Publications

• Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis.

  Yaffe D., Vergara-Jaque A., Forrest L.R., Schuldiner S.

  Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and stor ... >> More

  Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H⁺ per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders. During the transport cycle, VMAT is expected to occupy at least three different conformations: cytoplasm-facing, occluded, and lumen-facing. The lumen-to cytoplasm-facing transition, facilitated by protonation of at least one of the essential membrane-embedded carboxyls, generates a binding site for reserpine. Here we have identified residues in the cytoplasmic gate and show that mutations that disrupt the interactions in this gate also shift the equilibrium toward the cytoplasm-facing conformation, emulating the effect of protonation. These experiments provide significant insight into the role of proton translocation in the conformational dynamics of a mammalian H⁺-coupled antiporter, and also identify key aspects of the mode of action and binding of two potent inhibitors of VMAT2: reserpine binds the cytoplasm-facing conformation, and tetrabenazine binds the lumen-facing conformation. << Less

  Proc Natl Acad Sci U S A 113:E7390-E7398(2016) [PubMed] [EuropePMC]

• Proton: substrate stoichiometries during active transport of biogenic amines in chromaffin ghosts.

  Johnson R.G., Carty S.E., Scarpa A.

  The relationship between the electrochemical proton gradient (delta mu-H+) and the electrochemical gradient for biogenic amines (delta mu-A) was investigated in isolated chromaffin ghosts free of endogenous components and gradients. The addition of ATP to a ghost suspension resulted in the generat ... >> More

  The relationship between the electrochemical proton gradient (delta mu-H+) and the electrochemical gradient for biogenic amines (delta mu-A) was investigated in isolated chromaffin ghosts free of endogenous components and gradients. The addition of ATP to a ghost suspension resulted in the generation of a large proton concentration gradient (delta pH), acidic inside (measured by [14C]-methylamine distribution), and a large proton electrical gradient (delta psi), positive inside (measured by [14C]-thiocyanate distribution). In the presence of this large electrochemical proton gradient, the accumulation of [14C]5-hydroxytryptamine (serotonin) and other biogenic amines rapidly reached an apparent steady state level. Collapse of the proton gradients after steady state levels were achieved resulted in the efflux of the accumulated amines. Uptake in the presence of a delta psi alone produced an amine gradient equal to the magnitude of the delta psi, while in the presence of a delta pH alone biogenic amine distribution was equal to twice the magnitude of the delta pH. Using additions of ammonia or thiocyanate, it was possible to vary the magnitude of the electrochemical proton gradient over a wide range of values; the driving force for amine accumulation under these conditions was found to be equal to delta psi--2Z delta pH (where Z = 2.3 RT/F). The results, which provide unequivocal evidence for the primary role of the electrochemical proton gradient in the active transport of biogenic amines, are consistent with a model based on the chemiosmotic hypothesis, of an obligatory coupling of amine influx and proton efflux, mediated via a putative reserpine-sensitive translocator. Measurement of the stoichiometry of this coupling of H+ and amines permits conclusions concerning the molecular mechanism of amino accumulation. << Less

  J Biol Chem 256:5773-5780(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and functional expression of a tetrabenazine sensitive vesicular monoamine transporter from bovine chromaffin granules.

  Howell M.L., Shirvan A., Stern-Bach Y., Steiner-Mordach S., Dean G.E., Schuldiner S.

  Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b-VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal ... >> More

  Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b-VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N-terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin > dopamine = norepinephrine > epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N-terminus sequence of the purified vesicular amine transporter [Stern-Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730-9733]. << Less

  FEBS Lett. 338:16-22(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression cloning of a reserpine-sensitive vesicular monoamine transporter.

  Erickson J.D., Eiden L.E., Hoffman B.J.

  A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells ... >> More

  A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules. << Less

  Proc. Natl. Acad. Sci. U.S.A. 89:10993-10997(1992) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Functional genetic variants in the vesicular monoamine transporter 1 modulate emotion processing.

  Lohoff F.W., Hodge R., Narasimhan S., Nall A., Ferraro T.N., Mickey B.J., Heitzeg M.M., Langenecker S.A., Zubieta J.K., Bogdan R., Nikolova Y.S., Drabant E., Hariri A.R., Bevilacqua L., Goldman D., Doyle G.A.

  Emotional behavior is in part heritable and often disrupted in psychopathology. Identification of specific genetic variants that drive this heritability may provide important new insight into molecular and neurobiological mechanisms involved in emotionality. Our results demonstrate that the presyn ... >> More

  Emotional behavior is in part heritable and often disrupted in psychopathology. Identification of specific genetic variants that drive this heritability may provide important new insight into molecular and neurobiological mechanisms involved in emotionality. Our results demonstrate that the presynaptic vesicular monoamine transporter 1 (VMAT1) Thr136Ile (rs1390938) polymorphism is functional in vitro, with the Ile allele leading to increased monoamine transport into presynaptic vesicles. Moreover, we show that the Thr136Ile variant predicts differential responses in emotional brain circuits consistent with its effects in vitro. Lastly, deep sequencing of bipolar disorder (BPD) patients and controls identified several rare novel VMAT1 variants. The variant Phe84Ser was only present in individuals with BPD and leads to marked increase monoamine transport in vitro. Taken together, our data show that VMAT1 polymorphisms influence monoamine signaling, the functional response of emotional brain circuits and risk for psychopathology. << Less

  Mol. Psychiatry 19:129-139(2014) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Identification of a mammalian vesicular polyamine transporter.

  Hiasa M., Miyaji T., Haruna Y., Takeuchi T., Harada Y., Moriyama S., Yamamoto A., Omote H., Moriyama Y.

  Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon dep ... >> More

  Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H(+). SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT). << Less

  Sci. Rep. 4:6836-6836(2014) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.