A kinetic and allosteric model for the acetylcholine transporter-vesamicol receptor in synaptic vesicles

Abstract

The ligand binding relationship between the acetylcholine transporter (AcChT) and the vesamicol receptor (VR) and the kinetics of active transport were studied in synaptic vesicles purified from the Torpedo electric organ using analogues of AcCh and vesamicol. Methoxyvesamicol, which should exhibit better equilibration properties for kinetics measurements than the more potent parent, inhibits active transport in a nonlinear noncompetitive manner. AcCh analogues competitively inhibit binding of [3H]vesamicol with higher affinity in hyposmotically lysed vesicle ghosts than in intact vesicles, apparently due to removal of a competing internal, osmotically active factor. AcCh and actively transported analogues of AcCh that are up to 57% larger in van der Waals volume exhibit up to a 200-fold ratio for the dissociation constant measured by inhibition of vesamicol binding to ghosts (KIAg) compared to the Michaelis constant for transport (KM) or the IC50 value for inhibition of [3H]AcCh active transport. In contrast, two AcCh analogues that are about 120% larger and that almost surely are not transported exhibit a KIAg/IC50 ratio of about 1. The data demonstrate that the vesamicol family of compounds binds to an allosteric site in the AcChT. Initiation of active transport has no apparent effect on the affinities of vesamicol and AcCh analogues, which suggests that most of the AcChT-VR in purified vesicles is transport incompetent. Vesicle ghosts actively transport [3H]AcCh nearly as well as intact vesicles, which suggests that internal factor does not affect transport-competent AcChT-VR. A kinetics model is proposed that predicts that AcCh analogues exhibiting a KIAg/IC50 ratio significantly greater than 1 are actively transported. Some of the microscopic constants in the model are estimated. The AcChT binds AcCh very weakly with a dissociation constant of about 20-50 mM, but it transports substrates rapidly in a process exhibiting remarkably little selectivity for the detailed shape and volume of the transported ion.

Comparison of Noninvasive Quantification Methods of in Vivo Vesicular Acetylcholine Transporter Using [123I]-IBVM SPECT Imaging

“…In ex vivo studies, [ 123 I]-IBVM binds with high affinity (subnanomolar range) and high selectivity to the vesicular ACh transporter (VAChT), with a good specific-to-nonspecific ratio and with a binding in ACh-rich brain areas that is both saturable and specific (Altar and Marien, 1988;Bahr et al, 1992;Zea-Ponce et al, 2005).…”

Transmembrane Reorientation of the Substrate-Binding Site in Vesicular Acetylcholine Transporter

“…Vesicular acetylcholine transporter (VAChT) 1 translocates acetylcholine (ACh) from cytoplasm to the lumen of synaptic vesicles for evoked release from nerve terminals. Vesicular protons provided by V-ATPase are used by VAChT to power ACh transport.…”

“…When external ACh binds to outwardly oriented transporter, vesamicol cannot bind (1). External ACh presumably cannot bind to inwardly oriented transporter because the ACh-binding site is not exposed.…”

Mutational Analysis of Basic Residues in the Rat Vesicular Acetylcholine Transporter

“…In this process, it is suggested that the loss of a proton leads to a conformational change which makes the transporter active (2). Vesamicol ((-)-trans-2-(4-phenylpiperidino)cyclohexanol) has been shown to be a specific inhibitor of ACh transport by VAChT (3,4). This compound acts in a noncompetitive manner; however ACh blocks vesamicol binding to the transporter.…”