α-BGT Binding Research Articles

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

Quantitative changes of nicotinic receptors in the hippocampus of dystrophin-deficient mice

Lack of dystrophin in Duchenne muscle dystrophy (DMD) and in the mutant mdx mouse results in progressive muscle degeneration, structural changes at the neuromuscular junction, and destabilization of the nicotinic acetylcholine receptors (nAChRs). One-third of DMD patients also present non-progressive cognitive impairments. Considering the role of the cholinergic system in cognitive functions, the number of nAChR binding sites and the mRNA levels of α4, β2, and α7 subunits were determined in brain regions normally enriched in dystrophin (cortex, hippocampus and cerebellum) of mdx mice using specific ligands and reverse-transcription polymerase chain reaction assays, respectively. Membrane preparations of these brain regions were obtained from male control and mdx mice at 4 and 12 months of age. The number of [³H]-cytisine (α4β2) and [¹²⁵I]-α-bungarotoxin ([¹²⁵I]-αBGT, α7) binding sites in the cortex and cerebellum was not altered with age or among age-matched control and mdx mice. A significant reduction in [³H]-cytisine (48%) and [¹²⁵I]-αBGT (37%) binding sites was detected in the hippocampus of mdx mice at 12 months of age. When compared with the age-matched control groups, the mdx mice did not have significantly altered [³H]-cytisine binding in the hippocampus, but [¹²⁵I]-αBGT binding in the same brain region was 52% higher at 4 months and 20% lower at 12 months. mRNA transcripts for the nAChR α4, β2, and α7 subunits were not significantly altered in the same brain regions of all animal groups. These results suggest a potential alteration of the nicotinic cholinergic function in the hippocampus of dystrophin-deficient mice, which might contribute to the impairments in cognitive functions, such as learning and memory, that have been reported in the dystrophic murine model and DMD patients.

Olfactory discrimination varies in mice with different levels of α7-nicotinic acetylcholine receptor expression

Previous studies have shown that schizophrenics have decreased expression of α7-nicotinic acetylcholine (α7) receptors in the hippocampus and other brain regions, paranoid delusions, disorganized speech, deficits in auditory gating (i.e., inability to inhibit neuronal responses to repetitive auditory stimuli), and difficulties in odor discrimination and detection. Here we use mice with decreased α7 expression that also show a deficit in auditory gating to determine if these mice have similar deficits in olfaction. In the adult mouse olfactory bulb (OB), α7 expression localizes in the glomerular layer; however, the functional role of α7 is unknown. We show that inbred mouse strains (i.e., C3H and C57) with varying α7 expressions (e.g., α7 wild-type [α7+/+], α7 heterozygous knock-out [α7+/−] and α7 homozygous knock-out mice [α7−/−]) significantly differ in odor discrimination and detection of chemically-related odorant pairs. Using [125I] α-bungarotoxin (α-BGT) autoradiography, α7 expression was measured in the OB. As previously demonstrated, α-BGT binding was localized to the glomerular layer. Significantly more expression of α7 was observed in C57 α7+/+ mice compared to C3H α7+/+ mice. Furthermore, C57 α7+/+ mice were able to detect a significantly lower concentration of an odor in a mixture compared to C3H α7+/+ mice. Both C57 and C3H α7+/+ mice discriminated between chemically-related odorants sooner than α7+/− or α7−/− mice. These data suggest that α7-nicotinic-receptors contribute strongly to olfactory discrimination and detection in mice and may be one of the mechanisms producing olfactory dysfunction in schizophrenics.

Investigating the role of protein folding and assembly in cell-type dependent expression of α7 nicotinic receptors using a green fluorescent protein chimera

To test the hypothesis that cell-dependent expression of α7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat α7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of α7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. 125I-α-bungarotoxin (αBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of α7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant α-BGT binding compared to transfection with GFP. In contrast, α7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without αBGT binding. Flow cytometry of cells transfected with α7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with α7-GFP did not correlate with amounts of cell surface or immunoprecipitable αBGT binding. Therefore, GFP folding at the C-terminal of the α7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of α7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled α7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for α7 receptor assembly.

Pharmacological and immunological identification of native α7 nicotinic receptors: Evidence for homomeric and heteromeric α7 receptors

Controversy surrounds the expression of α7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, α7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [ 125I]α-bungarotoxin (αBGT) binding reaches equilibrium within 4 h and is saturable with a K d value of 4.2 nM. Using homologous competition experiments, the K i for binding of αBGT was 1.9 nM. These data are consistent with the expression of homomeric α7 nAChRs. Methyllycaconatine (MLA), which binds α7 nAChRs with high affinity, inhibits [ 125I]αBGT binding in a concentration-dependent manner with a K i of 30.6 nM; this value is ∼ 10 fold higher than the reported affinity of MLA for α7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate α7 nAChRs, as has been previous demonstrated for bovine adrenal α3β4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes α7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of ∼ 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes α3 and α5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of ∼ 53 kDa is identified. Together, these results support the expression of α7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric α7 nAChRs.

Exploring the pharmacological properties of insect nicotinic acetylcholine receptors

Insect nicotinic acetylcholine (nACh) receptors are molecular targets of insecticides such as neonicotinoids that are used to control disease-carrying insects and agricultural pests. To date, several insect nACh receptor subunits have been identified, indicating different nACh receptor subtypes and pharmacological profiles. Because of the difficulty in expressing functional insect nACh receptors in heterologous systems, new research tools are needed. Studies on insects resistant to the insecticide imidacloprid and on laboratory-generated hybrid and chimaeric nACh receptors in vitro have provided information about the molecular basis of receptor diversity, neonicotinoid resistance and selectivity. Additionally, recent results indicate that the sensitivity of insect nACh receptors to imidacloprid can be modulated by intracellular phosphorylation mechanisms, which offers a new approach to studying insect nACh receptor pharmacology.

Dual Role of the RIC-3 Protein in Trafficking of Serotonin and Nicotinic Acetylcholine Receptors

The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of alpha7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of alpha4beta2 nicotinic and 5-HT3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of alpha7 and 5-HT3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of alpha7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the alpha7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.

Charged Amino Acids of the N-terminal Domain Are Involved in Coupling Binding and Gating in α7 Nicotinic Receptors

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the alpha7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.

Expression and channel properties of alpha-bungarotoxin-sensitive acetylcholine receptors on chick ciliary and choroid neurons.

Cell-specific expression of nicotinic acetylcholine receptors (AChRs) was examined using ciliary and choroid neurons isolated from chick ciliary ganglia. At embryonic days 13 and 14 (E13,14) the neurons can be distinguished by size, with ciliary neuron soma diameters exceeding those of choroid neurons by about twofold. Both neuronal populations are known to express two major AChR types: alpha3*-AChRs recognized by mAb35, that contain alpha3, alpha5, beta4, and occasionally beta2 subunits, and alpha-bungarotoxin (alphaBgt)-AChRs recognized and blocked by alphaBgt, that contain alpha7 subunits. We found that maximal whole cell current densities (I/C(m)) mediated by alphaBgt-AChRs were threefold larger for choroid compared with ciliary neurons, while alpha3*-AChR current densities were similar in the two populations. Different densities of total cell-surface alphaBgt-AChRs could not explain the distinct alphaBgt-AChR response densities associated with ciliary and choroid neurons. Ciliary ganglion neurons display abundant [(125)I]-alphaBgt binding ( approximately 10(6) sites/neuron), but digital fluorescence measurements revealed equivalent site densities on both populations. AChR channel classes having single-channel conductances of approximately 30, 40, 60, and 80 pS were present in patches excised from both ciliary and choroid neurons. Treating the neurons with alphaBgt selectively abolished the 60- and 80-pS events, identifying them as arising from alphaBgt-AChRs. Kinetic measurements revealed brief open and long closed durations for alphaBgt-AChR channel currents, predicting a very low probability of being open (p(o)) when compared with 30- or 40-pS alpha3*-AChR channels. None of the channel parameters associated with the 60- and 80-pS alphaBgt-AChRs differed detectably, however, between choroid and ciliary neurons. Instead calculations based on the combined whole cell and single-channel results indicate that choroid neurons express approximately threefold larger numbers of functional alphaBgt-AChRs (N(F)) per unit area than do ciliary neurons. Comparison with total surface [(125)I]-alphaBgt-AChR sites (N(T)), reveals that N(F)/N(T) << 1 for both neuron populations, suggesting that "silent" alphaBgt-AChRs predominate. Choroid neurons may therefore express a higher density of functional alphaBgt-AChRs by recruiting a larger fraction of receptors from the silent pool than do ciliary neurons.

Nicotinic acetylcholine receptor chimeras of rat α7 andDrosophilaSAD reveal species-specific agonist binding regions

Species-specific agonist binding regions of nicotinic acetylcholine receptors (nAChR) were examined. Imidacloprid and physostigmine (Phy) selectively activated insect nAChR composed of Drosophila second alpha-like subunit (SAD) and chick β2, in contrast to rat α7 nAChR. The Phy-activated currents were α-bungarotoxin (α-BGT) sensitive, suggesting activation at the agonist binding loop C. Several SAD-α7 chimeras were constructed, by switching agonist binding regions, and expressed in oocytes. Though none of the chimeras was activated by a range of nicotinic agonists, [125I]α-BGT binding revealed homomeric assembly of all chimeric cDNAs. Phy differentially displaced [125I]α-BGT from the nAChR chimeras, suggesting that the β subunit is not involved in Phy binding, and that Phy targets the insect agonist binding loop C.

Improved method for heterologous expression of ion channels inXenopusOocyte: A PCR shortcut to Oocyte expression

Xenopus oocyte is one of the widely used heterologous expression systems of ion channels for electrophysiological studies. Here we describe a new method in which cRNA produced by polymerase chain reaction (PCR) and in vitro transcription is injected to express ion channels in oocytes. This method enables us (1) to eliminate all or a part of the untranslated region of the cDNA and to replace it with a known sequence which helps increase the expression level in oocytes, and (2) to use the PCR product for in vitro transcription without subcloning. Using this method, the expression level of one of the neuronal nicotinic acetyfcholine receptors (nAChRs) α7 subtype in oocytes was systematically increased by more than 100‐fold, which was confirmed both by the α‐Bungarotoxin (αBgt) binding assay and the current measurement

Structure-activity relationships of acyclic nicotinoids and neonicotinoids for insect nicotinic acetylcholine receptor/ion channel complex

The insect nicotinic acetylcholine (Ach) receptor (nAChR) is a target site for the neonicotinoid insecticides such as imidacloprid and its acyclic derivative acetamiprid. The structure–activity relationships of acetamiprid homologues and 3-pyridylmethylamines (known as the essential structural requirement of nicotinoid) are compared in terms of the affinity to the [3H]α-bungarotoxin (α-BGT) site (designated as Ach site) and the [3H]phencyclidine (PCP) site [designated as noncompetitive blocker (NCB) site] of the insect nAChR from the honeybee heads. Increasing the chain length of alkyl substituents (from methyl to n-butyl) on an amino nitrogen atom of acetamiprid homologue and 3-pyridylmethylamine reduces the potency as inhibitors of [3H]α-BGT binding, whereas it confers the enhanced potency as inhibitors of [3H]PCP binding in the insect nAChR. Scatchard analysis reveals that homologues of acetamiprid and 3-pyridylmethylamine having n-butyl substituents interact with the high-affinity binding site for [3H]PCP, which is considered to be the NCB site located in the ion channel of the insect nAChR. The interaction of acetamiprid homologues with the Ach or NCB site of nAChR is selective for insects, while that of the 3-pyridylmethylamines is effective for both insect and Torpedo [Tomizawa et al., J Pesticide Sci 21:412–418 (1996)]. The explorations in further structural modification of neonicotinoid compounds may facilitate development of new insecticides or probes for the ion channel of insect nAChR. Arch. Insect Biochem. Physiol. 37:17–23, 1998. © 1998 Wiley-Liss, Inc.

Mice Deficient in the α7 Neuronal Nicotinic Acetylcholine Receptor Lack α-Bungarotoxin Binding Sites and Hippocampal Fast Nicotinic Currents

The alpha7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding alpha-bungarotoxin (alpha-BGT). A null mutation for the alpha7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]alpha-BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the alpha-BGT binding sites are equivalent to the alpha7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the alpha7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene product in vivo.

Conversion of the sodium channel activator aconitine into a potent α7-selective nicotinic ligand

Methyllycaconitine (MLA) is a competitive antagonist of nicotinic acetylcholine receptors, with a remarkable preference for neuronal [ 125I]αBgt binding sites. We have begun to investigate the structural basis of its potency and subtype selectivity. MLA is a substituted norditerpenoid alkaloid linked to a 2-(methylsuccinimido)benzoyl moiety. Hydrolysis of the ester bond in MLA to produce lycoctonine diminished affinity for rat brain [ 125I]αBgt binding sites 2500-fold and abolished affinity for [ 3H]nicotine and muscle [ 125I]αBgt binding sites. The voltage-gated Na + channel activator aconitine, also a norditerpenoid alkaloid, but with significant structural differences from lycoctonine, displayed comparable weak or absent nicotinic activity. Addition of a 2-(methylsuccinimido)benzoyl sidechain to O-demethylated aconitine, to mimic MLA, abolished Na + channel activation and conferred nanomolar affinity for brain [ 125I]αBgt binding sites, comparable to that of MLA. We propose that the ester-linked 2-(methylsuccinimido)benzoyl group is necessary for nicotinic potency, but α7 selectivity resides in the norditerpenoid core of the molecule.

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [ 3H]nicotine or [ 3H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [ 3H]QNB were present at a density of 0.75 pmol/mg protein. [ 125I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B max of 128 fmol/mg protein and a K d of 0.6 nM; cortical cultures expressed five times fewer [ 125I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10 4 and 2 × 10 4 binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [ 125I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.

Pharmacological Characteristics of Insect Nicotinic Acetyicholine Receptor with Its Ion Channel and the Comparison of the Effect of Nicotinoids and Neonicotinoids

ミツバチ頭部膜画分を用い, [3H]α-ブンガロトキシン (α-BGT) および [3H] フェンサイクリジン (PCP) をプローブとしたラジオレセプターアッセイにより各種リガンドの作用を検討した結果, 昆虫ニコチン性アセチルコリンレセプター (nAChR)/イオンチャンネル複合体の薬理的性質はシビレエイのそれと大きく異なることを認めた. すなわち昆虫では供試したコリン作動性アゴニストは [3H]PCP結合を増加させず, また脊椎動物でイオンチャンネル内のアロステリック部位にのみ結合するPCPが [3H]α-BGT結合をも阻害した. [3H]α-BGT結合の飽和実験の結果は, ニコチン, PCPがα-BGTと同様アセチルコリン (ACh) 認識部位に結合することを示したが, さらにニコチンは [3H]α-BGT結合のみならず [3H]PCP結合をも強く阻害した. [3H]-PCPはシビレエイでは単一部位に結合したが, 昆虫では高親和性部位と低親和性部位の二つの結合部位が存在し, 前者はイオンチャンネル内のアロステリック部位, 後者はACh認識部位に相当することを推定した. ニコチン, アナバシン, ニテンピラムはACh認識部位とアロステリック部位の双方に結合する一方, イミダクロプリド, 6-Cl-PMNI, アセトアミプリドはACh認識部位に選択的に結合した. イエバエに対してPCPはニコチンと同程度の殺虫力を示したが, 中毒症状は異なり, ニコチンやイミダクロプリドは興奮作用を示したが, PCPは麻酔作用を示した.

Phorbol esters and K + up-regulate α-bungarotoxin binding sites in cultured chromaffin cells through a related mechanism

Nicotinic α-bungarotoxin (α-BGT) receptors in cultured chromaffin cells are up-regulated in response to long term (days) exposure to nicotinic antagonists, elevated K +, and activators of protein kinase C (PKC), such as the phorbol ester, 4β-phorbol 12-myristate 13-acetate (PMA). The present experiments were done to determine whether their was any interaction in the ability of PMA and K + to up-regulate the α-BGT receptors. Chromaffin cells were treated for 3 days with both 100 nM PMA and 20 mM K −, concentrations which produce maximal responses on their own. The increase in α-BGT binding after the combined treatment was the same as that seen with K + alone suggesting that K + inhibited the PMA-mediated effect. The K +-induced increase in toxin binding was partially prevented by polymyxin B, an agent which completely inhibited the PMA induced increase. The time courses of the increases in binding induced by both K + and PMA were similar in that the most marked increases in binding were observed at the later time points. The PMA-induced up-regulation was partially inhibited by an activator of adenylate cyclase, a result similar to that previously seen with K +. The present studies suggest that the up-regulation of α-BGT receptors induced by K + shares similarities with that induced by phorbol esters. The observations that K + inhibited the PMA induced increase and that a PKC inhibitor partially blocked the K + response suggest that the K + and PMA mediated induction of the α-BGT sites may be linked with the effects of K + preceding those of PMA.

Combined effects of a thymic peptide, thymopoietin and myasthenic patient sera in rat myotube culture

We investigated in a rat myotube assay the combined effect of 26 myasthenic (MG) patient sera and a thymic peptide, thymopoietin (Tpo) which had previously been shown to bind Torpedo and human AChR and to complete with α-bungarotoxin (α-Bgt) binding. Cultures were first exposed to Tpo alone for 3 h (0.3, 7.5, 15 nM), then MG sera (5% final dilution) were added for an additional 18 h. Reduction in the amount of 125I-α-Bgt binding sites in the presence of various concentrations of Tpo were similar with control sera and in all the patients with low or undetectable anti-AChR Ab (11 cases). In cultures exposed to Tpo and sera with high anti-AChR Ab titre (15 cases), Tpo and anti-AChR Ab have an additive capacity to reduce the number of α-Bgt binding sites. The results are compatible with the hypothesis that anti-AChR Ab and Tpo could impair neuromuscular transmission by complementary mechanisms.

Brain α-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily

α-Bungarotoxin (αBgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity αBgt binding proteins (αBgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding αBgtBP α1 and α2 subunits define αBgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed αBgtBP α1 and α2 subunit fragments reveal the existence of at least two different αBgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all αBgtBPs have the α1 subunit, but no α2 subunit, and a minor αBgtBP subtype (∼15%) has both the α1 and α2 subunits.

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.