Vesicular acetylcholine transporter (VAChT) is inhibited by (−)-vesamicol [(−)-trans-2-(4-phenylpiperidino)cyclohexanol], which binds tightly to an allosteric site. The tertiary alkylamine center in (−)-vesamicol is protonated and positively charged at acidic and neutral pH and unprotonated and uncharged at alkaline pH. Deprotonation of the amine has been taken to explain loss of (−)-vesamicol binding at alkaline pH. However, binding data deviate from a stereotypical bell shape, and more binding occurs than expected at alkaline pH. The current study characterizes the binding of (−)-vesamicol from pH 5 to pH 10 using filter assays, (−)-[ 3 H]vesamicol (hereafter called [ 3 H]vesamicol), and human VAChT expressed in PC12 A123.7 cells. At acidic pH, protons and [ 3 H] vesamicol compete for binding to VAChT. Pre-or long term-exposure of VAChT to high pH does not affect binding, thus eliminating potential denaturation of VAChT and failure of the filter assay. The dissociation constant for the complex between protonated [ 3 H]vesamicol and VAChT decreases from 12 nM at neutral pH to 2.1 nM at pH 10. The simplest model of VAChT that explains the behavior requires a proton at Site 1 to dissociate with pK 1 = 6.5 ± 0.1, a proton at Site A to dissociate with pK A = 7.6 ± 0.2, and a proton at Site B to dissociate with pK B = 10.0 ± 0.1. Deprotonation of the Site 1 proton is obligatory for [ 3 H]vesamicol binding. Deprotonation of Site A decreases affinity (2.2 ± 0.5)-fold and deprotonation of Site B increases affinity (18 ± 4)-fold. Time-dependent dissociation of bound [ 3 H]vesamicol is biphasic, but equilibrium saturation curves are not. The contrasting phasicity suggests that the pathway to and from the [ 3 H]vesamicol binding site exists in open and at least partially closed states. The potential significance of the findings to development of PET and SPECT ligands based on (−)-vesamicol for human diagnostics also is discussed.Vesicular acetylcholine transporter (VAChT 1 ) moves the neurotransmitter acetylcholine (ACh) from the cytoplasm of nerve terminals to the inside of synaptic vesicles (1,2). It belongs to the major facilitator superfamily (MFS) of transporters, most of which contain twelve transmembrane (TM) helices (3). Three-dimensional structures for four bacterial members of the MFS have been determined. They are lactose permease (4), glycerol phosphate transporter (5), oxalate-formate antiporter (6), and the multidrug resistance transporter EmrD (7). The structures all exhibit similar TM nearest neighbors (4,5,6). The first six TMs in the amino acid sequence pack into one bundle, and the second TMs pack into another bundle. The bundles are † This research was supported by Grant NS15047 from the National Institute of Neurological Disorders and Stroke. *To whom correspondence should be addressed: Department of Chemistry and Biochemistry, University of California, Santa Barbara, 893-2252. Fax: (805) 893-4120.. 1 Abbreviations: (±)-ABV, (±)-aminobenzovesamicol; ACh, acetylcholine; AMPSO, (N-(1,1-dimethy...

J. Neurochem. (2010) 115, 984–993. Abstract Vesicular acetylcholine transporter (VAChT; TC 2.A.1.2.13) mediates storage of acetylcholine (ACh) by synaptic vesicles. A three‐dimensional homology model of VAChT is available, but the binding sites for ACh and the allosteric inhibitor (−)‐trans‐2‐(4‐phenylpiperidino)cyclohexanol (vesamicol) are unknown. In previous work, mutations of invariant W331 in the lumenal beginning of transmembrane helix VIII (TM VIII) of rat VAChT led to as much as ninefold loss in equilibrium affinity for ACh and no loss in affinity for vesamicol. The current work investigates the effects of additional mutations in and around W331 and the nearby lumenal end of the substrate transport channel. Mutants of human VAChT were expressed in the PC12A123.7 cell line and characterized using radiolabeled ligands and filtration assays for binding and transport. Properties of a new and a repeat mutation in W331 are consistent with the original observations. Of 16 additional mutations in 13 other residues (Y60 in the beginning of lumenal Loop I/II, F231 in the lumenal end of TM V, W315, M316, K317, in the lumenal end of TM VII, M320, A321, W325, A330 in lumenal Loop VII/VIII, A334 in the lumenal beginning of TM VIII, and C388, C391, F392 in the lumenal beginning of TM X), only A334F impairs binding. This mutation decreases ACh and vesamicol equilibrium binding affinities by 14‐ and 4‐fold, respectively. The current results, combined with previous results, demonstrate existence of a spatial cluster of residues close to vesicular lumen that decreases affinity for ACh and/or vesamicol when the cluster is mutated. The cluster is composed of invariant W331, highly conserved A334, and invariant F335 in TM VIII and invariant C391 in TM X. Different models for the locations of the ACh and vesamicol binding sites relative to this cluster are discussed.

The method describes production and the selection of neurosecretory PC12A123.7 cells stably transfected with human vesicular acetylcholine transporter (hVAChT). Transfected cells provide postnuclear supernatant used to characterize equilibrium binding of the neurotransmitter acetylcholine (ACh), the pH dependence for transport of ACh, and the rate behavior for dissociation of the allosteric, high-affinity inhibitor vesamicol. Retention of radiolabeled ACh or vesamicol, mediated by hVAChT in synaptic-like microvesicles of postnuclear supernatant, is measured using filter assays. The procedure for regression analysis of data also is described.

Invariant E309 is in contact with critical and invariant D398 in a three-dimensional homology model of vesicular acetylcholine transporter (VAChT, TC# 2.A.1.2.13). In the work reported here, E309 and D398 in human VAChT were mutated singly and together to test their functions, assign pK values to them, and determine whether the residues are close to each other in three-dimensional space. Mutants were stably expressed in the PC12 A123.7 cell line, and transport and binding properties were characterized at different pH values using radiolabeled ligands and filtration assays. Contrary to a prior conclusion, the results demonstrate that most D398 mutants do not bind the allosteric inhibitor vesamicol even weakly. Earlier work showed that most D398 mutants do not transport ACh. D398 therefore probably is the residue that must deprotonate with pK = 6.5 for binding of vesamicol and with pK ~5.9 for transport of ACh. Because E309Q has no effect on VAChT functions at physiological pH, E309 has no apparent critical role. However, radical mutations in E309 cause decreases in ACh and vesamicol affinities and total loss of ACh transport. Unlike wild-type VAChT, which exhibits a peak of [ 3 H]vesamicol binding centered at pH 7.4, the mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH ≥9. The combination of high pH and mutated E309 apparently produces a relaxed (in contrast to tense) conformation of VAChT that binds vesamicol exceptionally tightly. No compensatory interactions between E309 and D398 in double mutants were discovered. Proof of a close spatial relationship between E309 and D398 was not found. Nevertheless, the data are more consistent with the homology model than an alternative hydropathy model of VAChT that likely locates E309 far away in three-dimensional space from D398 and the ACh binding site. Also, a probable network of interactions involving E309 and an unknown residue having pK = 10 has been revealed. Vesicular acetylcholine transporter (VAChT, TC# 2.A.1.2.13) 1 is found in the membranes of synaptic vesicles inside of nerve terminals that release acetylcholine (ACh). It transports ACh from cytoplasm to the inside of the vesicles, thereby preparing ACh for exocytotic release. Protons translocate from inside the vesicle through VAChT to cytoplasm and drive the transport cycle (1,2). Residues that could mediate translocation of protons thus are of special interest in the ACh transport mechanism. A man-made compound called vesamicol binds with high affinity to a saturable allosteric site and thereby inhibits ACh binding and transport. It has been very useful in elucidating VAChT properties (3).Based on hydropathy analysis of the amino acid sequence, twelve transmembrane helices (TMs) are predicted for VAChT. A number of the TMs contain invariant ionic or potentially ionic residues (4). VAChT is closely related to vesicular monoamine transporters (VMATs) 1 † This research was supported by Grant NS15047 from the National Institute of Neurological Disorders and Stroke. * T...