Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.     

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.     

Key amino acids in the gamma subunit of the gamma-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site

Pharmacological analyses of gamma-aminobutyric acidA (GABAA) receptor subtypes have suggested that both the alpha and gamma subunits, but not the beta subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different gamma subunits in the receptor macromolecule to identify amino acids gamma2Phe77 and gamma2Met130 as key determinants of the benzodiazepine binding site. gamma2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-beta-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, gamma2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218, 872, or methyl-beta-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of gamma1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that gamma2Phe77 and gamma2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the alpha subunit, we demonstrated a critical role for the gamma subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, gamma2Phe77 is homologous to alpha1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.     

Ligand-gated ion channel subunit partnerships: GABAA receptor alpha6 subunit gene inactivation inhibits delta subunit expression

Cerebellar granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact alpha6 protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse alpha6 subunit gene was disrupted by homologous recombination. In alpha6 -/- granule cells, the delta subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with delta subunit-specific antibodies. The delta subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the delta subunit. These results provide genetic evidence for a specific association between the alpha6 and delta subunits. Because in alpha6 -/- neurons the remaining alpha1, beta2/3, and gamma2 subunits cannot rescue the delta subunit, certain potential subunit combinations may not be found in wild-type cells.     

Direct interaction of the alpha and gamma subunits of the G proteins. Purification and analysis by limited proteolysis

The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.     

Topology of ligand binding sites on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of both alpha subunits there exist the binding sites for agonists such as the neurotransmitter acetylcholine (ACh) and for competitive antagonists such as d-tubocurarine. Agonists trigger the channel opening upon binding while competitive antagonists compete for the former ones and inhibit its pharmacological action. Identification of all residues involved in recognition and binding of agonist and competitive antagonists is a primary objective in order to understand which structural components are related to the physiological function of the AChR. The picture for the localisation of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are mainly located on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are sequentially identical, the observed high and low affinity for agonists on the receptor is conditioned by the interaction of the alpha subunit with the delta or the gamma chain, respectively. This relationship is opposite for curare-related drugs. This molecular interaction takes place probably at the interface formed by the different subunits. The principal component for the agonist/competitive antagonist binding sites involves several aromatic residues, in addition to the cysteine pair at 192-193, in three loops-forming binding domains (loops A-C). Other residues such as the negatively changed aspartates and glutamates (loop D), Thr or Tyr (loop E), and Trp (loop F) from non-alpha subunits were also found to form the complementary component of the agonist/competitive antagonist binding sites. Neurotoxins such as alpha-, kappa-bungarotoxin and several alpha-conotoxins seem to partially overlap with the agonist/competitive antagonist binding sites at multiple point of contacts. The alpha subunits also carry the binding site for certain acetylcholinesterase inhibitors such as eserine and for the neurotransmitter 5-hydroxytryptamine which activate the receptor without interacting with the classical agonist binding sites. The link between specific subunits by means of the binding of ACh molecules might play a pivotal role in the relative shift among receptor subunits. This conformational change would allow for the opening of the intrinsic receptor cation channel transducting the external chemical signal elicited by the agonist into membrane depolarisation. The ion flux activity can be inhibited by non-competitive inhibitors (NCIs). For this kind of drugs, a population of low-affinity binding sites has been found at the lipid-protein interface of the AChR. In addition, several high-affinity binding sites have been found to be located at different rings on the M2 transmembrane domain, namely luminal binding sites. In this regard, the serine ring is the locus for exogenous NCIs such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222, phencyclidine, and trifluoromethyliodophenyldiazirine. Trifluoromethyliodophenyldiazirine also binds to the valine ring, which is the postulated site for cembranoids. Additionally, the local anaesthetic meproadifen binding site seems to be located at the outer or extracellular ring. Interestingly, the M2 domain is also the locus for endogenous NCIs such as the neuropeptide substance P and the neurotransmitter 5-hydroxytryptamine. In contrast with this fact, experimental evidence supports the hypothesis for the existence of other NCI high-affinity binding sites located not at the channel lumen but at non-luminal binding domains. (ABSTRACT TRUNCATED)     

Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.     

Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase

Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26-156, bcyt) and other membrane peripheral subunits including alpha, beta, gamma, delta, epsilon, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and delta subunit fused to the DNA binding domain resulted in the strong expression of the beta-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the delta subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed delta subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the delta subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of delta, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyt together with alpha, beta, gamma, delta, and epsilon, or with the same combination except epsilon. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and delta was stabilized by F1 subunits other than epsilon and also suggested that b-delta interaction was important for F1-F0 interaction.     

Replicon typing characterization of plasmids encoding resistance to gentamicin and apramycin in Escherichia coli and Salmonella typhimurium isolated from human and animal sources in Belgium

cDNA sequences encompassing the full coding region for the human muscle acetylcholine receptor (AChR) epsilon and gamma subunits have been isolated. The deduced amino-acid sequences indicate that the mature epsilon subunit contains 473 amino acids and is preceded by a 20-amino-acid signal peptide. As predicted from genomic clones, the gamma subunit contains 495 amino acids preceded by a 22-amino-acid signal peptide. In common with the human alpha, beta, gamma and delta subunits the epsilon subunit is highly conserved between mammalian species. The epsilon subunit gene is not closely linked to the gamma and delta subunits on chromosome 2 but rather is located with the beta subunit on chromosome 17. Expression of the alpha-, beta-, gamma-, delta- and epsilon-subunit cRNAs in rabbit-reticulocyte lysates followed by analysis on SDS/PAGE show glycosylated proteins with apparent molecular masses of 44-60 kDa.     

The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Positron-emission tomography with fluorine-18-deoxyglucose in the staging and control of patients with lymphoma. Comparison with clinico-radiologic assessment

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+ coupled to the translocation of protons across the cytoplasmic membrane. It is composed of two subunits (alpha, beta) organized as an alpha 2 beta 2 tetramer. This brief review describes the use of site-directed mutagenesis to investigate the structure, mechanism and assembly of the transhydrogenase. This technique has located the binding sites for NAD(H) and NADP(H) in the alpha and beta subunits, respectively. Mutagenesis has shown that the cysteine residues of the enzyme are not essential for its function, and that inhibition of the enzyme by sulfhydryl-specific reagents must be due to perturbation of the three-dimensional structure. The sites of reaction of the inhibitors N,N'-dicyclohexylcarbodiimide and N-(1-pyrene)maleimide have been located. Selective mutation and insertion of cysteine residues followed by cupric o-phenanthrolinate-induced disulfide crosslinking has defined a region of interaction between the alpha subunits in the holoenzyme. Determination of the accessibility of selectively inserted cysteine residues has been used to determine the folding pattern of the transmembrane helices of the beta subunit. Site-directed mutagenesis of the transmembrane domain of the beta subunit has permitted the identification of histidine, aspartic acid and asparagine residues which are part of the proton-pumping pathway of the transhydrogenase. Site-directed mutagenesis and amino acid deletions have shown that the six carboxy terminal residues of the alpha subunit and the two carboxy terminal residues of the beta subunit are necessary for correct assembly of the transhydrogenase in the cytoplasmic membrane.     

Mechanism of auxiliary subunit modulation of neuronal alpha1E calcium channels

Voltage-gated calcium channels are composed of a main pore-forming alpha1 moiety, and one or more auxiliary subunits (beta, alpha2 delta) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of alpha1E calcium channels in combination with a beta (beta1-beta4) and/or the alpha2 delta subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting alpha2delta with beta subunits, of transfecting two different beta subunits simultaneously, and of COOH-terminal truncation of alpha1E to remove a second beta binding site. The main results are as follows. (a) The alpha2delta and beta subunits modulate alpha1E in fundamentally different ways. The sole effect of alpha2 delta is to increase current density by elevating channel density. By contrast, though beta subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different beta isoforms produce nearly indistinguishable effects on activation. However, beta subunits produced clear, isoform-specific effects on inactivation properties. (b) All the beta subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different beta subunits. (d) The modulatory features found here for alpha1E do not generalize uniformly to other alpha1 channel types, as alpha1C activation gating shows marked beta isoform dependence that is absent for alpha1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of alpha1E calcium channels.     

Modification of domains of alpha and beta subunits of F1-ATPase from the thermophylic bacterium PS3, in their isolated and associated forms, by 3''-O-(4-benzoyl)benzoyl adenosine 5''-triphosphate (BzATP)

Photoaffinity labeling by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) of the adenine nucleotide binding site(s) on isolated and complexed alpha and beta subunits of F1-ATPase from the thermophilic bacterium PS3 (TF1) is described. BzATP binds to both isolated alpha and beta subunits, to complexed beta subunit but not to complexed alpha subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [gamma-32P]BzATP-labeled alpha subunit indicates that residues on both the amino-terminal (residues A41-E67) and carboxy-terminal (residues Q422-Q476) were modified by BzATP. One of the residues in the carboxy-terminal modified by BzATP is most probably alpha Q422. Although the binding stoichiometry of 1 mol of BzATP incorporated by either isolated or complexed beta subunit was maintained, the spatial conformation of the polypeptide determines which amino acid residue(s) is more accessible to the reactive radical. CNBr derived fragments beta G10-M64, beta E75-M233, and beta D390-M469 were labeled with the isolated beta subunit. With complexed beta subunit the label was found only in CNBr fragments: beta E75-M233 and beta G339-M389. The locations where the covalently bound BzATP was found, in the soluble and assembled subunits, indicate that different conformational states exist. In the isolated form of the alpha and beta subunits the amino- and carboxy-termini can fold and reach the central domain of the polypeptide, the domain containing the adenine nucleotide binding site. When alpha combines with beta to form the alpha 3 beta 3 core complex the new conformation of the subunits is such that covalent labeling by BzATP of alpha and of the amino terminal of beta subunit is excluded.     

Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1)

The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.     

Immediate implantation of pure titanium implants into extraction sockets of Macaca fascicularis. Part I: Clinical and radiographic assessment

The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

Restoration of 125I-alpha-bungarotoxin binding activity to the alpha subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate

The four subunits (alpha, beta, gamma, delta) of the acetylcholine receptor from Torpedo californica have been isolated by preparative gel electrophoresis in sodium dodecyl sulfate. After removal of the sodium dodecyl sulfate by dialysis of the polypeptides against a cholate-containing buffer, the alpha subunit, but not the other chains, binds 125I-alpha-bungarotoxin in a saturable manner. The binding affinity, 0.1-0.2 microM, is approximately 10(4)-fold lower than that observed for native acetylcholine receptor. For three preparations of alpha subunit, 1 mol of subunit bound 0.87, 0.38, and 0.33 mol of 125I-alpha-bungarotoxin at saturation. The binding was inhibited by cholinergic ligands, although the apparent affinities of these ligands for alpha were 50-100-fold lower than that found for the native receptor. These results indicate that at least part of the alpha-bungarotoxin binding site resides on the alpha subunit.     

Epibatidine binds with unique site and state selectivity to muscle nicotinic acetylcholine receptors

Ligand binding sites in fetal (alpha2betagammadelta) and adult (alpha2betadeltaepsilon) muscle acetylcholine receptors are formed by alphadelta, alphagamma, or alphaepsilon subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the delta, gamma, or epsilon subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to alphagamma, alphadelta, and alphaepsilon intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is alphaepsilon > alphagamma > alphadelta, whereas for carbamylcholine the rank order is alphadelta congruent with alphaepsilon > alphagamma. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components differing by more than 170-fold. Studies of subunit-omitted receptors (alpha2betadelta2, alpha2betagamma2, and alpha2betaepsilon2) reveal that in the desensitized state, the alphadelta interface forms the low affinity epibatidine site, whereas the alphagamma and alphaepsilon interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding sites.     

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.     

Veno-right ventricular extracorporeal membrane oxygenation for thoracic surgery: an experimental study in dogs

Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N-hydroxysuccinimide esters of p-azidobenzoic, p-azidotetraflourobenzoic, p-benzoylbenzoic and p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acids. The ion-exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross-linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross-linking yield (up to 16% of the bound toxin) was observed for azidobenzoyl-Lys23-CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group: the azido derivatives labelled the gamma and delta subunits, benzoylbenzoyl derivative labelled the alpha and delta subunits, while p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl derivative reacted with alpha, gamma and delta subunits. The cross-linking experiments in the presence of varying concentrations of (+)-tubocurarine demonstrated that the Lys23-attached diazirinyl group contacts the delta and alpha subunits in one ligand-binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23-diazirinyl are with gamma and alpha subunits. This means that the central loop in the two CTX molecules binds at the alpha/gamma and alpha/delta interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X-ray structure of CTX, showed that this value does not exceed 13 A. The results obtained favor the disposition of the ligand-binding sites at the subunit interfaces, with the distance between alpha and delta, or alpha and gamma subunits at these sites being not more than 13 A.