Structure-activity relationship and site of binding of polyamine derivatives at the nicotinic acetylcholine receptor

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amid es, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylch oline receptor (nAChR). Effects of varying the structure of PhTXs and poly( methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay K-app values of these compounds have been determined. We found that an increase in size of the P hTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In g eneral, poly(methylene tetramine)s showed higher binding affinities than Ph TX analogues. The stoichiometry of PhTX binding was determined to be two PhTX molecules p er receptor monomer. PhTXs appeared to bind to a single class of nonalloste rically interacting binding sites and bound PhTX was found to be completely displaced by well-characterized luminal NCIs. To elucidate the site of PhT X binding, a photolabile, radioactive PhTX derivative was photocross-linked to the nAChR in its closed channel conformation resulting in labeling yiel ds for the two alpha and the beta, gamma and delta subunits of 10.4, 11.1, 4.0 and 7.4%, respectively. Based on these findings we suggest that PhTXs and poly(methylene tetramine) s enter the receptor's ionic channel from the extracellular side. The hydro phobic head groups most likely bind to the high-affinity NCI site, while th e positively charged polyamine chains presumably interact with the negative ly charged selectivity filter located deep in the channel lumen.