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In vitro characterization of [³H]VAT in cells, animal and human brain tissues for vesicular acetylcholine transporter.

ABSTRACT: Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [¹⁸F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [³H]VAT, a tritiated counterpart of [¹⁸F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [³H]VAT with K_d = 6.5 nM and B_max = 22.89 pmol/mg protein. In the [³H]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [³H]VAT was observed using VAChT ligands, the K_i values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [³H]VAT autoradiography of rat brain sections showed strong signals in the striatum, moderate to high signals in vermis, thalamus, cortex, and hippocampus, and weak signals in cerebellum. Strong [³H]VAT ARG signals were also observed from striatal sections of normal nonhuman primates and human brains. Competitive ARG study with human striatal sections demonstrated strong ARG signals of [³H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (-)-vesamicol, but not by the σ₁ receptor ligand Yun-122. ARG study also indicated that signal in the striatal sections from PSP human brains was lower than normal human brains. These data provide solid evidence supporting [¹⁸F]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo.

SUBMITTER: Liang Q

PROVIDER: S-EPMC8605764 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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In vitro characterization of [3H]VAT in cells, animal and human brain tissues for vesicular acetylcholine transporter.

Liang Qianwa Q Joshi Sumit S Liu Hui H Yu Yanbo Y Zhao Haiyang H Benzinger Tammie L S TLS Perlmutter Joel S JS Tu Zhude Z

European journal of pharmacology 20211007

Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [18F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [3H]VAT, a tritiated counterpart of [18F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [3H]VA ...[more]

PMID: 34627806

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Project description: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) [Vardy, E., et al. (2004) Protein Sci. 13, 1832-1840]. 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 a pK of 6.5 for binding of vesamicol and with a pK of approximately 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, mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH >or=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 from D398 and the ACh binding site in three-dimensional space. Also, a probable network of interactions involving E309 and an unknown residue having a pK of 10 has been revealed.

Dataset Information

In vitro characterization of [³H]VAT in cells, animal and human brain tissues for vesicular acetylcholine transporter.

Publications

In vitro characterization of [<sup>3</sup>H]VAT in cells, animal and human brain tissues for vesicular acetylcholine transporter.

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