Membrane-bound Acetylcholine Receptor Research Articles

The Role of Serine/Threonine and Tyrosine Protein Kinases in Depression of the Cholinosensitivity of Neurons in the Common Snail in a Cellular Analog of Habituation

The experiments reported here showed that the adenylate cyclase inhibitor SQ 22,536, inhibitors of serine/threonine protein kinases – protein kinase A (Rp-cAMPS), protein kinase G (H-Arg-Lys-Arg-Ala-Arg-Lys-Glu-OH), calcium/calmodulin-dependent protein kinase II (KN-93), p38 mitogen-activated protein kinase (PD 169316) – and an inhibitor of tyrosine protein kinases, including the Src (PP2) family (genistein), weaken depression of the acetylcholine-induced influx current in common snail defensive behavior command neurons in conditions of rhythmic local applications of acetylcholine to the cell body in a cellular model of habituation. The selective protein kinase C inhibitor chelerythrin did not alter depression of the acetylcholine current. Mathematical modeling of the effects of these inhibitors on the number of membrane-bound cholinoreceptors yielded calculated curves consistent with the experimental curves showing depression of the acetylcholine current. The experimental data and calculation results led to the following suggestion. Reversible depression of the cholinosensitivity of the bodies of defensive behavior command neurons in common snails in the cellular analog of habituation is associated with a decrease in the number of membranebound acetylcholine receptors in the test zone due to activation of several serine/threonine protein kinases: protein kinase A, protein kinase G, calcium/calmodulin-dependent protein kinase II, mitogen-activated protein kinases (excluding protein kinase C), as well as tyrosine protein kinases, including members of the Src kinase family. The main action of all these kinases (except protein kinase C) in command neurons is targeted to cellular cytoskeleton proteins (actin microfilaments and microtubules). Phosphorylation of these proteins leads to copolymerization and stabilization of actin filaments, stabilization of tubulin, which is the main microtubule protein, changes in the activity of motor proteins, and, thus, changes in the rates of transport processes, i.e., the rates of receptor endocytosis and exocytosis. The action of protein kinase G is (indirectly) on the interaction of actin with myosin. Protein kinase A, calcium/calmodulin-dependent protein kinase II and Src kinase also phosphorylate proteins activating the displacement of receptors into clathrin-coated pits in endocytosis.

A NOVEL MAGNESIUM-CREATINE CHELATE AND A LOW DOSE CREATINE SUPPLEMENTATION REGIMEN IMPROVE WORK

Creatine (Cr) is transported into skeletal muscle fibers through a Na+ dependent Cr transport protein that operates against a 200:1 gradient. The Cr transporter is subject to saturation as it is limited by the Na+ gradient. PURPOSE: The aim of the investigation was to evaluate the efficacy of a novel Mg2+-Cr chelate that may function to permit enhanced intracellular Cr levels via entry through membrane-bound acetylcholine (Ach) receptors, which function as ligand-gated cation channels. Increased levels of Cr in muscle fibers may translate into enhanced muscle function. We tested the hypothesis that when compared with both placebo and Cr groups, the Mg2+-Cr chelate group would: A.) demonstrate greater improvements in the 1 RM for flat bench press; and B.) perform more work at 70% of the 1 RM for flat bench press. METHODS: Trained male weight lifters (n = 31) were randomly assigned in a double blind manner to either a placebo group (multidextran), a Cr group (2.5-g Cr daily), or a Mg2+-Cr group (2.5-g Cr daily). On day 0, baseline data was collected for the flat bench press 1 RM, and maximal work completed during a fatigue set at 70% of the 1 RM. During the subsequent 10-day supplement regimen, subjects were instructed to continue their normal resistance training programs. On day 11, follow up tests were completed. RESULTS: There was no difference between groups when the preto post-test 1 RM values were evaluated. When work was evaluated, however, compared with the placebo group, both the Cr and the Mg2+-Cr groups demonstrated an improved performance. CONCLUSION: Partial support for the hypothesis suggests that low doses of Cr may be effective at increasing muscle fiber Cr content levels, and consequently, performance. As there were no differences for either performance test between the Cr and Mg2+-Cr group, the proposed mechanism of Cr entry via Ach receptors may not play a significant role in enhancing Cr transport over the Cr transport protein. Supported by Albion Laboratories, Inc. (RAD, STD), and an OSU Seed Grant (STD).

Benzophenone-Type Photoactivatable Derivatives of α-Neurotoxins and α-Conotoxins in Studies on Torpedo Nicotinic Acetylcholine Receptor

By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom α-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of α-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that α-neurotoxins and α-conotoxins bind at the subunit interfaces. Detecting the labeled α-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and α-conotoxin molecule.

Agonist binding to the Torpedo acetylcholine receptor. 1. Complexities revealed by dissociation kinetics.

Examination of the kinetics of dissociation of [3H]acetylcholine and [3H]suberyldicholine from the membrane-bound acetylcholine receptor from Torpedo californica has revealed complexities in the high-affinity binding of nicotinic agonists. Each agonist binds to two high-affinity sites per receptor with an equilibrium dissociation constant of approximately 15 nM. When dissociation of [3H]acetylcholine from the receptor complex was triggered by dilution, dissociation occurred as a monophasic process with an apparent rate of 0.023 +/- 0.010 s(-1). However, when micromolar concentrations of unlabeled agonists (acetylcholine, carbamylcholine or suberyldicholine) were included in the dilution buffer this rate increased about 5-fold. This accelerating effect occurred even when the two high-affinity sites were initially saturated with the radioligand. This suggested the presence of an additional site (or subsite) for agonist with affinity in the micromolar range. However, at concentrations of 0-20 microM, no additional sites for [3H]acetylcholine were detected at equilibrium. To explain these results, we propose that each high-affinity site is made up of two subsites, A and B, which are mutually exclusive at equilibrium. With [3H]acetylcholine initially occupying site A, occupancy of site B by unlabeled ligand reduces the affinity for site A and accelerates the dissociation of the radioligand. Studies of dissociation of [3H]suberyldicholine, a large bis-quaternary agonist, provide some clue as to the possible physical nature of these subsites. Whereas its dissociation rate was similar to that of [3H]acetylcholine (0.028 +/- 0.012 s(-1)), this rate was only marginally, if at all, affected by the presence of unlabeled ligands. These results, in addition to those presented in the accompanying manuscript, lead to the proposal that [3H]suberyldicholine is able to cross-link the two subsites or at least sterically occlude the second site.

The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica.

Cross-linking an alpha-neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with alpha-neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.

Biotechnology-based recognition elements

The author discusses technology, applications, and opportunities for biosensors. The following topics are considered: the shape of the myoglobin molecule; a schematic structure of an antibody showing the different regions of the molecule; the form of the membrane-bound acetylcholine receptor protein; forces responsible for interactive behavior between receptors and ligands; a photolithographic technique for the production of a peptide library on a chip; the principle of affine chromatography; the structure of a glucose electrode developed by Leland Clark; interferences with an enzyme-based glucose biosensor; components of biosensors; a glucose sensor based on fiber optics. >

A combined ELISA-immunoelectron microscopic approach for topological mapping of membrane protein epitopes: application to the nicotinic acetylcholine receptor.

Identification of epitope localization on either side of the lipid membrane by immunoelectron microscopy constitutes an intrinsic powerful method of structure determination for membrane proteins. We have developed a method allowing measurement and observation, under almost identical experimental conditions, of the binding of monoclonal antibodies (MAb) to membrane-bound acetylcholine receptor from Torpedo marmorata electric tissue. This method, based on ELISA and electron microscopy of negatively stained specimens, was developed with MAb of known epitope specificity. With native membrane fragments, we found that MAb bound to extracellular epitopes in a stoichiometric manner, whereas almost no binding was detected for intracellular epitopes. The treatment based on tissue homogenization in the presence of Zn2+ ions and sucrose resulted in the formation of large, stable openings, rendering accessible about 25% of intracellular epitopes. Electron microscopic observations showed a clear distinction between antibody binding to either intracellular or extracellular epitopes, both with native and Zn(2+)-treated membranes. In addition, the binding of one antibody directed against an extracellular epitope was strikingly dependent on the packing density of acetylcholine receptor molecules, thus enabling us to further distinguish between two levels of accessibility for extracellular epitopes. The method presented here is of general application for studies of epitope mapping in membrane proteins.

Mapping of the polypeptide chain organization of the main extracellular domain of the alpha-subunit in membrane-bound acetylcholine receptor by antipeptide antibodies spanning the entire domain.

To study the organization of the polypeptide chain of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) alpha-subunit, we examined the ability of the native membrane-bound AChR of Torpedo californica (T-AChR) to bind a panel of antibodies against overlapping synthetic peptides which collectively encompassed this entire domain. Antibodies against the alpha-chain peptides alpha 1-16, alpha 89-104 and alpha 158-174 were able to bind to membrane-bound T-AChR. Other anti-peptide antibodies showed little or no binding to T-AChR in the membrane. It is concluded that regions alpha 1-16, alpha 89-104 and alpha 158-174 are highly exposed on the surface of the alpha subunit of membrane-bound AChR.

Mapping the extracellular topography of the α-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain

The extracellular surface of the alpha-chain of Torpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptides alpha 1-16, alpha 89-104, alpha 158-174, alpha 262-276, and alpha 388-408. Lower, but significant, levels of reactivity were obtained with antibodies against peptides alpha 67-82, alpha 78-93, alpha 100-115, and alpha 111-126. On the other hand, free AChR bound high levels of antibodies against peptides alpha 34-49, alpha 78-93, alpha 134-150, alpha 170-186, and alpha 194-210. It also bound moderate levels of antibodies against peptides alpha 262-276 and alpha 388-408. Low, yet significant, levels of binding were exhibited by antibodies against peptides alpha 45-60, alpha 111-126, and alpha 122-138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).

Ligand binding to the membrane-bound acetylcholine receptor from Torpedo marmorata: a complete mathematical analysis.

We have studied by means of equilibrium binding and kinetic experiments the interaction of the membrane-bound nicotinic acetylcholine receptor (nACHR) from Torpedo marmorata with [3H]acetylcholine and the fluorescent agonist NBD-5-acylcholine. In agreement with previous studies by others, we observed the preexistence, in the absence of ligand, of an equilibrium between two states of the nAChR, one with high affinity and the other with low affinity for agonist. As additional requirements for a minimal reaction scheme, we recognized (i) the existence of two ligand-binding sites, each of which may exist in two conformational states when occupied, and (ii) ligand-induced transitions between these conformations. Employing a special form of the allosteric model which considers these requirements, we then developed a suitable algorithm in order to simultaneously fit the whole set of equilibrium binding and kinetic data obtained for the two ligands. In this way we determined for a minimal model of the mechanism of action of the nAChR the complete set of rate constants and KD values involved. With these values available, we were able to simulate the rise and fall in the concentrations of individual receptor-ligand complexes and conformations occurring in the course of excitatory events at the electrocyte synapse. The membrane environment of the nAChR plays a decisive role with respect to the rates of conformational change of the nAChR occurring in the course of ligand interaction. Thus, artificial changes in membrane structure and composition can speed up by several orders of magnitude the rate of conformational change ("desensitization"). A proper structure of the surrounding membrane hence is a prerequisite for the physiological function of the membrane-embedded nAChR.

Role of histidine residues in the α-bungarotoxin binding site of the nicotonic acetylcholine receptor

This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopyge tschudii on its specific α-bungarotoxin binding. The acylating reagent ethoxyformic anhydride (diethyl pyrucarbonate, DEP), was used. DEP-treatment induces a loss of binding capacity, time and DEP-concentration dependent. After a 30 min period of derivatization with 2 mM final DEP-concentration, at pH 7.4, the decrease reaches 70%; the loss of binding capacity is faster at pH 7.4 than at pH 6.0, as expected, since the amount of unprotonated species is higher under the first condition. Moreover, when ethoxyformylation is carried out at different pH values, the most important neurotoxin binding decrease occurs between pH 6.0 and 8.0. Furthermore, ethoxyformylation reversion restores such capacity. Consistent with the modification of a binding site, the ethoxyformylation does not bear on the affinity but reduces the number of receptors. Ethoxyformylation in the presence of carbamylcholine shows some ligand protective effect. These results, as a whole, strongly indicate a relevant role for histidine residues at the α-bungarotoxin binding site of the nicotinic acetylcholine receptor.

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.

The noncompetitive blocker [ 3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α-subunit

The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [ 3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The α-subunit was purified digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by [ 3H]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the α-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the δ-chain [1] and the Ser-254 and Leu-257 in the β-chain [2]. Extended sequence analysis of the hydrophobic segment M 1 further showed that no labeling occurred in this region.

Symmetry and dimensions of membrane-bound nicotinic acetylcholine receptors from Torpedo californica electric tissue: rapid rearrangement to two-dimensional ordered lattices.

Computer-aided image-averaging methods are applied to different preparations of membrane-bound nicotinic acetylcholine receptor. Circular harmonic averaging (CHA), a novel, reference-independent averaging method developed by W. Kunath and H. Sack-Kongehl [1989) Ultramicroscopy 27:171-184) allows analyzing images of single molecules of the receptor in its native membrane-bound state. The five subunits of the receptor are clearly resolved. At the resolution obtained (approximately 20 A) no differences were observed with resting and agonist-desensitized receptors. A method is proposed for rapidly arranging the acetylcholine receptors to ordered lattices. Depending on the conditions, tetragonal or hexagonal, two-dimensional lattices can be obtained within 2 to 6 days at 4 degrees C. Analysis by CHA shows that the receptor molecules preserve their gross structure and dimensions in these membranes, but that they are randomly oriented. Both lattices, therefore, do not represent true two-dimensional crystals.

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration.

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.

Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site.

The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.

Recognition of inter-transmembrane regions of acetylcholine receptor α subunit by antibodies, T cells and neurotoxins implications for membrane-subunit organization

Three regions of the α chain of Torpedo californica acetylcholine receptor (AChR), corresponding to residues α262–276, α388–408 and α427–437 were synthesized, purified and characterized. The first two peptides have been proposed to occupy inter-transmembrane regions while the third represented the C-terminal segment, proposed by various models to be either extracellular or intracellular. Peptide α388–408 stimulated a good response in the AChR-primed T cells of H-2 s haplotype mice, a low response in the H-2 q haplotype and no response in the H-2 b haplotype. Peptide α427–437 stimulated AChR-primed T cells of the H-2 s haplotype, but caused no response in the q and b haplotypes. Peptide α262–276 evoked no in vitro stimulation in any of the s, q or b haplotypes. In antibody binding studies, peptide α388–408 bound antibodies raised against free AChR or against membrane-bound AChR. The other two peptides showed little or no binding activity. Further, peptide α388–408 bound specifically both 125-I-labelled bungarotoxin and cobratoxin, while the other two peptides had no binding activity. These results were consistent with only one of the models for subunit organization with-in the membrane.

Phospholipase A 2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the α-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A 2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of α-bungarotoxin-binding sites are shifted to lower temperaturex by approx. 7 and 5 °C, respectively, upon exposure to phospholipase A 2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A 2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3( n −3 ) causes detectable destabilization of the α-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A 2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A 2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A 2 hydrolysis products.

Myasthenogenicity of human acetylcholine receptor synthetic alpha-subunit peptide 125-147 does not require intramolecular disulfide cyclization.

This study reports the synthesis of a disulfide-looped peptide corresponding to residues 125-147 (Cys 128-Cys 142) of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle, H alpha 125-147 (Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln- Asn-Cys-Ser-Nle-Lys Leu-Gly), and a nondisulfide-looped analogue, H alpha 125-147(S) (Lys-Ser-Tyr-Ser-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu- Gln-Asn-Cys-Ser-Nle-Lys-Leu-Gly), in which the amino acid Cys 128 was replaced with serine. Both peptides induced antigen-specific helper T cell responses, as evidenced in vitro by lymph node cell proliferation and in vivo by production of anti-AChR antibodies. Rats immunized with 100 micrograms of either synthetic peptide, without conjugation to a carrier, produced anti-peptide antibodies which bound to native AChR in immunoprecipitation assays and induced modulation of membrane-bound AChR from cultured human myotubes. Both peptides also induced electrophysiologic and biochemical signs of experimental autoimmune myasthenia gravis. Thus, region 125-147 of the AChR alpha-subunit is at least partly exposed extracellularly in human muscle and contains one or more autoantigenic sites capable of stimulating T cells and B cells. Disulfide-linkage between residues Cys 128 and Cys 142 is not essential for myasthenogenicity.

Thermal perturbation studies of membrane-bound acetylcholine receptor from Torpedo: Effects of cholinergic ligands and membrane perturbants

Thermal perturbation techniques have been used to probe structural features of the nicotinic acetylcholine receptor (AcChR). The information obtained from differential scanning calorimetry (DSC) of AcChR membranes (M. C. Farach and M. Martinez-Carrion (1983) J. Biol. Chem. 258, 4176) in the absence and in the presence of cholinergic ligands and local anesthetics, is comparable to that obtained from a simpler technique of heat inactivation of the α-bungarotoxin (α-Bgt) binding sites on the AcChR protein in similar samples. When AcChR membranes are heated at ~ 1 °C/min, heat inactivation of toxin binding sites has a characteristic T 50 value (temperature at which 50% of the initial capacity to bind α-Bgt remains) of ~60 °C. When heated at a constant temperature during increasing periods of time, the rate at which heat inactivation occurs is also characteristic of the temperature chosen for the experiment. The above thermal parameters are also sensitive to perturbation of the AcChR membrane matrix by the presence of subsolubilizing concentrations of detergents. Moreover, elimination of detergents by dialysis allows us to evaluate the reversibility or irreversibility of AcChR thermal destabilization induced by detergents or other membrane perturbants. Under the experimental conditions used, structural destabilization induced by octylglucoside or cholate can be fully reversed by detergent dialysis, while that exerted by deoxycholate cannot. “Thermal gel” analysis of the aggregation of AcChR subunits induced by heat (G. Soler, J. R. Mattingly, and M. Martinez-Carrion (1984) Biochemistry 23, 4630) has also been used to assess the effects of detergent presence on the AcChR protein. When deoxycholate is used as the perturbing agent, there is a particularly effective sulfhydryl-mediated aggregation of the γ-δ subunit group, which appears to correlate with the irreversible destabilization of α-Bgt binding sites induced by that detergent.