AH5183 and Cetiedil: Two Potent Inhibitors of Acetylcholine Uptake into Isolated Synaptic Vesicles from Torpedo marmorata

Abstract

Synaptic vesicles purified on a sucrose-KCl sedimentation gradient were tested for their ability to accumulate [1-14C]acetylcholine ([1-14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein-modifying agents N-ethylmaleimide and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 +/- 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 +/- 9.5 nM. Neither ATP-dependent calcium uptake nor Mg2+-ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L-[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of approximately 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 +/- 4.1 nM under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Active Transport of Acetylcholine by the Human Vesicular Acetylcholine Transporter

“…However, the number of human VAChT transporters expressed was greatly increased, and the B max (8.9 Ϯ 0.6 pmol/mg) was similar to that found for [ (27). Uptake was specifically inhibited by L-vesamicol, a noncompetitive inhibitor of vesicular ACh uptake in Torpedo (28,29), exhibiting an IC 50 of 14.7 Ϯ 1.5 nM (Fig. 3, inset).…”

Cloning and expression of the vesamicol binding protein from the marine ray Torpedo

“…Expression of the Torpedo protein in mammalian fibroblasts confers high-affinity vesamicol binding to membranes isolated from transfected cells similar to that described on synaptic vesicles (7-20 nM( [19,35]. While the binding of vesamicol to synaptic vesicles from C. eleguns has never been reported, UNC-17 expressed separately in the same heterologous system demonstrated specific vesamicol binding as well, with lower affinity (-124 n&I) than that of Torpedo.…”

“…The inhibition constant of the transport of acetylcholine is similar to the dissociation constant of the drug, suggesting that binding and inhibition are directly related. However, inhibition is of the mixed non-competetive type [19,36] indicating that vesamicol does not bind to the a~etylcholine uptake site. Two models to account for this data have been proposed by Parsons and co-workers [37]: (i) vesamicol binds to an allosteric site on the vesicular acetylcholine transporter or (ii) vesamico1 binds to a different protein that acts by an unknown mechanism to inhibit acetylcholine uptake.…”

“…Its action involves inhibition of acetylcholine uptake into synaptic vesicles by binding to a single population of sites on the cholinergic vesicle [ 18,191. Pharmacological studies on the interaction of acetylcholine and vesamicol with Torpedo vesicles and vesicle membrane preparations suggest that the vesamicol binding site is within the acetylcholine vesicular transporter [19,20], although a single polypeptide binding both vesamicol and acetylcholine has not yet been identified.…”

Analysis of Point Mutants in the Caenorhabditis elegans Vesicular Acetylcholine Transporter Reveals Domains Involved in Substrate Translocation

“…For VAChT, a reserpine-like competitive antagonist that can identify the high affinity acetylcholine recognition site does not exist. However, vesamicol acts as a specific noncompetitive inhibitor of vesicular acetylcholine transport with an inhibition constant (K i ) in the nanomolar range, similar to its dissociation constant (K d ) (51)(52)(53). Acetylcholine can competitively displace the binding of vesamicol with a K i 10 -100 times greater than the K m for acetylcholine transport (20,54).…”