Chain length dependence of the interactions of bisquaternary ligands with the Torpedo nicotinic acetylcholine receptor

The interactions of a series of bisholine esters [(CH3)3N+CH2CH2OCO–(CH2)n–COOCH2CH2N+(CH3)3] with the Torpedo nicotinic acetylcholine receptor have been investigated. In equilibrium binding studies, [3H]-suberyldicholine (n = 6) binds to an equivalent number of sites as [3H]-acetylcholine and with similar affinity (KD ∼ 15 nM). In competition studies, all bischoline esters examined displaced both radioligands in an apparently simple competitive manner. Estimated dissociation constants (KI) showed clear chain length dependence. Short chain molecules (n ≤ 2) were of lower affinity (KI's of 150–300 nM), whereas longer ligands (n > 6) had high affinity similar to suberyldicholine. Functional responses were measured by either rapid flux techniques using Torpedo membrane vesicles or voltage-clamp analyses of recombinant receptors expressed in Xenopus oocytes. Both approaches revealed that suberyldicholine (EC50 ∼ 3.4 μM) is 14–25-fold more potent than acetylcholine. However, suberyldicholine elicited only about 45% of the maximum response of the natural ligand, i.e., it is a partial agonist. The potency of this bischoline series increased with chain length. Whereas the shorter ligands (n ≤ 3) displayed potencies similar to acetylcholine, longer ligands (n ≥ 4) had similar (or higher) potency to suberyldicholine. Ligand efficacy had an approximately bell-shaped dependence on chain length and compounds where n ≤ 3 and ≥8 were very poor partial agonists. Based on estimates of interonium distances, we suggest that bisquaternary ligands can interact with multiple binding sites on the nAChR and, depending on the conformational state of the receptor, these sites are 15–20 Å apart.