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An Analog of Lophotoxin Reacts Covalently with Tyr190 in the α-Subunit of the Nicotinic Acetylcholine Receptor
- Source :
- Journal of Biological Chemistry. 264:12666-12672
- Publication Year :
- 1989
- Publisher :
- Elsevier BV, 1989.
-
Abstract
- Lophotoxin and lophotoxin analog-1 are natural diterpenes from coral that inhibit nicotinic acetylcholine receptors by covalent reaction with the acetylcholine recognition sites on the alpha-subunits. Although both toxins contain potentially reactive epoxides and alpha,beta-unsaturated aldehydes, the mechanism of their covalent reaction with the receptor is not known. The role of the alpha,beta-unsaturated aldehyde in analog-1 was investigated by reduction of the aldehyde to an alcohol with [3H]NaBH4. The reduced [3H]analog-1 bound selectively and covalently to the alpha-subunit of the receptor. Covalent binding was inhibited by agonists and antagonists, but not by noncompetitive allosteric inhibitors. The apparent dissociation constant of the reduced [3H]analog-1 was approximately 1.5 x 10(-6) M. These results demonstrate that the alpha,beta-unsaturated aldehyde in analog-1 is not an absolute requirement for covalent reaction with the receptor. Receptors were treated with the reduced-[3H]analog-1, and the labeled alpha-subunits were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with staphylococcal V8 protease. A labeled 20-kDa V8 protease fragment was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography and subjected to sequence analysis. A peptide beginning at Ser173 was identified, and the label appeared in the 18th step corresponding to Tyr190. This assignment was confirmed by digestion of the labeled 20-kDa V8 protease fragment with cyanogen bromide, followed by purification of the labeled cyanogen bromide peptide on reverse-phase high performance liquid chromatography. A peptide beginning at Lys179 was identified, and the label appeared in the 12th step, again corresponding to Tyr190. Tyr190 may react with the coral toxin by nucleophilic addition at one of the carbons associated with an epoxide, and may form part of the alkylammonium-binding subsite of the acetylcholine recognition site.
- Subjects :
- chemistry.chemical_classification
Gel electrophoresis
Protease
Chemistry
Stereochemistry
medicine.medical_treatment
Peptide
Cell Biology
Biochemistry
Nicotinic acetylcholine receptor
chemistry.chemical_compound
Nicotinic agonist
medicine
Cyanogen bromide
Molecular Biology
G alpha subunit
Acetylcholine receptor
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 264
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi...........c314edcf57bd35ae60e6092721edd617