Primary structure of delta subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence

Clones carrying cDNA sequences for the delta subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has indicated that this polypeptide consists of 516 amino acids including a hydrophobic prepeptide of 21 amino acids. The delta subunit of the calf muscle acetylcholine receptor, like the alpha, beta and gamma subunits of the same receptor as well as the alpha and gamma subunits of its human counterpart, exhibits structural features common to all four subunits of the Torpedo electroplax receptor, apparently being oriented across the membrane in the same manner as proposed for the fish receptor subunits. The degree of amino acid sequence homology between the calf and Torpedo delta subunits (60%) is comparable to that between the beta subunits (59%) and to that between the gamma subunits (56%), but is lower than that between the alpha subunits of the two species (81%). This suggests that the alpha subunit evolved more slowly than the three other subunits. A dendrogram representing the sequence relatedness among the four subunit precursors of the mammalian and fish acetylcholine receptors has been constructed. Some regions of the delta subunit molecule, including the region containing the putative disulphide bridge and that encompassing the clustered putative transmembrane segments M1, M2 and M3, are relatively well conserved between calf and Torpedo. The relative pattern of regional homology is similar for all four subunit precursors.     

Interactions between alpha-conotoxin MI and the Torpedo marmorata receptor alpha-delta interface

The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.     

Phosphorylation sites of the nicotinic acetylcholine receptor. A novel site detected in position delta S362.

The delta-subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue isolated form receptor purified in the absence of protein phosphatase inhibitors contains a total of four phosphate groups. Three of these are shown to represent phosphoserine groups. The fourth possible represents phosphotyrosine. The phosphate groups are localized within the primary structure: We found phosphoserine in positions delta S361 and delta S377, the predicted sites phosphorylated by PKA and PKC, respectively. In addition, we found that position delta S362 is also phosphorylated. Phosphorylation experiments with the synthetic peptide delta L357-delta K368 show that phosphorylation of this novel site can be catalyzed by PKA and by PKC. It is concluded that the delat-subunit of the acetylcholine receptor is stably and not transiently phosphorylated. Implications for the physiological functions of receptor phosphorylation are discussed.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Segment alpha 182-198 of Torpedo californica acetylcholine receptor contains second toxin-binding region and binds anti-receptor antibodies

The area around Cys-192 and Cys-193 is thought to be a functionally important part of the alpha-subunit of the acetylcholine receptor. We have synthesized peptide alpha 182-198 of the alpha-chain of the Torpedo californica acetylcholine receptor and investigated the binding to the peptide of alpha-bungarotoxin, cobratoxin and antibodies raised against acetylcholine receptor. The results showed that the synthetic peptide alpha 182-198 contains a second toxin-binding region and also binds a considerable fraction of anti-receptor antibodies. We also report here the toxin-binding activity of synthetic peptide alpha 125-148 of the human acetylcholine receptor which has been previously localized as a toxin-binding region in the alpha-chain of the Torpedo receptor.     

Biochemical properties of acteylcholine receptor subunits from Torpedo californica.

Four polypeptide chains composing acetylcholine receptors from the electric organ of Torpedo californica were purified by preparative electrophoresis in sodium dodecyl sulfate. Their apparent mole ratio alpha/beta/gamma/delta is 2:1:1:1. These chains are not readily distinguished by amino acid or carbohydrate composition but are distinguished by apparent molecular weight and polypeptide maps. By peptide maps, no extensive homology is evident between these chains or between any of these chains and higher molecular weight chains found in receptor-enriched membrane fragments.     

A universal oligonucleotide probe for acetylcholine receptor genes. Selection and sequencing of cDNA clones for the mouse muscle beta subunit

A region of 25 nucleotides is highly conserved in genes coding for the alpha, beta, gamma, and delta subunits of the nicotinic acetylcholine receptor (AChR) of human, mouse, calf, chicken, and Torpedo. Based on this observation, a 2-fold degenerate oligonucleotide was synthesized and used as a probe to screen a cDNA library made from a mouse myogenic cell line. Clones coding for the beta, gamma, and delta subunits were identified by the probe. The protein sequence deduced from the beta subunit clones codes for a precursor polypeptide of 501 amino acids with a calculated molecular weight of 56,930 daltons, which includes a signal peptide of 23 amino acids. The protein sequence and structural features of the beta subunits of mouse, calf, and Torpedo are conserved. A clone coding for the mouse gamma subunit was isolated, and its identity was confirmed by alignment of its sequence to previously published cDNA sequences for the mouse and calf gamma subunits. The clone contained approximately 200 nucleotides more at its 3' end untranslated region than a mouse gamma clone recently described. Northern blot analysis, utilizing as probes these beta and gamma subunit cDNAs and previously characterized alpha and delta subunit cDNAs, shows that the steady-state levels of the four AChR mRNAs increase coordinately during terminal differentiation of cultured C2 and C2i mouse myoblasts. The increase in mRNA levels can account for the rise of cell surface receptors during myogenesis and suggests that the muscle AChR genes may be regulated during development by a common mechanism. Utilization of this oligonucleotide probe should prove useful for screening a variety of libraries made from different species and tissues which are known to express AChRs.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.     

Anti-acetylcholine receptor response achieved by immunization with a synthetic peptide from the receptor sequence

A synthetic peptide corresponding to the first twenty amino acids of the N-terminal region from the alpha-subunit of the Torpedo acetylcholine receptor cross reacts with antibodies to the receptor. A conjugate of this peptide to bovine serum albumin elicits in rabbits an immune response towards the synthetic peptide as well as towards the acetylcholine receptor. Blotting experiments demonstrate that the antipeptide antibodies react exclusively with the alpha-subunit of the acetylcholine receptor. Antibodies against synthetic peptides from various regions of the receptor sequence may provide useful reagents for structural and developmental analysis of the acetylcholine receptor as well as for the regulation of experimental autoimmune myasthenia gravis.     

Structural and dynamic studies of the gamma-M4 trans-membrane domain of the nicotinic acetylcholine receptor

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the gamma-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear alpha-helical conformation with the helix aligned within 15 degrees of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.     

Solution conformation of alpha-conotoxin EI, a neuromuscular toxin specific for the alpha 1/delta subunit interface of torpedo nicotinic acetylcholine receptor

A high resolution structure of alpha-conotoxin EI has been determined by (1)H NMR spectroscopy and molecular modeling. alpha-Conotoxin EI has the same disulfide framework as alpha 4/7 conotoxins targeting neuronal nicotinic acetylcholine receptors but antagonizes the neuromuscular receptor as do the alpha 3/5 and alpha A conotoxins. The unique binding preference of alpha-conotoxin EI to the alpha(1)/delta subunit interface of Torpedo neuromuscular receptor makes it a valuable structural template for superposition of various alpha-conotoxins possessing distinct receptor subtype specificities. Structural comparison of alpha-conotoxin EI with the gamma-subunit favoring alpha-conotoxin GI suggests that the Torpedo delta-subunit preference of the former originates from its second loop. Superposition of three-dimensional structures of seven alpha-conotoxins reveals that the estimated size of the toxin-binding pocket in nicotinic acetylcholine receptor is approximately 20 A (height) x 20 A (width) x 15 A (thickness).     

Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells

When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.     

Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor alpha subunit: effects of cysteine/cystine modification and species-specific amino acid substitutions

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.     

The alphaM1 segment of the nicotinic acetylcholine receptor exhibits conformational flexibility in a membrane environment

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) is predominantly alpha-helical, and of the four distinctly different transmembrane M-segments, only the helicity of M1 is ambiguous. In this study, we have investigated the conformation of a membrane-embedded synthetic M1 segment by solid-state nuclear magnetic resonance (NMR) methods. A 35-residue peptide representing the extended alphaM1 domain 206-240 of the Torpedo californica nAChR was synthesized with specific 13C - and 15N-labelled amino acids, and was incorporated in different phosphatidylcholine model membranes. The chemical shift of the isotopic labels was resolved by magic angle spinning (MAS) NMR and could be related to the secondary structure of the alphaM1 analog at the labelled sites. Our results show that the membrane-embedded alphaM1 segment forms an unstable alpha-helix, particularly near residue Leu18 (alphaLeu223 in the entire nAChR). This non-helical tendency was most pronounced when the peptide was incorporated in fully hydrated phospholipid bilayers, with an estimated 40-50% of the peptides having an extended conformation at position Leu18. We propose that the conserved proline residue at position 16 in the alphaM1 analog imparts a conformational flexibility on the M1 segments that could enable membrane-mediated modulation of nAChR activity.     

Characterization and three-dimensional structure determination of psi-conotoxin Piiif,a novel noncompetitive antagonist of nicotinic acetylcholine receptors

A novel inhibitor of nicotinic acetylcholine receptors (nAChRs), psi-conotoxin Piiif, was isolated from the venom of Conus purpurascens and found to have the sequence GOOCCLYGSCROFOGCYNALCCRK-NH2. The sequence is highly homologous to that of psi-conotoxin Piiie, a previously identified noncompetitive inhibitor of Torpedo electroplax nAChR, also isolated from C. purpurascens. Both psi-conotoxins block Torpedo and mouse nicotinic acetylcholine receptors (nAChRs), but psi-Piiif is less potent by a factor of 10(1)-10(2). A high-resolution structure of psi-Piiif was determined by NMR and molecular modeling calculations. Psi-Piiif analogues containing [(13)C]-labeled cysteine at selected positions were synthesized to resolve spectral overlap of Cys side chain proton signals. The structures are well-converged, with backbone atom position RMSDs of 0.21 A for the main body of the peptide between residues 4 and 22 and 0.47 A for all residues. The overall backbone conformation is closely similar to psi-Piiie, the main difference being in the degree of conformational disorder at the two termini. Psi-Piiie and psi-Piiif have similar locations of positive charge density, although psi-Piiif has a lower overall charge. One disulfide bridge of psi-Piiif appears to undergo dynamic conformational fluctuations based on both the model and on experimental observation. Chimeras in which the three intercysteine loops were swapped between psi-Piiie and psi-Piiif were tested for inhibitory activity against Torpedo nAChRs. The third loop, which contains no charged residues in either peptide, is the prime determinant of potency in these psi-conotoxins.     

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit

Monoclonal antibodies (mAbs) to the main immunogenic region (MIR) bind to fusion proteins containing region 37-200 of the alpha chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse alpha chain, these mAbs react with sequence 61-216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse alpha chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The conformational dependence and epitope specificity of the mAbs are discussed.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor

Fourteen clonal hybridoma lines that secrete monoclonal antibodies (mabs) to the Torpedo acetylcholine receptor (AChR) have been isolated. When analyzed by an immunoreplica technique, two mabs recognized the alpha subunit, three reacted with the beta subunit, one reacted with the gamma chain, and five recognized the delta subunit. One mab failed to react with any of the subunits using this assay and two mabs recognized determinants found on both the gamma and the delta subunits. These were classified according to their reactivities with the membrane-bound Torpedo AChR. One category is comprised of mabs (including both anti-alpha mabs) that recognize extracellular epitopes. A second classification included mabs that are unable to bind the membrane-associated AChR. The third category is comprised of mabs directed against cytoplasmic epitopes of the AChR. The latter mabs, all of which recognize the gamma or delta subunits or both, bind only slightly to sealed, outside-out Torpedo vesicles. The binding is increased 10-20-fold by either alkaline extraction or treatment of the vesicles with 10 mM lithium diiodosalicylate but not by permeabilization of the vesicles with saponin. Three of the six mabs in this category react with frog muscle endplates but only if the cytoplasmic surface of the membrane is accessible.