The binding site for α-bungarotoxin resides in the sequence 188–201 of the α-subunit of acetylcholine receptor: Structure, conformation and binding characteristics of peptide [Lys] 188–201

Gotti, Cecilia; Mazzola, G.; Longhi, Renato; Fornasari, Diego; Clementi, Francesco

doi:10.1016/0304-3940(87)90180-7

Cited by 25 publications

(15 citation statements)

References 10 publications

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“…For example, in the nAcChRs, a1 (Neumann et al, 1987a) and Tyr-189 (Gotti et al, 1987) have been implicated as important for binding to aBgTx, which is consistent with the sequence analysis of Figure 2. These aromatic residues are substituted in the nonligand binding subunits and in the neuronal a subunits, which interact with aBgTx at low affinity.…”

Section: Non-aligned and Family-specific Cholinergic Subtype Residuessupporting

confidence: 71%

“…The nAcChR subunits are predicted to traverse the membrane five times; four on the basis of hydropathic analysis (Claudio et al, 1983;Devillers-Thiery et al, 1983;Noda et al, 1983b) and a fifth by amphipathic analysis (Finer-Moore and Stroud, 1984). 01173-204 (Wilson et al, 1985), a185-196 (Neumann et al, 1986a), a172-205, a185-199 (Ralston et al, 1987, and a188-201 (Gotti et al, 1987), and bovine and human a173-204 (Wilson and Lentz, 1988). Reduction and alkylation of a (Kao and Karlin, 1986;Ralston et al, 1987) blocks 1251-aBgTx binding to the native receptor (Kao et al, 1984;Ralston et al, 1987) and to synthetic peptides Torpedo a172- 205, al85-196, and al88-201 (Neumann et al, 1986a;Gotti et al, 1987;Ralston et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

“…01173-204 (Wilson et al, 1985), a185-196 (Neumann et al, 1986a), a172-205, a185-199 (Ralston et al, 1987, and a188-201 (Gotti et al, 1987), and bovine and human a173-204 (Wilson and Lentz, 1988). Reduction and alkylation of a (Kao and Karlin, 1986;Ralston et al, 1987) blocks 1251-aBgTx binding to the native receptor (Kao et al, 1984;Ralston et al, 1987) and to synthetic peptides Torpedo a172- 205, al85-196, and al88-201 (Neumann et al, 1986a;Gotti et al, 1987;Ralston et al, 1987). The primary AcCh binding site has also been located to within residues 01166-216 by expression cloning in Escherichia coli (Barkas et al, 1987;Gershoni, 1987) and residues a152-3 13 by peptide mapping (Neumann et al, 1986b).…”

Section: Introductionmentioning

confidence: 99%

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Consensus residues at the acetylcholine binding site of cholinergic proteins

Peterson

1989

J of Neuroscience Research

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The nicotinic (nAcChR) and muscarinic (mAcCh) acetylcholine receptors and acetylcholinesterase (AcChEase) are structurally unrelated but share a common functional property: interaction with acetylcholine (AcCh). Alignment of the probable AcCh binding site regions of the nAcChR and mAcChR protein sequences revealed the presence of ten nearly identically spaced consensus residues, six of which contain potentially ligand-interactive side chains. Important elements of the consensus residues also were found in one unique sequence region of the AcChEases. Alignments among the two receptors and AcChEase outside the apparent binding region were rare, and the consensus AcCh binding residues were largely substituted in the homologous proteins, which do not bind AcCh. The consensus residues include two possible anionic subsite Asp residues and a Ser that may hydrogen bond to the AcCh carbonyl in the receptors. These residues correspond to positions Asp-166, Ser-173, and Asp-200 in the neuromuscular nAcChR; Asp-71, Ser-78, and Asp-105 in the M1 mAcChR; and Asp-93 and Asp-128 in Torpedo AcChEase. No corresponding consensus Ser is found in the AcChEase sequence; this is expected because of a downstream esterase active-site Ser-200 (Torpedo). A receptor-conserved and disulfide-linked Cys corresponding to neuromuscular nAcChR residue 193 and M1 mAcChR residue 97 may be important in energy transduction associated with agonist-mediated events. The presence of additional binding-site aromatic residues that may form a hydrophobic environment near the anionic subsite are aligned within, but not between, the three cholinergic protein groups. These observations target specific regions and residues within these proteins for structure-function studies of the cholinergic binding domain.

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Section: Non-aligned and Family-specific Cholinergic Subtype Residuessupporting

confidence: 71%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Consensus residues at the acetylcholine binding site of cholinergic proteins

Peterson

1989

J of Neuroscience Research

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“…We have shown that these two cysteines as well as tryptophan at position 187, which is also conserved in muscle AcChoR, are crucial for retaining toxin binding (7). Studies by other groups using CNBr-derived fragments of affinity-labeled a subunit (8), proteolytic fragmentation (9), synthetic peptides (10)(11)(12), and genetic constructs (13)(14)(15) have also localized the cholinergic binding site in close proximity or contiguous to the two tandem cysteine residues 192 and 193. Interestingly, the a subunit of the brain nicotinic AcChoR, which does not bind a-bungarotoxin (a-BTX), also contains the two tandem cysteines at positions 192 and 193 (16), although it differs in other amino acid residues from muscle AcChoRs.…”

mentioning

confidence: 99%

Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

Neumann

Barchan

Horowitz

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind abungarotoxin. Numerous studies indicate that the ligandbinding site of the AcChoR includes cysteine residues at positions 192 and 193 of the a subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR a subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion ofthe snake AcChoR a subunit. First, a mouse AcChoR a-subunit cDNA probe was used to screen a size-selected snake (Natrix tesseUata) genomic library. A genomic done was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR a subunit. The domain of the a subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake a subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR a subunits. Sequence comparison revealed that the cloned region of the snake a subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind a-bungarotoxin. In the presumed ligand-binding site, in the vicinity of , four major substitutions occur in the snake sequence-at positions 184 (Trp, Phe), 185

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“…In contrast, if any effect was seen, DTT caused a slight increase in [ 1251]NBT binding in homogenates of chick retina (a more detailed description of the effects of reduction and oxidation on [ 1251]NBT binding is in preparation). However, nicotinic ligands retain the ability to bind to the reduced nicotinic receptor, judging from the ability of these ligands to displace ['251]NBT binding (Loring, unpublished Gotti et al, 1987). The possibility that nicotinic receptors in chick retina differ dramatically in terms of ionic properties and the ability to bind to DEAE-cellulose from those of muscle, or those recognized in brain by monoclonal antibodies, has not yet been rigorously investigated.…”

Section: Discussionmentioning

confidence: 99%

Pharmacological and Biochemical Properties of Nicotinic Receptors from Chick Retina

Loring

Zigmond

1990

Eur J of Neuroscience

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Previous work has established that functional nicotinic receptors in the chick retina are blocked by neuronal bungarotoxin (NBT), and that the binding of radio-iodinated NBT to retinal homogenates is displaced by nicotinic ligands. In the present study, we examined the desensitizing effects of agonists on nicotinically-mediated depolarizations recorded from chick retina. The concentrations of five agonists necessary to reduce the amplitude of these depolarizations by 50% were found to correlate closely with the concentrations of these same agonists previously found necessary to displace 50% of NBT binding. In addition, bromoacetylcholine (BAC), a selective affinity alkylating agent for the agonist binding site, irreversibly inactivated the functional responses of intact chick retina with an inhibiting concentration for 50% block (IC50) near 10-6 M, the same concentration of BAC that displaced 50% of labelled NBT binding from alkylated retinal homogenates. These data suggest that NBT acts at the receptor agonist binding site. Furthermore, this binding site has a relatively low affinity for agonists, in the micromolar range, even in the desensitized state. Multiple subtypes of nicotinic receptors are known to exist in neuronal tissue, and receptors that bind agonists in the nanomolar range have been detergent-solubilized and purified using monoclonal antibodies. Under similar conditions, detergent-solubilization of chick retinal homogenates interfere with the interaction between NBT and the low-affinity neuronal nicotinic receptors. These data suggest that the conditions used to purify high-affinity neuronal nicotinic receptors may denature the subtype(s) of neuronal receptors recognized by NBT.

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The binding site for α-bungarotoxin resides in the sequence 188–201 of the α-subunit of acetylcholine receptor: Structure, conformation and binding characteristics of peptide [Lys] 188–201

Cited by 25 publications

References 10 publications

Consensus residues at the acetylcholine binding site of cholinergic proteins

Consensus residues at the acetylcholine binding site of cholinergic proteins

Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

Pharmacological and Biochemical Properties of Nicotinic Receptors from Chick Retina

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