Referenced in: none

Automatically associated channels: Kv10.1

Title: Synthesis and characterization of a heterobifunctional mercurial cross-linking agent: incorporation into cobratoxin and interaction with the nicotinic acetylcholine receptor.

Authors: E R Wohlfeil, R A Hudson

Journal, date & volume: Biochemistry, 1991 Jul 23 , 30, 7231-41

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/1854732

Abstract
The heterobifunctional organomercurial reagents 3-(acetoxymercurio)- and 3-(chloromercurio)-5-nitrosalicylaldehyde were prepared, characterized in model studies, and used to probe the interaction between cobratoxin, purified from the venom of the Thailand cobra (Naja naja siamensis), and the affinity-purified nicotinic acetylcholine receptor (AcChR) from Torpedo california electroplax. These reagents may also be useful in introducing chemically well-defined heavy metal atoms into proteins containing no reactive thiols. Model reagent adducts were prepared in situ by reductive amination with N-butylamine and N alpha-acetyllysine-N-methylamide. The nitrophenolic pKaS of the amine adducts were similar to those of the aldehyde reagents through reduced by 1.3-1.5 units when compared with the hydroxylmethyl reduction product. Reaction of either mercuriosalicylaldehyde with cobratoxin led to a single major modification product incorporating 1 mol of the reagent into cobratoxin at Lys 23. The Lys 23 modified toxin had a reduced binding affinity for the AcChR over that of the native toxin (Kd 2.75 nM cf. 0.3 nM). Reduction of the purified AcChR with 1 mM dithiothreitol (DTT) followed by removal of excess thiol led to cross-linking reactions with the Lys 23 modified cobratoxin to both the alpha and beta subunits of the AcChR complex. Reaction of DTT-treated AcChR with N-ethylmaleimide (NEM) blocked cross-linking, while treatment of the initially cross-linked toxin-AcChR complex with mercaptoethanol leads to reversal of cross-linking. These observations strongly support cross-linking mediated by the formation of a mercury-sulfur bond and further lend support the identity of the respective interacting sites in AcChR and toxin.