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Lophotoxin and related coral toxins covalently label the alpha-subunit of the nicotinic acetylcholine receptor
- Source :
- Journal of Biological Chemistry. 263:18568-18573
- Publication Year :
- 1988
- Publisher :
- Elsevier BV, 1988.
-
Abstract
- Lophotoxin and lophotoxin analog-1 are uncharged cyclic diterpenes obtained from gorgonian corals. They have been shown to block the function of nicotinic acetylcholine receptors. Inhibition results from blockade of the agonist recognition site and appears irreversible in that extensive washing does not restore receptor function. This study was undertaken to determine whether this apparently irreversible inhibition involves covalent labeling at a selective site and to further characterize this site directly. Incubation of membranes prepared from the electric organ of Torpedo californica with analog-1 followed by reduction with NaB3H4 resulted in the incorporation of radioactivity into several membrane proteins. The incorporation of radioactivity into the alpha-subunit of the receptor was blocked by prior incubation with agonists and antagonists. [3H]Lophotoxin and [3H]analog-1 were prepared by reduction with NaB3H4 and back-oxidation with CrO3. The radiolabeled coral toxins reacted selectively and covalently with the alpha-subunit of the receptor. Their binding was prevented by prior exposure to agonists and antagonists. In contrast to the site-directed alkylating agent 4-(N-maleimido)benzyltrimethylammonium iodide, prior reduction of the receptor was not required for covalent binding of 3H-labeled coral toxins. Selective reduction of Cys192 and Cys193 followed by alkylation with 4-(N-maleimido)benzyltrimethylammonium iodide blocked the binding of [3H]analog-1, whereas alkylation with iodoacetic acid or iodoacetamide did not. Thus, the binding site for the coral toxins does not overlap the binding surface near Cys192 and Cys193. Digestion of isolated labeled alpha-subunits with endoglycosidase H revealed that the polypeptide portion of the protein retained the covalently bound [3H]analog-1. Digestion with staphylococcal V8 protease revealed two major peptides of approximately 19 and 20 kDa, along with several smaller peptides. Only the 20-kDa peptide retained the covalently bound [3H]analog-1, localizing the site of covalent attachment between Ser173 and Glu335. The unique chemical structure and covalent reactivity of these gorgonian coral toxins will undoubtedly allow further insights into the structure of the agonist recognition site.
- Subjects :
- chemistry.chemical_classification
Electric Organ
Iodoacetic acid
Macromolecular Substances
Terpenes
Chemistry
Cell Membrane
Peptide
Borohydrides
Cell Biology
Receptors, Nicotinic
Torpedo
Biochemistry
chemistry.chemical_compound
Nicotinic acetylcholine receptor
Cnidarian Venoms
Nicotinic agonist
Iodoacetamide
Animals
Binding site
Oxidation-Reduction
Molecular Biology
Acetylcholine receptor
G alpha subunit
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 263
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....6cb3a98ff81cab69c7ea5b847ab41c2b