organ.

Isolation and expression of cDNAS for the human muscle nicotinic acetylcholine receptor

Isolation and expression of cDNAS for the human muscle nicotinic acetylcholine receptor