Sidedness and chemical and kinetic properties of the vesamicol receptor of cholinergic synaptic vesicles

Abstract

Cholinergic synaptic vesicles isolated from Torpedo electric organ contain a receptor for the compound l-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183), which when occupied blocks storage of acetylcholine (AcCh). The inside or outside orientation of the receptor and its chemical and ligand binding kinetics characteristics were studied. Binding of [3H]vesamicol to the receptor is inhibited efficiently by the protein modification reagents 4-(chloromercuri)benzenesulfonate and N,N''-dicyclohexylcarbodiimide and by protease treatment of cholate-solubilized receptor. The receptor in native vesicles in resistant to irreversible inactivation by proteases, elevated temperature, or pH extremes. [3H]Vesamicol binding depends on deprotonation of a group of pKa1 = 6.26 .+-. 0.03 and protonation of a group of pKa2 = 10.60 .+-. 0.04, which is probably the tertiary amine of the drug molecule itself. The membrane-impermeant zwitterionic vesamicol analogue dl-trans-4-oxo-4-[[5,6,7,8-tetrahydro-6-hydroxy-7-(4-phenyl-1-piperidinyl)-1-naphthalenyl]amino]butanoic acid (TPNB) is an effective inhibitor of AcCh active transport with an IC50 value of (51 .+-. 8) .times. 10-9 M. At 23.degree. C, [3H]vesamicol bound to the receptor at a rate of (1.74 .+-. 0.06) .times. 105 M-1 s-1, and excess unlabeled vesamicol displaced a low concentration of bound [3H]vesamicol at 0.29 .+-. 0.01 min-1. At 0.degree. C, 10 .mu.M unlabeled vesamicol displaced 36 .+-. 2% of a low concentration of bound [3H]vesamicol at 0.16 .+-. 0.02 min-1 and 64 .+-. 2% at 0.013 .+-. 0.001 min-1. One micromolar unlabeled vesamicol behaved similarly. In a different preparation of vesicles, 10 .mu.M TPNB displaced 22 .+-. 2% at 0.28 .+-. 0.05 min-1 and 78 .+-. 1% at 0.013 .+-. 0.001 min-1. Several types of receptor heterogeneity are consistent with the data. It is concluded that the vesamicol receptor is a stable protein often exhibiting heterogeneity, which faces the cytoplasmic compartment of the cholinergic nerve terminal. It probably contains a binding site carboxylate in a hydrophobic environment, which ion pairs with the protonated tertiary ammonium group of the drug. It also contains a cytoplasmically oriented sulfhydryl group, which is linked to but not part of the binding site.