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G Rudnick

Publications and source records attributed to G Rudnick.

At least 19 recordsLinked to original sources
PubMed2006

Structure/function relationships in serotonin transporter: new insights from the structure of a bacterial transporter.

Serotonin transporter (SERT) serves the important function of taking up serotonin (5-HT) released during serotonergic neurotransmission. It is the target for important therapeutic drugs and psychostimulants. SERT catalyzes the influx of 5-HT together with Na+ and Cl- in a 1:1:1 stoichiometry. In the same catalytic cycle, there is coupled efflux of one K+ ion. SERT is one member of a large family of amino acid and amine transporters that is believed to utilize similar mechanisms of transport. A bacterial member of this family was recently crystallized, revealing the structural basis of these transporters. In light of the new structure, previous results with SERT have been re-interpreted, providing new insight into the substrate binding site, the permeation pathway, and the conformational changes that occur during the transport cycle.

Amino Acid Sequence↗
PubMed2003

Serotonin transporter missense mutation associated with a complex neuropsychiatric phenotype.

Two common serotonin transporter (SERT) untranslated region gene variants have been intensively studied, but remain inconclusively linked to depression and other neuropsychiatric disorders. We now report an uncommon coding region SERT mutation, Ile425Val, in two unrelated families with OCD and other serotonin-related disorders. Six of the seven family members with this mutation had OCD (n=5) or obsessive-compulsive personality disorder (n=1) and some also met diagnostic criteria for multiple other disorders (Asperger's syndrome, social phobia, anorexia nervosa, tic disorder and alcohol and other substance abuse/dependence). The four most clinically affected individuals--the two probands and their two slbs--had the I425V SERT gene gain-of-function mutation and were also homozygous for 5'-UTR SERT gene variant with greater transcriptional efficacy.

Amino Acid Sequence↗
PubMed2003

Functional consequences of homo- but not hetero-oligomerization between transporters for the biogenic amine neurotransmitters.

Before this study, the human norepinephrine transporter (hNET) was the only member of the biogenic amine neurotransmitter transporter family that had not been demonstrated to be a functional homo-oligomer. Here, using two forms of the transporter, I155C and hNET-myc, with distinct antigenicity and inhibitor sensitivity, we demonstrated that hNET exists as a homo-oligomer. hNET I155C is a functional mutant and is sensitive to inactivation by the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate, while hNET-myc is resistant to inactivation by this reagent. Coimmunoprecipitation of these two forms demonstrated that a physical interaction exists between norepinephrine transporter monomers. Further characterization of this physical interaction has revealed that the activity of norepinephrine transporters depends on interactions between monomers. Because norepinephrine transporters and serotonin transporters are the only two members of the neurotransmitter transporter family endogenously expressed in the cell membrane of the same cells, placental syncytiotrophoblasts, we tested the ability of norepinephrine transporters and serotonin transporters to associate and function in a hetero-oligomeric form. Similarly, coexpression of hNET-myc with serotonin transporter-FLAG showed a physical interaction in coimmunoprecipitation assays. However, coexpression of serotonin and norepinephrine transporters did not sensitize norepinephrine transporter activity to inhibition by citalopram, a selective serotonin transport inhibitor. Thus, the norepinephrine transporter-serotonin transporter physical association did not produce functional consequences. Based on this, we propose that the transporters for biogenic amine neurotransmitters interact functionally in homo- but not hetero-oligomeric forms.

Amino Acid Sequence↗
PubMed2001

A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter.

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained approximately 32% of wild type transport activity and approximately 56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na(+) and at 4 degrees C, their ability to protect Cys-357 required Na(+) and was prevented at 4 degrees C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K(+) ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity.

Animals↗
PubMed2001

A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109.

Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity. Replacing Na(+) with N-methyl-d-glucamine (NMDG(+)) did not reduce the affinity of cocaine for SERT, as measured by displacement of a high affinity cocaine analog, but replacement of Na(+) with Li(+) led to a 2-fold increase in the K(D) for cocaine. The addition of either cocaine or serotonin (5-HT) protected SERT against MTSET inactivation. When SERT was expressed in Xenopus oocytes, inward currents were elicited by superfusing the cell with 5-HT (in the presence of Na(+)) or by replacing Na(+) with Li(+) but not NMDG(+). MTSET treatment of oocytes in Li(+) but not in Na(+) decreased both 5-HT and Li(+) induced currents, although 5-HT-induced currents were inhibited to a greater extent. Na(+) antagonized the effects of Li(+) on both inactivation and current. These results are consistent with Li(+) inducing a conformational change that exposes Cys-109, decreases cocaine affinity, and increases the uncoupled inward current.

Animals↗
PubMed2001

The NH(2)-terminus of norepinephrine transporter contains a basolateral localization signal for epithelial cells.

When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity. To identify possible localization signals responsible for this difference, DAT-NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH(2)-terminal regions. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH(2)-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH(2)-terminal region. However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane.

Amino Acid Sequence↗
PubMed2000

Functional role of critical stripe residues in transmembrane span 7 of the serotonin transporter. Effects of Na+, Li+, and methanethiosulfonate reagents.

Mutations at critical residue positions in transmembrane span 7 (TM7) of the serotonin transporter affect the Na(+) dependence of transport. It was possible that these residues, which form a stripe along one side of the predicted alpha-helix, formed part of a water-filled pore for Na(+). We tested whether cysteine substitutions in TM7 were accessible to hydrophilic, membrane-impermeant methanethiosulfonate (MTS) reagents. Although all five cysteine-containing mutants tested were sensitive to these reagents, noncysteine control mutants at the same positions were in most cases equally sensitive. In all cases, MTS sensitivity could be traced to changes in accessibility of a native cysteine residue in extracellular loop 1, Cys-109. Moreover, none of the TM7 cysteines reacted with the biotinylating reagent MTSEA-biotin when tested in the C109A background. It is thus unlikely that the critical stripe forms part of a water-filled pore. Instead, studies of the ion dependence of the reaction between Cys-109 and MTS reagents lead to the conclusion that TM7 is involved in propagating conformational changes caused by ion binding, perhaps as part of the translocation mechanism. The critical stripe residues on TM7 probably represent a close contact region between TM7 and one or more other TMs in the transporter's three-dimensional structure.

Carrier Proteins↗
PubMed2000

Evidence for a Black Hole and Accretion Disk in the LINER NGC 4203.

We present spectroscopic observations from the Hubble Space Telescope that reveal for the first time the presence of a broad pedestal of Balmer line emission in the LINER galaxy NGC 4203. The emission-line profile is suggestive of a relativistic accretion disk and is reminiscent of double-peaked transient Balmer emission observed in a handful of other LINERs. The very broad line emission thus constitutes clear qualitative evidence for a black hole, and spatially resolved narrow-line emission in NGC 4203 can be used to constrain its mass, MBH</=6x106 M middle dot in circle, at 99.7% confidence. This value implies a ratio of black hole mass to bulge mass of less, similar7x10-4 in NGC 4203, which is less by a factor of approximately 3-9 than the mean ratio obtained for other galaxies. The availability of an independent constraint on central black hole mass makes NGC 4203 an important testbed for probing the physics of weak active galactic nuclei. Assuming MBH near the detection limit, the ratio of the observed luminosity to the Eddington luminosity is approximately 10-4. This value is consistent with advection-dominated accretion and hence with scenarios in which an ion torus irradiates an outer accretion disk that produces the observed double-peaked line emission. Follow-up observations will make it possible to improve the black hole mass estimate and study variability in the nuclear emission.

Journal Article↗
PubMed2000

Oligomerization of serotonin transporter and its functional consequences.

Two forms of serotonin transporter (SERT) were prepared with different epitope tags. When co-expressed in HeLa cells, the form containing a FLAG tag (Res-FLAG) was associated with the form containing a c-myc tag (Sens-myc). Antibody against c-myc precipitated Res-FLAG from detergent extracts of cells expressing both forms, but not when Res-FLAG was expressed alone. The specificity of the interaction was demonstrated by the observation that anti-myc antibodies did not precipitate the unrelated vesicular stomatitis virus coat glycoprotein when it was co-expressed with Sens-myc. Sens-myc contained a reactive cysteine at position 172, which reacted with both (2-aminoethyl)methanethiosulfonate and N-biotinylaminoethyl methanethiosulfonate on the surface of intact cells. Sens-myc, but not Res-FLAG, was inactivated by these reagents. When co-expressed with Sens-myc, functionally active Res-FLAG was precipitated by immobilized streptavidin from digitonin-solubilized cells that had been treated with N-biotinylaminoethyl methanethiosulfonate. In cells co-expressing mixtures of Sens-myc and Res-FLAG, the amount of inactivation by (2-aminoethyl)methanethiosulfonate was less than expected if the two forms were independent. The results are consistent with a dimeric form of SERT with functional interactions between subunits, and with association of dimers into a higher order complex, possibly a tetramer.

Biopolymers↗
PubMed2000

Permeation and gating residues in serotonin transporter.

The third transmembrane domain (TM3) of serotonin transporter (SERT) contains two isoleucine residues previously proposed to be involved in binding and transport of serotonin. When Ile-172 was replaced with cysteine, SERT became sensitive to inactivation by externally added [2-(trimethylammonium)ethyl]methanethio-sulfonate (MTSET). The disulfide product of this inactivation was not sensitive to reduction by externally added sulfhydryl compounds, but apparently reacted with intracellular reducing agents to spontaneously regenerate active SERT. The apparent accessibility of this residue to both external and cytoplasmic reagents is consistent with its localization near a serotonin binding site that is alternately exposed to both internal and external media. In another SERT mutant, I179C, transport also was inactivated by MTSET but substrate binding was resistant. External substrate bound to the inactivated I179C and enhanced its reactivation by free thiols. In norepinephrine transporter (NET), cysteine replacement of Ile-155 (corresponding to SERT Ile-179) also rendered the transporter sensitive to MTSET inactivation. In NET I155C, cocaine enhanced this inactivation, and the substrate, dopamine, apparently protected against inactivation. The characteristics of this protection suggest that dopamine was transported, converting NET to a form in which Ile-155 was occluded. The results support the proposal that TM3 of SERT and NET constitute part of the substrate permeation pathway, and that Ile-172 in SERT resides close to the substrate binding site. They also suggest that Ile-179 in SERT (and Ile-155 in NET) is in a conformationally sensitive part of TM3, which may act as part of an external gate.

Allosteric Regulation↗
PubMed1999

The role of external loop regions in serotonin transport. Loop scanning mutagenesis of the serotonin transporter external domain.

Chimeric transporters were constructed in which the predicted external loops of the serotonin transporter (SERT) were replaced one at a time with a corresponding sequence from the norepinephrine transporter (NET). All of the chimeric transporters were expressed at levels equal to or greater than those of wild type SERT, but the transport and binding activity of the mutants varied greatly. In particular, mutants in which the NET sequence replaced external loops 4 or 6 of SERT had transport activity 5% or less than that of wild type, and the loop 5 replacement was essentially inactive. In some of these mutants, binding of a high affinity cocaine analog was less affected than transport, suggesting that the mutation had less effect on the initial binding steps in transport than on subsequent conformational changes. The more severely affected mutants also displayed an altered response to Na(+). In contrast to the dramatic reduction in transport and binding, the specificity of ligand binding was essentially unchanged. Chimeric transporters did not gain affinity for dopamine, a NET substrate, or desipramine, an inhibitor, at the expense of affinity for serotonin or paroxetine, a selective SERT inhibitor. The results suggest that external loops are not the primary determinants of substrate and inhibitor binding sites. However, they are not merely passive structures connecting transmembrane segments but rather active elements responsible for maintaining the stability and conformational flexibility of the transporter.

Base Sequence↗
PubMed1999

Molecular cloning, expression and characterization of a bovine serotonin transporter.

The serotonin transporter (SERT) is a member of a highly homologous family of sodium/chloride dependent neurotransmitter transporters responsible for reuptake of biogenic amines from the extracellular fluid. SERT constitutes the pharmacological target of several clinically important antidepressants. Here we report the molecular cloning of SERT from the bovine species. Translation of the nucleotide sequence revealed 44 amino acid differences compared to human SERT. When transiently expressed in HeLa cells and compared with rat and human SERTs the K(m) value for uptake was increased 2-fold. V(max) and B(max) were both increased about 4-fold indicating the turnover number is conserved. The pharmacological profile revealed a decreased sensitivity towards imipramine, desipramine, citalopram, fluoxetine and paroxetine compared with human SERT, while the sensitivity towards 3, 4-methylenedioxymethamphetamine (MDMA) was mainly unchanged. RT-PCR amplification of RNA from different tissues demonstrated expression of SERT in placenta, brain stem, bone marrow, kidney, lung, heart, adrenal gland, liver, parathyroid gland, thyroid gland, small intestine and pancreas.

Amino Acid Sequence↗
PubMed1998

Critical amino acid residues in transmembrane span 7 of the serotonin transporter identified by random mutagenesis.

Transmembrane span 7 of the rat brain serotonin transporter was subjected to random mutagenesis. Of the 27 amino acid residues mutated, six were identified as functionally important by their sensitivity to nonconservative mutations. These residues were Asn-368 and Tyr-385, where substitutions that retained hydrogen-bonding ability were preferred; Gly-376 and Gly-384, where only glycine was accepted; Phe-380, where a phenyl ring was preferred; and Met-386, where hydrophobic substitutions were preferred. Mutations that did not preserve these structural characteristics were highly detrimental to serotonin transport activity. These six residues form a stripe that runs at an angle down the side of the putative alpha-helix, lending support to this structural prediction. Mutations at some of these positions also specifically impaired transport activity under low Na+ conditions. Other mutations at nearby positions in transmembrane span 7 also impaired activity in low Na+, although the activity of the mutants in high Na+ was similar to wild type. These results suggest that at least some of the six critical residues play a role in Na+ binding or perhaps in the coupling of Na+ binding to later steps in the transport cycle. These residues may be important in other aspects of the transporter's function as well.

Amino Acid Sequence↗
PubMed1998

Determination of external loop topology in the serotonin transporter by site-directed chemical labeling.

The transmembrane topology of the serotonin transporter (SERT) has been examined by measuring the reactivity of selected lysine and cysteine residues with extracellular reagents. An impermeant biotinylating reagent, sulfosuccinimidyl 2-(biotinamido)ethyl-1, 3-dithiopropionate (NHS-SS-biotin), was shown to label SERT transiently expressed in cultured cells. Replacement of four lysine residues that were predicted to lie in external hydrophilic loops (eK-less) largely prevented the biotinylation reaction. Likewise, the cysteine-specific biotinylation reagent N-biotinylaminoethylmethanethiosulfonate (MTSEA-biotin) labeled wild type SERT but not a mutant in which Cys-109, predicted to lie in the first external loop, was replaced with alanine. These two mutant transporters reacted with the biotinylating reagents in digitonin-permeabilized cells, demonstrating that the abundant lysine and cysteine residues predicted to lie in intracellular hydrophilic domains were reactive but not accessible in intact cells. Mutants containing a single external lysine at positions 111, 194, 243, 319, 399, 490, and 571 reacted more readily with NHS-SS-biotin than did the eK-less mutant. Similarly, mutants with a single cysteine at positions 109, 310, 406, 489, and 564 reacted more readily with MTSEA-biotin than did the C109A mutant. All of these mutants were active and therefore likely to be folded correctly. These results support the original transmembrane topology and argue against an alternative topology proposed recently for the related glycine and gamma-aminobutyric acid transporters.

Biotin↗
PubMed1998

Bioenergetics of neurotransmitter transport.

Neurotransmitter transporters are essential components in the recycling of neurotransmitters released during neuronal activity. These transporters are the targets for important drugs affecting mood and behavior. They fall into at least four gene families, two encoding proteins in the plasma membrane and two in the synaptic vesicle membrane, although the known vesicular transporters have not all been cloned. Each of these transporters works by coupling the downhill movement of small ions such as Na+, Cl-, K+, and H+ to the uphill transport of neurotransmitter. Plasma membrane transporters move the transmitter into the cytoplasm by cotransport with Na+. Many transporters also couple Cl- cotransport to transmitter influx and these all belong to the NaCl-coupled family, although within the family the coupling stoichiometry can vary. Transporters for glutamate couple influx of this excitatory amino acid to Na+ and H+ influx and K+ efflux. Transporters in synaptic vesicles couple H+ efflux to neurotransmitter transport from the cytoplasm to the vesicle lumen.

Animals↗
PubMed1997

The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding.

Twenty residues in the third transmembrane domain of the serotonin transporter (SERT) were mutated, one at a time, to cysteine. Almost all of these mutants were fully active for serotonin (5-HT) transport and insensitive to inactivation by the positively charged cysteine reagent [2-(trimethylammonium)ethyl]methanethiosul-fonate (MTSET). Two active mutants, I172C and I179C, were sensitive to rapid inactivation by MTSET but were relatively insensitive to the negatively charged reagent (2-sulfonatoethyl)methanethiosulfonate (MTSES). Inactivation of I172C was blocked by 5-HT and cocaine, but I179C was not similarly protected. Replacement of Tyr-175 with cysteine resulted in a mutant with low transport activity, and, at the neighboring Tyr-176, cysteine replacement completely blocked transport. The Y175C and Y176C mutants were expressed on the cell surface at levels 84% and 69%, respectively, that of wild type (C109A) SERT. Mutants Y175C and Y176C had lower cocaine affinity than C109A, as measured by displacement of the high affinity cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT). For Y176C, 5-HT affinity also was decreased. MTSET inactivated beta-CIT binding to I172C and Y176C, but only slightly inhibited binding to I179C and C109A. The MTSET sensitivity of cysteine replacements at positions 172, 176, and 179 was not observed when these positions were replaced with alanine, serine, or methionine. The results suggest that Ile-172, Tyr-176 and Ile-179 are on one face of an alpha-helical transmembrane element, and that Ile-172 and Tyr-176 are in proximity to the binding site for 5-HT and cocaine.

Asparagine↗