Vesamicol Binding Sites Research Articles

Search for the acetylcholine and vesamicol binding sites in vesicular acetylcholine transporter: the region around the lumenal end of the transport channel

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 PC12(A123.7) 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.

Multiple Protonation States of Vesicular Acetylcholine Transporter Detected by Binding of [3H]Vesamicol

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, (-)-[3H]vesamicol (hereafter called [3H]vesamicol), and human VAChT expressed in PC12(A123.7) cells. At acidic pH, protons and [3H]vesamicol compete for binding to VAChT. Preexposure 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 [3H]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 pK1 = 6.5 +/- 0.1, a proton at site A to dissociate with pKA = 7.6 +/- 0.2, and a proton at site B to dissociate with pKB = 10.0 +/- 0.1. Deprotonation of the site 1 proton is obligatory for [3H]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 [3H]vesamicol is biphasic, but equilibrium saturation curves are not. The contrasting phasicity suggests that the pathway to and from the [3H]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.

Synthesis and in vitro evaluation of new benzovesamicol analogues as potential imaging probes for the vesicular acetylcholine transporter

Our goal was to synthesize new stereospecific benzovesamicol analogues, which could potentially be used as SPECT or PET radioligands for the vesicular acetylcholine transporter (VAChT). This paper describes the chemical synthesis, resolution and determination of binding affinity for four enantiomeric pairs of derivatives. Their intrinsic affinities were determined by competition against binding of [ 3H]vesamicol to human VAChT. Of the eight enantiomers, ( E)-( R, R)-5-AOIBV [( R, R)- 3], and ( R, R)-5-FPOBV [( R, R)- 4] displayed the highest binding affinities for VAChT ( K d = 0.45 and 0.77 nM, respectively), which indicated that an elongation of the chain from 5-idodo as in the case of 5-iodobenzovesamicol (5-IBVM), to a 5-( E)-3-iodoallyloxy or 5-fluoropropoxy substituent, as in 5-AOIBV and 5-FPOBV, respectively, was very well tolerated at the vesamicol binding site. The enantiomer ( R, R)-4-MAIBV [( R, R)- 16], which retains the basic structure of (−)-5-IBVM but possess an additional aminomethyl substituent in the 4-position of the piperidine ring, displayed lower binding affinity ( K d = 8.8 nM). Nevertheless, the result suggests that substitution at this position may be an interesting alternative to investigate for development of new benzovesamicol analogues. As expected, the corresponding ( S, S) enantiomers displayed lower K d values, they were approximately 10-fold lower in the case of ( S, S)-5-FPOBV ( K d = 8.4 nM) and ( E)-( S, S)-5-AOIBV ( K d = 4.3 nM). ( R, R)- 3, and ( R, R)- 4 showed the same high affinity for VAChT as (−)-5-IBVM and may be suitable as imaging agents of cholinergic nerve terminals.

Acetylcholine binding site in the vesicular acetylcholine transporter.

This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently determined three-dimensional structures for other transporters in the major facilitator superfamily.

Regulation of vesicular acetylcholine transporter by the activation of excitatory amino acid receptors in the avian retina.

1. Previous studies have shown that phorbol esters induce protein kinase C (PKC) mediated phosphorylation of the vesicular acetylcholine transporter (VAChT) and change its interaction with vesamicol. However, it is not clear whether physiological activation of receptors coupled to PKC activation can alter VAChT behavior. 2. Here we tested whether activation of kaianate (KA) receptors alters VAChT. Several studies suggest that the cholinergic amacrine cells display KA/AMPA receptors that mediate excitatory input to these neurons. In addition, KA in the chicken retina can generate intracellular messengers with the potential to regulate cellular functions. 3. In cultured chicken retina (E8C11) KA reduced vesamicol binding to VAChT by 53%. This effect was potentiated by okadaic acid, a protein phosphatase inhibitor, and was totally prevented by BIM, a PKC inhibitor. 4. Phorbol myristate acetate (PMA), but not alpha-PMA, reduced in more than 85% the number of L-[3H]-vesamicol-specific binding sites in chicken retina, confirming that activation of PKC can influence vesamicol binding to chicken VAChT. 5. The data show that activation of glutamatergic receptors reduces [3H]-vesamicol binding sites (VAChT) likely by activating PKC and increasing the phosphorylation of the ACh carrier.

Autoradiographic comparison of [ 3H](−)nicotine, [ 3H]cytisine and [ 3H]epibatidine binding in relation to vesicular acetylcholine transport sites in the temporal cortex in Alzheimer's disease

The laminar binding distribution of three nicotinic receptor agonists, [ 3H](−)nicotine, [ 3H]cytisine, and [ 3H]epibatidine, and their relation to the [ 3H]vesamicol binding, which is known to represent the vesicular acetylcholine transport sites, was performed employing in vitro autoradiography on the medial temporal cortex (Brodmann area 21). Autopsied brain tissue from nine Alzheimer patients and seven age-matched controls were used. The binding pattern of the three nicotinic ligands in the normal cortex was in general similar, showing binding maxima in the cortical layers I, III and V. The binding of [ 3H](−)nicotine, [ 3H]cytisine, and [ 3H]epibatidine was lower in the older controls and more uniform throughout the layers as compared with younger controls. There was a significant age-related decrease in the binding of the three nicotinic ligands within the controls (age range: 58 to 89 years; P [3H](−)nicotine=0.002, P [3H]epibatidine=0.010, P [3H]cytisine=0.037). In the older controls, the [ 3H]epibatidine binding was much decreased as compared with that of [ 3H](−)nicotine and [ 3H]cytisine. This may indicate a higher selectivity of [ 3H]epibatidine for a nicotinic receptor subtype that is particularly affected by aging. The laminar binding pattern of [ 3H]vesamicol showed one maximum in the outer cortical layers II/III. The [ 3H]vesamicol binding did not change with aging. The binding of all ligands was significantly decreased in all layers of the temporal cortex in Alzheimer's disease, but the [ 3H]vesamicol binding decreased only half as much as the nicotinic receptors. Also, choline acetyltransferase activity was percentually more reduced than [ 3H]vesamicol binding in Alzheimer's disease. The cortical laminar binding pattern of all 3H-ligands was largely absent in the Alzheimer's disease cases. The less severe loss of vesicular acetylcholine transport sites as compared with the loss of the nicotinic receptors and choline acetyltransferase activity may suggest that vesamicol binding sites might be more preserved in presynaptic terminals still existing and thereby expressing compensatory capacity to maintain cholinergic activity.

18F-labelled vesamicol derivatives: Syntheses and preliminary in vivo small animal positron emission tomography evaluation

As possible presynaptic tracers for cholinergic function in humans, three 18F-labelled vesamicol analogs were synthesized for use in positron emission tomography (PET): cis-[ 18F]-4-fluoromethylvesamicol (FMV), [ 18F]- N-fluoroacetamidobenzovesamicol (FAA) and [ 18F]- N-ethyl- N-fluoroacetamidobenzovesamicol (NEFA). Radiolabelling was accomplished using [ 18]fluoride and the corresponding tosylates, the syntheses of which are also described. Yields were on the order of 40–60, 5 and 40–60%, respectively. Dynamic studies of the biodistribution in rats of [ 18F]FAA and [ 18F]NEFA using PET were compared with those previously reported for [ 18F]FMV. Due to probable rapid metabolism, [ 18F]FAA was considered unsuitable as a ligand for in vivo imaging. [ 18F]NEFA, similar to [ 18F]FAA, displayed a more moderate cerebral uptake than that of [ 18F]FMV (2 vs 20–30%). Pretreatment with vesamicol blocked the cerebral uptake, indicating a specific interaction with the vesamicol binding site. The biodistribution of high specific activity [ 18F]NEFA with time could be described with a three-compartmental model. The evaluation of [ 18F]NEFA as a tracer for cholinergic function is currently being pursued in monkeys and humans.

Cholinergic regulation of hippocampal brain-derived neurotrophic factor mRNA expression: Evidence from lesion and chronic cholinergic drug treatment studies

Quantitative in situ hybridization and northern blot analysis techniques were used to determine the effects of removal of the cholinergic input on levels and topographical distribution of brain-derived neurotrophic factor mRNA in the hippocampus of adult rats. First, the effects of partial and full fimbrial transections, which result in partial and near-total cholinergic deafferentation respectively, were compared. Twenty-one days after partial unilateral fimbrial transections, there were significant decreases in brain-derived neurotrophic factor mRNA expression throughout the hippocampal formation. Decreased expression of brain-derived neurotrophic factor mRNA was evident in all areas of localization within the hippocampal formation. The decreases amounted to 22–36% reductions compared with unlesioned control animals. Brain-derived neurotrophic factor mRNA levels were decreased to a greater extent (50–69%) following full unilateral fimbrial transections. Quantitative northern blot analysis indicated that hippocampal BDNF mRNA was decreased by 29 and 68%, three weeks after partial or full unilateral fimbrial transections, respectively. The extent of the reductions in brain-derived neurotrophic factor mRNA levels correlated with reductions in acetylcholinesterase staining density and cholinergic terminal density determined by quantitative autoradiographic analysis of [ 3H]vesamicol binding sites. Second, we found that chronic treatment with atropine (20 mg/kg per day for 14 days) decreased (by 54%) brain-derived neurotrophic factor mRNA levels in all areas of localization within the hippocampus. In contrast, chronic treatment with nicotine (1.18 mg/kg per day for 14 days), a treatment known to desensitize nicotinic receptors, did not affect brain-derived neurotrophic factor mRNA expression in the hippocampal formation. The findings provide evidence for cholinergic muscarinic regulation of brain-derived neurotrophic factor mRNA expression in the adult rat hippocampal formation and they suggest the existence of a tonic stimulation of brain-derived neurotrophic factor synthesis by the cholinergic afferents.

Effect of N, N′-dicyclohexylcarbodiimide on the binding of vesamicol, an inhibitor of acetylcholine transport into synaptic vesicles

Vesamicol is a highly potent inhibitor of active acetylcholine transport into isolated cholinergic vesicles from Torpedo. On the basis of transport kinetics and vesamicol sensitivity, we have shown that the acetylcholine transporter could be in an activated state even in the absence of a stimulated ATPase. In this preparation, N, N′-dicyclohexylcarbodiimide (DCCD), an hydrophobic carbodiimide, inactivates both ACh transport and vesamicol binding. Inhibition of vesamicol binding by DCCD is time dependent, saturable and prevented by vesamicol. DCCD first affected the affinity constant for vesamicol. K i-value for DCCD lies in the micromolar range. These results imply that there is a DCCD reactive site within the ACh transporter and that it is located in an hydrophobic environment near the vesamicol binding site. SDS gel electrophoresis after labelling of the vesicle membrane proteins with [ 14C]DCCD shows that radioactivity is mainly incorporated in a 15 kDa subunit. Time-course and concentration dependence of [ 14C]DCCD labelling and vesamicol inhibition do not coincide. Hence, the two processes are probably unrelated and the result rather points to another inactivation mechanism which can be an intramolecluar cross link.

Photoaffinity labeling of the acetylcholine transporter

The acetylcholine (AcCh) binding site in the AcCh transporter-vesamicol receptor (AcChT-VR) present in synaptic vesicles isolated from the electric organ of Torpedo was characterized. A high-affinity analogue of AcCh containing an aryl azido group, namely, cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide (AzidoAcCh), was synthesized in nonradioactive and highly tritiated forms. AzidoAcCh was shown to be a competitive inhibitor of [3H]AcCh active transport and binding of [3H]-vesamicol to the allosteric site. The [3H]AzidoAcCh saturation curve was determined. In all cases the AcChT.AzidoAcCh complex exhibited an inhibition or dissociation constant of about 0.3 microM. Binding of [3H]AzidoAcCh was inhibited by vesamicol and AcCh. AzidoAcCh irreversibly blocked greater than 90% of the [3H]vesamicol binding sites after multiple rounds of photolysis and reequilibration with fresh ligand. Autofluorographs of synaptic vesicles photoaffinity-labeled with [3H]AzidoAcCh showed specific labeling of material exhibiting a continuous distribution from 50 to 250 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The result demonstrates that the AcChT has an unexpected structure highly suggestive of the synaptic vesicle proteoglycan.

Vesamicol Binding to Subcellular Membranes That Are Distinct from Catecholaminergic Vesicles in PC 12 Cells

We have examined PC12 cells for the localization of binding sites for vesamicol [l-2-(4-phenylpiperidino) cyclohexanol], a compound that has previously been shown to bind to cholinergic vesicles and to inhibit the uptake of acetylcholine. Initial studies presented in this article demonstrate the existence of a specific, saturable vesamicol binding site in PC12 cells. Subsequent experiments show that these binding sites reside in a membrane population that is distinct from catecholamine-containing compartments with respect to density and antigenic composition. In particular, vesamicol binding compartments have a lower density than catecholaminergic vesicles and, unlike these latter vesicles, do not appear to contain the vesicle-specific proteins synaptophysin and SV2 as part of the same membrane. These results suggest that vesicular transport proteins for acetylcholine and catecholamines are differentially sorted to distinct membrane compartments in PC12 cells.

3H]Vesamicol binding in human brain cholinergic deficiency disorders

We measured the binding of the vesicular acetylcholine transport blocker [ 3H]vesamicol (2-[4-phenylpiperidino] cyclohexanol; AH-5183) to autopsied frontal cortex and amygdala of patients from 4 disorders having a marked brain cholinergic reduction, namely Alzheimer's disease, Parkinson's disease with dementia, dominantly inherited olivopontocerebellar atrophy and Down's syndrome. Although mean activity of the specific cholinergic marker enzyme choline acetyltransferase (ChAT) was markedly reduced by about 60% in frontal cortex in the 4 patient groups and by 80% or greater in amygdala of the Alzheimer's and Down's syndrome patients, [ 3H]vesamicol binding density was, on average, either normal or only slightly reduced as compared with the controls. This discrepancy suggests that in human brain [ 3H]vesamicol binding is either not preferentially localized to cholinergic nerve endings or, in these cholinergic deficiency syndromes, a substantial proportion of the vesamicol binding sites persist on cholinergic nerve terminals despite loss of ChAT activity.

Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor

Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right-side-out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low-affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed.

3H]vesamicol binding in brain: autoradiographic distribution, pharmacology, and effects of cholinergic lesions.

An autoradiographic analysis of high-affinity binding sites for the vesicular acetylcholine transport blocker [3H]vesamicol (2-(4-phenylpiperidino) cyclohexanol; AH 5183) was conducted in rat brain. [3H]Vesamicol binding was displaced 52-99% by DPPN [( 2,3,4,8]-decahydro-3-(4-phenyl-1-piperidinyl)-2-napthalenol) (IC50 = 14 nM) and by ketanserin (500 nM), haloperidol (43 nM), and vesamicol analogs, but not by drugs selective for adenosine, adrenergic, amino acid, calcium channel, monoaminergic, opioid, PCP, sigma, or several other receptor classes. [3H]Vesamicol binding was most concentrated in the interpeduncular nucleus and fifth and seventh cranial nerve nuclei. Moderate binding was found in the lateral caudate-putamen, medial nucleus accumbens, olfactory tubercle, vertical and horizontal diagonal bands of Broca, and basolateral amygdala. The distribution of [3H]vesamicol binding was similar to distributions of acetylcholine (r = 0.88), acetylcholine esterase (r = 0.97), choline acetyltransferase (ChAT) (r = 0.97), and [3H]hemicholinium-3 binding sites (r = 0.95-0.99). Lower correlations were obtained between [3H]vesamicol and muscarinic receptor densities (r = 0.50-0.70). Few exceptions to the match between binding and cholinergic neuronal markers were found, e.g., the molecular layer of the cerebellum and the thalamus. Lesions of cholinergic neuronal projections to the neocortex or hippocampus reduced [3H]vesamicol binding in each of these regions, but to a lesser extent than reductions in ChAT. [3H]Vesamicol binding sites appear to be anatomically associated with brain cholinergic neurons, a locus that is consistent with the control by this site of vesicular acetylcholine uptake.