Aromatic trivalent arsenicals: covalent yet reversible reagents for the agonist binding site of nicotinic receptors

Loring, Ralph H.; Dou, Yamin; Lane, W. V.; Jones, Gordon E.; Stevenson, Kenneth J.

doi:10.1016/0169-328x(92)90158-8

Cited by 14 publications

(10 citation statements)

References 30 publications

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“…To test for juxtaposition of the cysteines at position 448, we first used the trivalent arsenical reagent p-aminophenyl-dichloroarsine (APA), which selectively interacts with nearby pairs of thiols, but not with single thiols (Loring et al, 1992). APA (100 M) had little effect on closed channels, but rapidly diminished currents when treatment was continued during 5 s depolarizations that opened and subsequently inactivated the channels (Figure 4A).…”

Section: Cysteines At the Deepest Position Can Be Crosslinked Between Subunits In The Inactivated Statementioning

confidence: 99%

“…We would like to thank Dr. Arthur Karlin for having kindly provided prepared from aminophenylarsine oxide (Aldrich), as described by us with the MTS reagents before they became commercially avail- Loring et al, (1992), and was dissolved in 100% dry ethanol before able, and Dr. Ralph Loring for his generous supply of APA early being added to the extracellular solution. The phenanthroline (Aldin this project.…”

Section: Electrophysiologymentioning

confidence: 99%

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Dynamic Rearrangement of the Outer Mouth of a K+ Channel during Gating

Liu

Jurman

Yellen

1996

Neuron

429

415

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With prolonged stimulation, voltage-activated K+ channels close by a gating process called inactivation. This inactivation gating can occur by two distinct molecular mechanisms: N-type, in which a tethered particle blocks the intracellular mouth of the pore, and C-type, which involves a closure of the external mouth. The functional motion involved in C-type inactivation was studied by introducing cysteine residues at the outer mouth of Shaker K+ channels through mutagenesis, and by measuring state-dependent changes in accessibility to chemical modification. Modification of three adjacent residues in the outer mouth was 130-10,000-fold faster in the C-type inactivated state than in the closed state. At one position, state-dependent bridging or crosslinking between subunits was also possible. These results give a consistent picture in which C-type inactivation promotes a local rearrangement and constriction of the channel at the outer mouth.

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Section: Cysteines At the Deepest Position Can Be Crosslinked Between Subunits In The Inactivated Statementioning

confidence: 99%

Section: Electrophysiologymentioning

confidence: 99%

Dynamic Rearrangement of the Outer Mouth of a K+ Channel during Gating

Liu

Jurman

Yellen

1996

Neuron

429

415

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“…Direct binding to arsenic is believed to be important in at least two aspects: 1) binding and resulting inhibition of enzymes and other proteins may cause the chemotherapeutic effects; 2) binding to certain proteins may be a detoxification process (14). It is known that trivalent arsenic is chemically more reactive than pentavalent species, and binds many proteins, for example, tubulin and actin (15), IKKα/β (16), E. coli arsR protein (17), E. coli RI methyltransferase (18), hemoglobin (19,20), nicotinic receptor (21), metallothionein (22), galectin 1 and thioredoxin peroxidase II (23,24), thioredoxin and protein disulfide isomerase (25), glucocorticoid receptor (26), and estrogen receptor α (27). Some other proteins were also shown to bind arsenic, yet their identities are still unknown (25,28).…”

Section: Introductionmentioning

confidence: 99%

Identification of arsenic-binding proteins in human breast cancer cells

et al. 2007

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As a cancer chemotherapeutic drug, arsenic acts on numerous intracellular signal transduction pathways in cancer cells. However, its mechanism of actions is still not fully understood. Previous studies suggest that arsenic reacts with closely spaced cysteine (Cys) residues of proteins with high Cys content and accessible sulfhydryl (SH) groups. In this study, human breast cancer cell line MCF-7 was examined as a cellular model to explore arsenic-binding proteins and the mechanism of binding. An arsenic-biotin conjugate was synthesized by coupling the pentafluorophenol ester of biotin with p-aminophenylarsenoxide. Arsenic-binding proteins were eluted with streptavidin resin from arsenic-biotin treated MCF-7 cells, separated by polyacrylamide gel electrophoresis, and identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Arsenic-binding properties of two of these proteins, beta-tubulin and pyruvate kinase M2 (PKM2), were studied further in vitro and the biological consequences of this binding was evaluated. Binding assay with Western blotting confirmed binding of beta-tubulin and PKM2 by arsenic in a concentration-dependent manner. Arsenic binding inhibited tubulin polymerization, but surprisingly had no effect on PKM2 activity. Molecular modeling showed that binding of Cys(12) alone or vicinal Cys residues (Cys(12) and Cys(213)) of beta-tubulin by arsenic blocked the active site for access of GTP, which is necessary for tubulin polymerization. On the contrary, all Cys residues of PKM2 were far away from the active site of the enzyme. In summary, this study confirmed beta-tubulin and PKM2 as arsenic-binding proteins in MCF-7 cells. Functional consequence of such binding may depend on whether arsenic binding causes conformational changes or blocks active sites of target proteins.

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“…The AChoR reduction by the site-directed reducing agent will also be an interesting tool for the use of aromatic arsenoxides as selective labeling reagents for the nicotinic receptor. In fact, the biological effect of arsenicals was previously investigated [21] and particularly for labeling the AChoR by forming covalent yet reversible complexes with pairs of thiols [22,23]. The use of an affinity reducing agent which specifically reduces the disulfide bond at the vicinity of the agonist binding site could greatly increase the efficacy of the arsenical labeling and so the detection of many subtypes of neuronal and muscular AChoR.…”

Section: Discussionmentioning

confidence: 99%

Site‐directed disulfide reduction using an affinity reagent: Application on the nicotinic acetylcholine receptor

1995

FEBS Letters

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Aromatic trivalent arsenicals: covalent yet reversible reagents for the agonist binding site of nicotinic receptors

Cited by 14 publications

References 30 publications

Dynamic Rearrangement of the Outer Mouth of a K+ Channel during Gating

Dynamic Rearrangement of the Outer Mouth of a K+ Channel during Gating

Identification of arsenic-binding proteins in human breast cancer cells

Site‐directed disulfide reduction using an affinity reagent: Application on the nicotinic acetylcholine receptor

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