Mutational Analysis of Aspartate Residues in the Transmembrane Regions and Cytoplasmic Loops of Rat Vesicular Acetylcholine Transporter

Kim, Myung‐Hee; Lu, Mengmeng; Lim, E.-J.; Chai, Young-Gyu; Hersh, Louis B.

doi:10.1074/jbc.274.2.673

Cited by 43 publications

(78 citation statements)

References 40 publications

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“…The results (Fig. 5 C and D) confirm previous observations for rVMAT1, rVMAT2, and VAChT (17,18,25) showing that position 427 does not tolerate even conservative replacements. In all cases, no significant transport activity into vesicles was measured.…”

Section: Identification Of a Hydrogen Bond Cluster Linking The N-and supporting

confidence: 81%

“…2 and 4A). D400 has been the subject of intensive research in all members of the vesicular amine transporter (SLC18) family (17,18,25). In all cases, a carboxyl group at this position was found to be crucial for proper transport activity.…”

Section: Discussionmentioning

confidence: 99%

“…Previous work suggested that D427 from TM11 interacts with K139 from TM2 via an ion pair or salt bridge (17). Position 427 (and equivalents in rVMAT1 and rVAChT) was identified as essential for transport activity, and even the most conservative replacement to glutamate dramatically reduced transport activity (17,18,25). Interestingly, however, the rVMAT1 mutant retains the ability to bind the competitive inhibitor reserpine, a process that is accelerated by the proton gradient and involves transport of one of the two H + ions exchanged with the transported substrates (18,26).…”

Section: Identification Of a Hydrogen Bond Cluster Linking The N-and mentioning

confidence: 99%

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Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Yaffe

Radestock

Shuster

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Vesicular monoamine transporter 2 (VMAT2) catalyzes transport of monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily (MFS) of secondary transporters. Here we present a homology model of VMAT2, which has the standard MFS fold, that is, with two domains of six transmembrane helices each which are related by twofold pseudosymmetry and whose axis runs normal to the membrane and between the two halves. Demonstration of the essential role of a membraneembedded glutamate and confirmation of the existence of a hydrogen bond probably involved in proton transport provide experimental evidence that validates some of the predictions inherent to the model. Moreover, we show the essential role of residues at two anchor points between the two bundles. These residues appear to function as molecular hinge points about which the two six transmembrane-helix bundles flex and straighten to open and close the pathways on either side of the membrane as required for transport. Polar residues that create a hydrogen bond cluster form one of the anchor points of VMAT2. The other results from hydrophobic interactions. Residues at the anchor points are strongly conserved in other MFS transporters in one way or another, suggesting that interactions at these locations will be critical in most, if not all, MFS transporters.ion coupling | multidrug resistance | membrane proteins | neurotransmitter transporter | homology modeling T ransport and storage of neurotransmitters in synaptic vesicles allow their regulated release from the presynaptic cell into the synaptic cleft. The neurotransmitter molecules are accumulated in synaptic vesicles by vesicular neurotransmitter transporters (1-3). Transport of monoamines (serotonin, dopamine, histamine, adrenaline, and noradrenaline) is carried out by the vesicular monoamine transporter (VMAT) family, which includes two isoforms, VMAT1 and VMAT2, in a process that involves the exchange of two protons for one substrate molecule (1-3). The proton electrochemical gradient necessary for transport is generated by the vesicular H + -ATPase (V-ATPase). The structural basis for the function of VMAT remains unknown. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily (MFS) of secondary transporters. Most MFS transporters contain 12 transmembrane (TM) helices, and crystal structures revealed that the 12 TM helices are arranged in two domains of six TMs each, which are related by a twofold pseudosymmetry with an axis that runs normal to the membrane and between the two halves (4-9). Furthermore, analysis of the lactose permease (LacY) crystal structure revealed the presence of inverted topology repeat units within each of the domains (10). That is, the first three helices of each domain are structurally related to the second three helices of that domain by a twofold pseudosymmetry ax...

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Section: Identification Of a Hydrogen Bond Cluster Linking The N-and supporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Identification Of a Hydrogen Bond Cluster Linking The N-and mentioning

confidence: 99%

See 1 more Smart Citation

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Yaffe

Radestock

Shuster

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Sequence conservation of charged and polar amino acids within the putative hydrophobic transmembrane domains of VAChT suggests functional importance. In a previous report (5), Asp-398 in TM10 and Asp-425 in TM11 were shown to possess important roles in acetylcholine transport.…”

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confidence: 99%

“…This compound acts in a noncompetitive manner; however ACh blocks vesamicol binding to the transporter. Recent mutagenesis studies have separated the vesamicol binding site from the ACh binding site (5).…”

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confidence: 99%

Mutational Analysis of Basic Residues in the Rat Vesicular Acetylcholine Transporter

Kim

Kelly

et al. 2000

Journal of Biological Chemistry

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Chemically distinct preganglionic inputs to iris-projecting postganglionic neurons in the rat: A light and electron microscopic study

et al. 1999

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Individual autonomic postganglionic neurons are surrounded by pericellular baskets of preganglionic terminals that are easily identifiable with the light microscope. It has been assumed that the target cell of a pericellular basket of preganglionic terminals is the neuron at the centre of the basket. This assumption has enabled the connectivity of preganglionic neurons to be determined at the light microscopic level. However, if the preganglionic terminals in a pericellular basket make synapses with the dendrites of nearby, but functionally different, postganglionic neurons, then the conclusions of light microscopic studies are far less certain. We have used a serial section ultrastructural study to determine the target of the preganglionic pericellular basket in a situation where the apparent target cell is surrounded by neurons of dissimilar function. In the rat superior cervical ganglion, postganglionic neurons projecting to the iris were identified, using retrograde tracers, as single neurons (i.e., not in clusters). We have used immunohistochemistry to show that iris‐projecting neurons are surrounded by preganglionic nerve terminals containing calcitonin gene‐related peptide (CGRP). We have demonstrated that the pericellular basket of CGRP‐immunoreactive preganglionic terminals provides inputs only to the soma at the centre of the basket and not to the dendrites of surrounding neurons. This suggests that, in autonomic ganglia, light microscopic identification of the preganglionic terminal baskets is likely to be a reliable method for identifying the targets of subclasses of preganglionic neurons. J. Comp. Neurol. 412:606–616, 1999. © 1999 Wiley‐Liss, Inc.

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Mutational Analysis of Aspartate Residues in the Transmembrane Regions and Cytoplasmic Loops of Rat Vesicular Acetylcholine Transporter

Cited by 43 publications

References 40 publications

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

Mutational Analysis of Basic Residues in the Rat Vesicular Acetylcholine Transporter

Chemically distinct preganglionic inputs to iris-projecting postganglionic neurons in the rat: A light and electron microscopic study

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