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V I Tsetlin

Publications and source records attributed to V I Tsetlin.

At least 19 recordsLinked to original sources
PubMed2004

Diversity of nicotinic receptors mediating Cl- current in Lymnaea neurons distinguished with specific agonists and antagonist.

Diversity of nicotinic acetylcholine receptors (nAChRs) mediating Cl- current in voltage-clamped identifiable Lymnaea stagnalis neurons was studied using acetylcholine (ACh), three agonists and alpha-conotoxin ImI (ImI). Cytisine, nicotine, and choline, full agonists at alpha7 subunit-containing nAChRs of vertebrates, were found to evoke at saturating concentration 84-92% of the maximal current elicited by ACh. ImI, known to block selectively alpha7 and alpha9 nAChRs, markedly diminished the responses to ACh. The average maximal ImI-induced block was 80%, leaving a residual current which had very slow kinetics. The choline-, cytisine-, and nicotine-induced currents were blocked by ImI almost completely, suggesting that they activate only ImI-sensitive receptors. Two groups of cells which differ in desensitization kinetics and in sensitivity to ImI were revealed. IC50 values for ImI against ACh were 10.3 and 288 nM, respectively, with the rapidly desensitizing current being the more sensitive to ImI. The data obtained suggest the existence of at least three pharmacologically distinct subtypes of nicotinic receptors in Lymnaea neurons. Two of the subtypes are similar to alpha7 nAChRs of vertebrates, but differ from each other in their affinity for ImI and in their desensitization kinetics. The third subtype is quite distinct, in that it is resistant to ImI, is not activated by nicotine, cytisine or choline, and mediates a very slowly developing current.

Animals↗
PubMed2004

Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications.

This review covers recent data on interactions of nicotinic acetylcholine receptors (AChR) with snake venom proteins (alpha- and kappa-neurotoxins, 'weak' toxins recently shown to act on AChRs), as well as with peptide alpha-conotoxins from Conus snails. Mutations of AChRs and toxins, X-ray/nuclear magnetic resonance structures of alpha-neurotoxin bound to AChR fragments, and the X-ray structure of the acetylcholine-binding protein were used by several groups to build models for the alpha-neurotoxin-AChR complexes. Application of snake toxins and alpha-conotoxins for pharmacological distinction of muscle, neuronal and neuronal-like AChR subtypes and for other medical purposes is briefly discussed.

Animals↗
PubMed2003

Direct cloning of a target gene from a pool of homologous sequences: complete cDNA sequence of a weak neurotoxin from cobra Naja kaouthia.

Selective cloning of the cDNA coding for a weak neurotoxin (WTX) from cobra N. kaouthia including the 5'- and 3'-non-translated regions (NTR) is described. The known amino acid sequence of WTX was used together with the nucleotide sequence of a weak neurotoxin NNAM2 from cobra Naja atra, to design WTX-specific primers for direct amplification of an internal WTX cDNA fragment by RT- PCR. The sequence of the complete WTX cDNA was determined in sequencing runs on internal PCR products, cloned 3'- and 5'-RACE-fragments and several full-length cDNA clones. The cDNA coding sequence is in excellent agreement with the previously determined WTX amino acid sequence, has a high homology with other known weak toxin cDNAs, whereas even higher homology (up to 96%) with several classes of 3-finger toxins was detected in the 59 bp 3'-NTR consensus sequence. A possible function of the highly conserved nucleotide sequence elements is discussed.

Amino Acid Sequence↗
PubMed2002

Studying the spatial organization of membrane proteins by means of tritium stratigraphy: bacteriorhodopsin in purple membrane.

The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10-29 of helix A. They turned out to be low (0.04+/-0.02 A(-1)) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15+/-0.05 A(-1)) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane.

Bacteriorhodopsins↗
PubMed2001

Nicotinic receptors in Lymnaea stagnalis neurons are blocked by alpha-neurotoxins from cobra venoms.

The influence of cobra neurotoxins on the Cl-dependent responses to acetylcholine (ACh) of Lymnaea neurons was studied by the voltage-clamp technique. It was found that a short chain neurotoxin II (NT II), a long chain cobratoxin (CTX) and weak neurotoxin (WTX) diminished the ACh-induced currents, the block being concentration-dependent and competitive. The IC(50) values of 130 nM for CTX, 11 microM for NT II, and 67 microM for WTX were determined. The block induced by NT II was quickly reversible upon toxin washout, whereas the action of CTX and WTX was only partially reversible even after an hour of intensive washing. The data obtained suggest that acetylcholine receptors (AChRs) in Lymnaea neurons have common features with cation-selective alpha 7 AChRs of vertebrates and one type of Aplysia Cl-conducting AChRs.

Acetylcholine↗
PubMed2001

"Weak toxin" from Naja kaouthia is a nontoxic antagonist of alpha 7 and muscle-type nicotinic acetylcholine receptors.

A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [(125)I]alpha-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC(50) of approximately 2.2 microm. In this respect, it is approximately 300 times less potent than neurotoxin II from Naja oxiana and alpha-cobratoxin from N. kaouthia, representing short-type and long-type alpha-neurotoxins, respectively. WTX and alpha-cobratoxin displaced [(125)I]alpha-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC(50) values of 4.3 and 9.1 microm, respectively, whereas for neurotoxin II the IC(50) value was >100 microm. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha7 AChRs, inhibiting the latter most efficiently (IC(50) of approximately 8.3 microm). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from alpha-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak alpha-neurotoxin. It differs from the short chain alpha-neurotoxins, which potently block the muscle-type but not the alpha7 AChRs, and is closer to the long alpha-neurotoxins, which have comparable potency against the above-mentioned AChR types.

Amino Acid Sequence↗
PubMed2001

First tryptophan-containing weak neurotoxin from cobra venom.

With the purpose of studying structure-function relationships among weak neurotoxins (called so because of their low toxicity), we have isolated a toxin (WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, Hoppe-Seyler's Z. Physiol. Chem., 361 (1980) 425). The differences include an extra residue (Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom. WTX was found to compete with radioiodinated alpha-bungarotoxin for binding to the membrane-bound nicotinic acetylcholine receptor from Torpedo californica.

Amino Acid Sequence↗
PubMed2001

Photoactivatable alpha-conotoxins reveal contacts with all subunits as well as antagonist-induced rearrangements in the Torpedo californica acetylcholine receptor.

Azidobenzoyl (AzBz) and benzoylbenzoyl (BzBz) derivatives of alpha-conotoxin MI and L-benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI were synthesized. All these compounds, similarly to native alpha-conotoxins, completely displaced the radioiodinated MI or GI from the membrane-bound nicotinic acetylcholine receptor (AChR) of Torpedo californica. However, the GI(Bpa11) analog was considerably less potent than GI in competing with radioiodinated alpha-bungarotoxin (alphaBgt). Irradiation of iodinated AzBz derivatives bound to AChR resulted in labeling of all AChR subunits. The BzBz and Bpa derivatives gave lower levels of specific cross-linking but considerable labeling at additional sites that was enhanced, rather than suppressed, by an excess of native alpha-conotoxins or alphaBgt. Both equilibrium binding of benzophenone-derivatized alpha-conotoxins and their cross-linking could be totally abolished by physostigmine. The results obtained demonstrate that (a) specific binding sites for alpha-conotoxins and alphaBgt are overlapping but not identical, (b) each of the AChR subunits can be labeled with photoactivatable alpha-conotoxins and (c) enhancement of benzophenone-derivatized alpha-conotoxins cross-linking at additional (physostigmine-related) sites by alphaBgt or GI indicates that these antagonists induce structural alterations in the AChR outside their binding sites.

Amino Acid Sequence↗
PubMed2001

Two forms of nerve growth factor from cobra venom prevent the death of PC12 cells in serum-free medium.

Nerve growth factor (NGF) from the venom of cobra Naja kaouthia is highly homologous to mouse NGF. However, the differences between these two factors include the sequence regions determining the specificity of NGF interaction with Trk A or p75 receptors. To test if these variations can bring about dissimilarity in biological activity between these two NGFs, we have studied the effect of cobra factor on the survival of the primed PC12 cells after serum withdrawal. It was found that in a serum-free medium, cobra NGF prevented the death of PC12 cells with efficacy comparable to that of NGF from mouse submaxillary glands. In the course of purification two forms of cobra NGF were observed, both acting as a survival and a differentiation factor for PC12 cells in a serum-free medium. The form, eluting later from a reversed-phase column, displays survival effect at lower concentrations than the earlier eluting one.

Animals↗
PubMed2000

Muscarinic toxin-like proteins from cobra venom.

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.

Amino Acid Sequence↗
PubMed2000

Structural organization of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.

Allosteric Regulation↗
PubMed1999

NMR spatial structure of alpha-conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors.

A 600 MHz NMR study of alpha-conotoxin ImI from Conus imperialis, targeting the alpha7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin-spin coupling constants, and amide protons hydrogen-deuterium exchange data: rmsd of the backbone atom coordinates at the 2-12 region is 0.28 A in the 20 best structures. The structure is described as a type I beta-turn (positions 2-5) followed by a distorted helix (positions 5-11). Similar structural patterns can be found in all neuronal-specific alpha-conotoxins. Highly mobile side chains of the Asp-5, Arg-7 and Trp-10 residues form a single site for ImI binding to the alpha7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.

Amino Acid Sequence↗
PubMed1999

Aromatic substitutions in alpha-conotoxin ImI. Synthesis of iodinated photoactivatable derivative.

Conotoxin ImI is a specific marker of alpha7 nicotinic acetylcholine receptors. To study the role of aromatic indole group of tryptophan 10 in biological activity of ImI, the analogue containing tyrosine at this position was synthesized by solid-phase peptide synthesis. The analogue obtained, as well as its iodinated derivatives, were shown to be active against rat brain alpha7 acetylcholine receptor expressed in Xenopus oocytes. Attachment of bulky aromatic p-benzoylbenzoyl group to N-terminal alpha-amino group of iodinated [Tyr10]ImI only slightly affected the biological activity of the analogue. The data obtained suggest that indole ring of tryptophan 10 is not absolutely necessary for biological activity of conotoxin ImI, and that the N-terminus can accommodate a large aromatic group without loss of biological activity.

Acetylcholine↗
PubMed1999

How do acetylcholine receptor ligands reach their binding sites?

The access pathway to the binding sites for large competitive antagonists of the nicotinic acetylcholine receptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with alpha-neurotoxins. Binding assays with [125I]alpha-bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor-rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically 'hindered' in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the alpha/gamma- but not to the alpha/delta-binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor's binding sites. Together, our data suggest (a) that alpha-neurotoxins approach their binding sites from the membrane-facing periphery of the receptor's extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.

Animals↗
PubMed1999

[alpha-Neurotoxins and alpha-conotoxins--nicotinic cholinoreceptor blockers].

The review is devoted to the competitive blockers of different nicotinic acetylcholine receptors, alpha-neurotoxins from snake venoms, and alpha-conotoxins from marine snails of the Conidae family. The relationship between the structure and function of these toxins is discussed. Recent data on the mechanism of alpha-neurotoxin and alpha-conotoxin interaction with the nicotinic acetylcholine receptor are presented.

Amino Acid Sequence↗
PubMed1998

Spatial structure of the M3 transmembrane segment of the nicotinic acetylcholine receptor alpha subunit.

The three-dimensional structure of a synthetic peptide corresponding to the putative transmembrane segment M3 (amino acid residues 277-301) of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by means of two-dimensional 1H-NMR spectroscopy in a chloroform/methanol (1:1) mixture containing 0.1 M LiClO4. Complete resonance assignment has been performed using double-quantum-filtered COSY (DQF-COSY), TOCSY and NOESY spectra. The spatial structure has been calculated using the Diana program on the basis of integrated intensities of NOESY spectra. HN-C(alpha)H and HC(alpha)-C(beta)H spin-spin coupling constants. Residues 279-297 of M3 form a right-handed helix (root mean square deviation is 0.032 nm for backbone atoms and 0.088 nm for all heavy atoms). The conformations of the 17 side chains have been unambiguously determined. The obtained structure is in accord with the photolabeling pattern of the membrane nicotinic acetylcholine receptor (nAChR) which suggests alpha-helical structure of M3 in the labeled portion [Blanton, M. P. & Cohen, J. B. (1994) Biochemistry 33, 2859-2872].

Amino Acid Sequence↗
PubMed1998

Two distinct structures of alpha-conotoxin GI in aqueous solution.

The detailed analysis of conformational space of alpha-conotoxin GI in aqueous solution has been performed on the basis of two-dimensional NMR spectroscopy data using multiconformational approach. As the result, two topologically distinct interconvertible sets of GI conformations (populations of 78% and 22%) have been found. A common feature of the two sets is the Asn4-Cys7 beta-turn. The Gly8 to Tyrll region has a structure of right-handed helical turn in the major set and two sequential bends in the minor one. N-terminus and C-terminus also have different orientations, anti-parallel in the major conformational set and parallel in the minor one. An average pairwise rmsd for backbone heavy atoms is 0.56 A in the major set, 0.23 A in the minor, and 1.85 A between the structures of the two sets. The X-ray structure of GI [Guddat, L. W., Martin, J. A., Shan, L., Edmundson, A. B. & Gray, W. R. (1996) Biochemistry 35, 11329 - 11335] has the same folding pattern as the major NMR set, the average backbone rmsd between the two structures being 0.77 A.

Amino Acid Sequence↗