Equilibrium Binding and Transport by Vesicular Acetylcholine Transporter

Khare, Parul; White, Aubrey R.; Mulakaluri, Anuprao; Parsons, Stanley M.

doi:10.1007/978-1-60761-700-6_10

Cited by 3 publications

(9 citation statements)

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“…Resuspended cells were broken open in a Potter-Elvehjam homogenizer (three to six strokes) using a motor driven stirrer until ~95% of them took up trypan blue as determined by microscopic examination (20). The suspension was centrifuged at 1500 g for 10 min, and the resulting postnuclear supernatant was stored at − 80°.…”

Section: Methodsmentioning

confidence: 99%

Possible Important Pair of Acidic Residues in Vesicular Acetylcholine Transporter

et al. 2010

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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...

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Section: Methodsmentioning

confidence: 99%

Possible Important Pair of Acidic Residues in Vesicular Acetylcholine Transporter

et al. 2010

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“…Homogenate was prepared (22). Briefly, resuspended cells were broken in a Potter-Elvehjam homogenizer (three to six strokes) using a motor driven stirrer until 95% of them took up trypan blue as determined by microscopic examination.…”

Section: Methodsmentioning

confidence: 99%

“…Clones of recombinant vector (hVAChT/pcDNA 6.2/V5) were obtained in XL-1 Blue supercompetent Escherichia coli cells selected on ampicillin and amplified. Isolated hVAChT/pcDNA 6.2/V5 was transfected into PC12 A123.7 cells with lipofectamine in antibiotic-free medium (22). Twenty-four hours later, cells were passaged at different dilutions in different culture plates, and blasticidin selection agent was added at 10 μg of blasticidin/mL to each plate.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were trypsinized and harvested upon growth to confluency, washed with cold phosphate-buffered saline, and resuspended in homogenization buffer [0.32 M sucrose, 10 mM N-(2-hydroxyethyl)piperazine-N 0 -2-ethanesulfonic acid (HEPES), 1 mM dithiothreitol, adjusted to pH 7.4 with KOH, fresh 100 μM phenylmethanesulfonyl fluoride (Sigma, St. Louis, MO), 100 μM diethyl p-nitrophenyl phosphate (paraoxon), and complete protease inhibitor cocktail (Roche, Mannheim, Germany)]. Homogenate was prepared (22). Briefly, resuspended cells were broken in a Potter-Elvehjam homogenizer (three to six strokes) using a motor-driven stirrer until 95% of them took up trypan blue as determined by microscopic examination.…”

Section: Methodsmentioning

confidence: 99%

“…Western Blot Analysis and Selection of Clonal Line. Expression of hVAChT by each of the clones was monitored by Western blot (22). Postnuclear supernatant (150 μg) was diluted to 500 μL with homogenization buffer containing complete protease inhibitor cocktail and pelleted by ultracentrifugation at 171500g for 1.5 h at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

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

2009

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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...

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The Concise Guide toPHARMACOLOGY2013/14: Transporters

Alexander

Benson

Faccenda

et al. 2013

British J Pharmacology

146

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The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full.Transporters are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets.It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates.

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Equilibrium Binding and Transport by Vesicular Acetylcholine Transporter

Cited by 3 publications

References 8 publications

Possible Important Pair of Acidic Residues in Vesicular Acetylcholine Transporter

Possible Important Pair of Acidic Residues in Vesicular Acetylcholine Transporter

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

The Concise Guide toPHARMACOLOGY2013/14: Transporters

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Equilibrium Binding and Transport by Vesicular Acetylcholine Transporter

Cited by 3 publications

References 8 publications

Possible Important Pair of Acidic Residues in Vesicular Acetylcholine Transporter

Possible Important Pair of Acidic Residues in Vesicular Acetylcholine Transporter

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

The Concise Guide toPHARMACOLOGY2013/14: Transporters

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