NBD-5-Acylcholine: Fluorescent analog of acetylcholine and agonist at the neuromuscular junction (acetylcholine receptor/ion channel/depolarizing agent/kinetics of interaction)

We have studied the properties of N-7{4-ni- trobenzo-2-oxa-1,3-diazole)-w-aminohexanoic acid 134N-tri- methylammonium)ethyl ester, a fluorescent analog of acetyl- choline at the cellular level by using oharmacological and electrophysiological techniques and at the molecular level by measuring the kinetics of interaction with solubilized acetyl- choline receptor and with acetylcholine esterase (EC 3.1.1.7). The fluorescent drug is a powerful agonist of acetylcholine at the neuromuscular junction and also strongly desensitizes muscle fibers. Interaction with acetylcholine receptor is ac- companied by large changes in the drug's fluorescence. From the kinetics of interaction studied by means of a stopped-flow fluorimeter with laser light source, we obtained a second-order forward rate constant in excess of 1 X 108 M-1 sec-' and an initial dissociation rate constant (k1) of 0.5 sec-1 for receptor from Electrophorus electricus. Interaction of this analog with acetylcholine esterase from E. electricus is accompanied by a transient decrease in fluorescence followed by an increase leading to a stable plateau value at a level near the original one. The initial decrease in fluorescence followed second-order ki- netics with k2 of the order of 109 M-1 sec-1. The slower con- secutive reaction, which could be blocked by phosphorylation of the esteratic site, was of first order with k1 = 0.05 sec-1.