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G Rudnick

Publications and source records attributed to G Rudnick.

At least 37 records · Page 2Linked to original sources
PubMed1997

An extracellular loop region of the serotonin transporter may be involved in the translocation mechanism.

The serotonin transporter (SERT) is a member of a highly homologous family of proteins responsible for the reuptake of biogenic amines from the synaptic cleft. We took advantage of native restriction sites in SERT to construct a chimeric transporter containing a small (34 amino acid) region of the norepinephrine transporter. The substituted region corresponds to about half of the largest extracellular loop. This chimera transports serotonin very slowly compared to wild type SERT. However, it binds serotonin and the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane with a high affinity indistinguishable from wild type. It has the same specificity as wild type SERT for the antidepressants paroxetine and desipramine. The low rate of transport does not appear to be due to poor expression, since the chimeric transporter is expressed at the membrane surface at close to wild type levels as measured by cell surface biotinylation. These observations lead us to conclude that, rather than playing a role in substrate or drug binding, this region of the large extracellular loop may be involved in the conformational changes associated with substrate translocation into the cell.

Amino Acid Sequence↗
PubMed1997

External cysteine residues in the serotonin transporter.

Hydropathy analysis predicts three cysteines (C109, C200, and C209) in extracellular loops of the rat serotonin transporter (SERT). We mutated these residues, singly and in combination, to either alanine or serine and expressed the mutant transporters in HeLa cells using the vaccinia-T7 transient expression system. Mutation of C109 to alanine had no effect on transport activity or surface expression of the transporter. In Na+-containing solutions, methanethiosulfonate (MTS) reagents had little effect on transport activity in the wild type or in the C109A mutant. When Na+ was replaced with Li+, inactivation of wild type by MTS reagents increased dramatically, but C109A was still resistant. The results suggest that C109 is exposed to the external medium in a manner dependent on cation binding. Replacing either C200 or C209 with serine resulted in either a partial (C200S) or almost total (C209S) loss of transport activity. MTS reagents rapidly inactivated transport activity in mutant C200S, suggesting increased accessibility of a previously unreactive cysteine residue. The double mutants C200S-C109A and C200S-C209S each retained partial activity. C200S-C109A was very sensitive to MTS reagents, but the C200S-C209S mutant was much less sensitive, similar to the wild type transporter. Replacement of C200 or C209 with serine dramatically decreased surface expression of the fully glycosylated transporter. Expression was normal, however, in the C200S-C209S double mutant. The Na+ dependence of transport and ligand binding was abnormal in both C200S and C200S-C209S mutants. Replacing C200 or C209 had similar effects on Na+ dependence and surface expression. Together with the increased MTS reactivity of C200S, these results support the possibility that C200 and C209 may be linked by a disulfide bond in the second external loop of SERT.

Animals↗
PubMed1997

Placental biogenic amine transporters: cloning and expression.

During intrauterine development, catecholamine turnover (production and clearance rates) is higher than under any other circumstances. This is mediated in large part by placental clearance of circulating catecholamines via a cocaine-sensitive, neuronal transporter-dependent mechanism. In order to confirm the molecular mechanisms for placental transport, we screened an ovine placental cDNA library for biogenic amine transporters. We report here the identification of two biogenic amine transporters with sequences very similar to their neuronal counterparts. One is an ovine serotonin transporter (oSERT) with > 90% homology to the human neuronal SERT. Expression studies confirm transport and competitive binding affinities consistent with a SERT transporter. We have also isolated a partial sequence for the ovine norepinephrine transporter (oNET). These results confirm the placental expression of plasma membrane biogenic amine transporters. We suggest the exaggerated fetal vulnerability to uptake inhibitors, like cocaine, may be due to blockade of placental biogenic amine transport.

Amino Acid Sequence↗
PubMed1996

Cell-specific sorting of biogenic amine transporters expressed in epithelial cells.

We have utilized polarized epithelial cells stably expressing neurotransmitter transporters to analyze the sorting behavior of these membrane proteins. The transporters for serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are expected to be present in situ in the most distal extremities of axonal membranes, where they terminate the action of their biogenic amine substrates. Both Madin-Darby canine kidney (MDCK) and LLC-PK1 cells were stably transfected with cDNAs encoding either the rat 5-HT transporter (SERT), the human NE transporter (NET), or the rat or human DA transporter (DAT). These cells were grown on permeable filter supports, and the transporters were localized by three independent techniques. Confocal immunofluorescence microscopy indicated that each of the transporters expressed in LLC-PK1 cells was sorted to the basolateral membrane, co-localizing with the Na+/K+-ATPase. In MDCK cells, however, DAT was located primarily on the apical surface, while SERT and NET were found on the basolateral membranes. Cell surface biotinylation using an impermeant biotinylating reagent confirmed the immunocytochemistry results. Thus, SERT and NET in MDCK cells were labeled more efficiently from the basolateral medium than the apical medium, and DAT in MDCK cells was labeled more efficiently from the apical side than the basolateral side. Transport measurements in transfected MDCK cells agreed with the immunocytochemistry and biotinylation results. These results suggest the existence of cell-specific mechanisms that discriminate between neurotransmitter transporters for surface expression and render unlikely any simple hypothesis that sorting mechanisms in neurons and epithelia are identical.

Animals↗
PubMed1996

Ion coupling stoichiometry for the norepinephrine transporter in membrane vesicles from stably transfected cells.

We prepared membrane vesicles from stable LLC-PK1 cells expressing serotonin (5-HT) gamma-aminobutyric acid (GABA) and norepinephrine (NE) transporters (SERT, GAT-1, and NET). These vesicles accumulate transport substrates when the appropriate transmembrane ion gradients are imposed. For NET, accumulation of [3H]dopamine (DA) was stimulated by imposition of Na+ and Cl- gradients (out > in) and of a K+ gradient (in > out). The presence of Na+ or Cl-, even in the absence of a gradient, stimulated DA accumulation by NET, but K+ had little or no effect in the absence of a K+ gradient. Stimulation by a K+ gradient was markedly enhanced by increasing the K+ permeability with valinomycin, suggesting that net positive charge is transported together with DA. Cationic DA is likely to be the major substrate for NET, since varying pH did not affect Km. We estimated the Na+:DA stoichiometry by measuring the effect of the transmembrane Na+ gradient on peak DA accumulation. The results suggest a 1:1 cotransport of Na+ with DA. Taken together, the results suggest that NET catalyzes cotransport of one cationic substrate molecule with one Na+ ion, and one Cl- ion, and that K+ does not participate directly in the transport process.

Animals↗
PubMed1996

Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons.

At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations.

Amino Acid Sequence↗
PubMed1995

Biogenic amine flux mediated by cloned transporters stably expressed in cultured cell lines: amphetamine specificity for inhibition and efflux.

LLC-PK1 cells have been stably transfected with cDNAs encoding the human norepinephrine transporter (NET), rat dopamine transporter (DAT), and rat serotonin transporter. Using these cell lines, the specificity of each transporter toward agents that inhibit substrate influx and stimulate substrate efflux across the plasma membrane was examined. With 1-methyl-4-phenylpyridinium as a substrate for DAT and NET and serotonin as a substrate for the serotonin transporter, each transporter demonstrated a distinct pattern of inhibition by a panel of amphetamine derivatives and analogs, including amphetamine, methamphetamine (also known as "ecstasy"), p-chloroamphetamine, 3,4-methylenedioxymethamphetamine, methylphenidate (ritalin), and 5-methoxy-6-methyl-2-aminoindan. For each cell line expressing a single biogenic amine transporter, efflux of the accumulated substrate was stimulated by amphetamine derivatives, and this efflux was blocked by mazindol, an inhibitor of all three transporters. Of the amphetamine derivatives tested, some caused efflux at concentrations similar to those that inhibited transport. Other derivatives were much less effective at stimulating efflux than at inhibiting uptake. Methylphenidate caused little or no efflux, although it blocked uptake mediated by both NET and DAT. Other inhibitors of transport, such as cocaine, mazindol, citalopram, and nisoxetine, failed to stimulate efflux from these cells at concentrations that inhibited influx. The results suggest that potency toward individual plasma membrane biogenic amine transporters and the ability to release accumulated amine substrates are independent properties of each amphetamine derivative.

1-Methyl-4-phenylpyridinium↗
PubMed1994

Ligand binding to the serotonin transporter: equilibria, kinetics, and ion dependence.

The effects of Na+ and Cl- on the binding of [3H]imipramine and the cocaine analog [125I]-beta-carbomethoxy-3 beta-(4-iodophenyl)tropane([125I]-beta-CIT) to the human platelet serotonin transporter have been measured. The ion dependence of beta-CIT binding is consistent with binding beta-CIT together with one Na+ ion, but not in an ordered sequence. Imipramine affinity, like beta-CIT affinity, is increased by Na+, but imipramine binding involves at least two Na+ ions. This conclusion is based on the observation that both imipramine association rate constants and equilibrium affinity constants show a sigmoidal Na+ dependence. As with beta-CIT, the imipramine and Na+ binding sequence is not strictly ordered. Cl- increases imipramine affinity, apparently by slowing dissociation. beta-CIT binding occurs even in the absence of Na+ and Cl-. This provided a means to measure substrate and inhibitor affinity in both the presence and absence of cotransported ions. Nontransported inhibitors, such as imipramine and citalopram, as well as the transport substrates serotonin and 3,4-(methylenedioxy)methamphetamine all displaced beta-CIT binding in the absence of NaCl. In the absence of Cl-, Na+ increased the affinity of nontransported inhibitors but not of substrates. The results suggest that Na+ and Cl- induce independent changes in the transporter binding site and that binding of substrates and inhibitors is affected differently by these changes.

Blood Platelets↗
PubMed1994

Human norepinephrine transporter. Biosynthetic studies using a site-directed polyclonal antibody.

Antibodies have been raised against synthetic peptides derived from the predicted primary sequence of the human cocaine- and antidepressant-sensitive norepinephrine (NE) transporter (NET). One antibody (N430), raised and purified against a putative intracellular human norepinephrine transporter (hNET) epitope, detects hNET expression in a stably transfected cell line (LLC-NET) by indirect immunofluorescence only in the presence of detergent, while no immunoreactivity is observed in either the parental cells (LLC-PK1) or in LLC-NET cells incubated with preimmune sera or peptide absorbed antibody. N430 immunoblots of LLC-NET cell extracts reveal two major immunoreactive hNET species in these cells, migrating at 80 and 54 kDa, respectively. Pulse-chase N430 immunoprecipitation studies confirm that the 54-kDa species is a transient, glycosylated intermediate of a longer lived, more highly glycosylated protein with an apparent M(r) of 80,000. In contrast, a 54-kDa species is the primary hNET product in vaccinia virus T7-infected HeLa cells, transiently transfected with hNET cDNA. PNGase F digestion of extracts prepared from LLC-NET- and hNET-transfected HeLa cells convert all immunoreactive species to a 46-kDa form, equivalent to that observed following incubation of whole cells with the glycosylation inhibitor tunicamycin. As transiently transfected HeLa and stable LLC-NET cells exhibit a pharmacologically similar NE transport activity, it appears likely that the additional glycosylation evident in the stable line does not contribute significantly to antagonist sensitivity. On the other hand, NE transport and antagonist ([125I]RTI-55) binding assays on whole LLC-NET cells treated with tunicamycin reveal a pronounced reduction in NE transport activity and hNET membrane density paralleled by an inability of NET proteins to replenish the higher M(r) hNET pool. These findings suggest an obligate role for N-linked glycosylation in hNET biosynthetic maturation, stability, and functional expression. In summary, N430 antibody is a useful tool for the visualization and characterization of hNET gene products and has permitted the first direct evaluation of biosynthetic steps leading to functional catecholamine transporter expression.

Animals↗
PubMed1994

Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence.

We have constructed stable cell lines expressing transporters for dopamine (DA), norepinephrine (NE), and serotonin (5-HT) by transfection with cloned cDNAs. The parental LLC-PK1 cell does not express any of these neurotransmitter transporters. Therefore, monoamine transport activities in each of these cell lines are due to the transfected DNA only, allowing comparison in the same background. Drug inhibition profiles for each cell line are distinct and as expected for each transporter. LLC-NET and LLC-DAT cells transported both NE and DA and both cell types exhibited a lower KM for DA transport than for NE transport. Analysis of Vmax data for LLC-NET cells suggests that substrate is bound to the NE transporter during the rate-limiting step(s) in transport. The cocaine analog 2-beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binds to each cell type, and is displaced by transport substrate in each case. Binding and transport measurements on parallel cell cultures allowed estimation of turnover numbers for norepinephrine, dopamine, and serotonin transporters. All three transporters require external Na+ and Cl-. The Na+ concentration dependence suggests that a single Na+ ion is involved in transport catalyzed by norepinephrine and serotonin transporters while more than one Na+ ion participate in transport mediated by the dopamine transporter.

Animals↗
PubMed1994

The axonal gamma-aminobutyric acid transporter GAT-1 is sorted to the apical membranes of polarized epithelial cells.

Recent studies suggest that epithelial cells and neurons employ similar mechanisms to target proteins to the distinct subdomains of their polarized cell surface membranes. We have examined the sorting behavior of the neuronal gamma-aminobutyric acid (GABA) transporter GAT-1 expressed by transfection in the polarized epithelial Madin-Darby canine kidney (MDCK) cell line. We find that the GABA transporters endogenously expressed by polarized hippocampal neurons in culture are restricted to axonal plasma membranes. In transfected MDCK cells, the GABA transporter is found to be localized primarily to the apical cell surface when examined by immunocytochemistry, cell surface biotinylation, and transport assay. MDCK cells exposed to hyperosmotic stress express a close relative of GAT-1, the betaine transporter (BGT-1). We find that BGT-1 expressed by transfection in MDCK cells accumulates predominantly at the basolateral cell surface. These observations suggest that the sorting information required for axonal targeting may be similar to that which mediates apical localization in epithelia. Furthermore, it would appear that despite their high degree of homology, the BGT-1 and GAT-1 transporters manifest sorting signals which specify their targeting to distinct cell surface domains.

Animals↗
PubMed1993

From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters.

Biogenic amine transport systems in the presynaptic plasma membrane and the synaptic vesicle provide a mechanism for rapidly terminating the action of released transmitters and for recycling neurotransmitters. Alterations in the activity of these transporters, either by endogenous regulatory mechanisms or by drugs, affect the regulation of synaptic transmitter levels. For drugs such as antidepressants and stimulants that interact with these transport systems, the therapeutic and behavioral consequences are profound. Now that the cDNAs encoding the transporters have been isolated, we can expect rapid progress in understanding how the individual proteins work at the molecular level to couple ion gradients to the reuptake and storage of biogenic amine neurotransmitters.

Amino Acid Sequence↗
PubMed1993

Amphetamine derivatives interact with both plasma membrane and secretory vesicle biogenic amine transporters.

The interaction of fenfluramine, 3,4-methylenedioxymethamphetamine (MDMA), and p-chloroamphetamine (PCA) with the platelet plasma membrane serotonin transporter and the vesicular amine transporter were studied using both transport and binding measurements. Fenfluramine is apparently a substrate for the plasma membrane transporter, and consequently inhibits both serotonin transport and imipramine binding. Moreover, fenfluramine exchanges with internal [3H]serotonin in a plasma membrane transporter-mediated reaction that requires NaCl and is blocked by imipramine. These properties are similar to those of MDMA and PCA as previously described. In adrenal chromaffin granule membrane vesicles containing the vesicular amine transporter, fenfluramine inhibited serotonin transport and dissipated the transmembrane pH difference (delta pH) that drives amine uptake. The use of [3H]reserpine-binding measurements to determine drug interaction with the vesicular amine transporter allowed assessment of the relative ability of MDMA, PCA, and fenfluramine to bind to the substrate site of the vesicular transporter. These measurements permit a distinction between inhibition of vesicular serotonin transport by directly blocking vesicular amine transport and by dissipating delta pH. The results indicate that MDMA and fenfluramine inhibit by both mechanisms but PCA dissipates delta pH without blocking vesicular amine transport directly.

3,4-Methylenedioxyamphetamine↗
PubMed1993

Binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane to serotonin and dopamine transporters: different ionic requirements for substrate and 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binding.

The iodinated cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane (beta-[125I]CIT) binds with high affinity to the platelet plasma membrane serotonin transporter, as previously reported for dopamine transporters from rat brain [Eur. J. Pharmacol. 194:133-134 (1991)]. Unlabeled beta-CIT also inhibits serotonin transport by platelet membrane vesicles. In both rat striatal membranes and platelet plasma membranes, beta-[125I]CIT binding was found to be pH dependent, with a pKa of 6.4-6.9, and did not require the presence of Cl-. Na+ dramatically stimulated beta-[125I]CIT binding to both serotonin and dopamine transporters, although a small fraction of beta-[125I]CIT binding to the serotonin transporter was observed in the absence of Na+. The substrates serotonin and dopamine competed with beta-[125I]CIT for binding to their respective transporters. However, substrate affinity was enhanced by Cl-, whereas beta-[125I]CIT binding affinity was not. [3H]Imipramine binding to the platelet serotonin transporter and [3H]GBR-12935 binding to the dopamine transporter were not inhibited by decreasing the pH from 8 to 6.5. Likewise, the ability of serotonin to compete with [3H]imipramine binding and that of dopamine to inhibit [3H]GBR-12935 binding were equal at pH 6.5 or 8. Thus, beta-[125I]CIT binding to biogenic amine transporters is distinct from serotonin or dopamine binding by virtue of its inhibition by H+ and its insensitivity to Cl-.

Animals↗
PubMed1993

Non-neurotoxic amphetamine derivatives release serotonin through serotonin transporters.

3,4-Methylenedioxymethamphetamine (MDMA) and several other amphetamine derivatives cause degeneration of serotonergic nerve terminals. These drugs also release serotonin from nerve terminals both in vivo and in vitro. Two non-neurotoxic derivatives of MDMA were tested in membrane vesicle model systems to determine whether they also lacked the ability to release serotonin. 3-Methoxy-4-methylamphetamine (MMA) and 5-methoxy-6-methyl-2-aminoindan (MMAI) both inhibited imipramine binding to serotonin transporters in platelet plasma membrane vesicles and both inhibited Na+ gradient-driven serotonin transport into those vesicles. Significantly, both MMA and MMAI released [3H]serotonin from plasma membrane vesicles, apparently by a process of exchange. The half-maximal concentrations for this effect were comparable to that reported for MDMA. In addition to their effects on plasma membrane transporters, MMA and MMAI both inhibited serotonin transport into chromaffin granule membrane vesicles catalyzed by the vesicular biogenic amine transporter. At higher concentrations, these compounds also caused release of [3H]serotonin from chromaffin granule membrane vesicles and dissipated the transmembrane pH difference (delta pH). Although MMAI effects on the serotonin transporter were similar to those of MDMA, the two compounds had different effects on dopamine transporters. MDMA and methamphetamine inhibited binding of a cocaine analog to the dopamine transporter and released dopamine accumulated by cells expressing dopamine transporters, but similar concentrations of MMAI were inactive.

Amphetamines↗
PubMed1992

p-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.

Binding, Competitive↗
PubMed1992

The molecular mechanism of "ecstasy" [3,4-methylenedioxy-methamphetamine (MDMA)]: serotonin transporters are targets for MDMA-induced serotonin release.

MDMA ("ecstasy") has been widely reported as a drug of abuse and as a neurotoxin. This report describes the mechanism of MDMA action at serotonin transporters from plasma membranes and secretory vesicles. MDMA stimulates serotonin efflux from both types of membrane vesicle. In plasma membrane vesicles isolated from human platelets, MDMA inhibits serotonin transport and [3H]imipramine binding by direct interaction with the Na(+)-dependent serotonin transporter. MDMA stimulates radiolabel efflux from plasma membrane vesicles preloaded with [3H]serotonin in a stereo-specific, Na(+)-dependent, and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, which contain the vesicular biogenic amine transporter, MDMA inhibits ATP-dependent [3H]serotonin accumulation and stimulates efflux of previously accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due to dissipation of the transmembrane pH difference generated by ATP hydrolysis and to direct interaction with the vesicular amine transporter.

3,4-Methylenedioxyamphetamine↗