LABELING STUDIES OF PHOTOLABILE PHILANTHOTOXINS WITH NICOTINIC ACETYLCHOLINE-RECEPTORS - MODE OF INTERACTION BETWEEN TOXIN AND RECEPTOR

Background: The nicotinic acetylcholine receptors (nAChRs) and glutama te receptors are ligand-gated cation channels composed of five separat e polypeptide chains. A 43 kDa protein of unknown function is noncoval ently associated with the cytoplasmic side of nAChR in vivo. The venom s of many wasps and spiders contain toxins that block the activity of these channels. Philanthotoxin-433 (PhTX-433) is a non-competitive cha nnel blocker found in the venom of the wasp Philanthus. We have used a photolabile derivative to investigate how PhTX-433 interacts with nAC hRs. Results: A radiolabeled PhTX analog, containing a photolabile gro up substituted on one of its aromatic rings, photocrosslinked to all f ive subunits (alpha, alpha', beta, gamma, delta) of purified nAChR in the absence of the 43 kDa protein. In the presence of the 43 kDa prote in, the alpha subunit was preferentially labeled. Proteolysis of the r eceptor after crosslinking indicated that the hydrophobic end (head) o f the PhTx-433 analog bound to the cytoplasmic loop(s) of the alpha-su bunit. Binding is inhibited by other non-competitive channel blockers such as the related polyamine-amide toxins from spiders and chlorproma zine. Conclusions: These results, coupled with previous structure/acti vity studies, lead to a putative model of the binding of PhTx and rela ted polyamine toxins to nAChRs in vivo. The 43 kDa protein appears to influence the orientation of toxin binding. Further binding studies ar e necessary to confirm the model and to define how toxins enter the re ceptor and how they are oriented within it. A precise understanding of ligand/receptor interaction is crucial for the design of drugs specif ic for a particular subtype of receptor.