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Louis J DeFelice

Publications and source records attributed to Louis J DeFelice.

14 recordsLinked to original sources
PubMed2007

Transporters as channels.

This review investigates some key aspects of transport mechanisms and recent advances in our understanding of this ubiquitous cellular process. The prevailing model of cotransport is the alternating access model, which suggests that large conformational changes in the transporter protein accompany cotransport. This model rests on decades of research and has received substantial support because many transporter characteristics are explained using its premises. New experiments, however, have revealed the existence of channels in transporters, an idea that is in conflict with traditional models. The alternating access model is the subject of previous detailed reviews. Here we concentrate on the relatively recent data that document primarily the channel properties of transporters. In some cases, namely, the observation of single-transporter currents, the evidence is direct. In other cases the evidence--for example, from fluctuation analysis or transporter currents too large to be described as anything other than channel-like--is indirect. Although the existence of channels in transporters is not in doubt, we are far from understanding the significance of this property. In the online Supplemental Material , we review some pertinent aspects of ion channel theory and cotransport physiology to provide background for the channels and transporters presented here. We discuss the existence of channels in transporters, and we speculate on the biological significance of this newly unveiled property of transport proteins.

Animals↗
PubMed2006

Getting the message across: a recent transporter structure shows the way.

Efforts to define the mechanisms governing neurotransmitter uptake and drug action have moved into high gear with the publication of a high-resolution structure of a leucine transporter from Aquifex aeolicus, a bacterial member of the SLC6 transporter family. Solved with the substrate leucine bound, the new structure corroborates extensive biochemical and mutagenesis studies performed with related mammalian neurotransmitter transporters and provides exciting suggestions as to how coupling arises between ions and substrates to permit efficient neurotransmitter clearance.

Animals↗
PubMed2005

Substrate binding stoichiometry and kinetics of the norepinephrine transporter.

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.

1-Methyl-4-phenylpyridinium↗
PubMed2005

Biogenic amine neurotransmitter transporters: just when you thought you knew them.

Plasma membrane transporters have long been known to support the reuptake of biogenic amine neurotransmitters following release in the central and peripheral nervous systems. Using high-resolution imaging, patch-clamp and amperometric approaches, as well as molecular manipulations of transporter-regulatory pathways, surprising new details have been uncovered as to how transporters work and are influenced by signaling pathways and psychostimulants.

Animals↗
PubMed2004

Dopamine transporters depolarize neurons by a channel mechanism.

Neurotransmitter transporters generate larger currents than expected if one assumes fixed stoichiometry models. It remains controversial, however, whether these depolarizing currents arise from high density and rapid turnover rates of a classical transporter, or whether transporters exhibit bona fide channel behavior. Although heterologously expressed transporters show single-channel behavior and noise analysis in native cells strongly suggests channel behavior, no directly observed single-channel events associated with transporters have been reported thus far in native cells. We describe single-channel events arising directly from the Caenorhabditis elegans dopamine transporter (DAT-1) as evidenced by DA-induced channel activity blocked by a high-affinity DAT-1 inhibitor, increased channel activity in neurons that overexpress DAT-1, and loss of channels in dat-1 knockout neurons. Our data indicate that authentic transporter channels underlie depolarizing whole-cell currents. Thus, DA transporters not only transport DA but also exhibit a channel mode of conduction that directly modulates membrane potential and neuronal function.

Animals↗
PubMed2004

A spontaneous, recurrent mutation in divalent metal transporter-1 exposes a calcium entry pathway.

Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe(2+) transporter mediating cellular iron uptake in mammals. Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn(2+), Co(2+), and Cu(2), but not for Mg(2+) or Ca(2+). A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca(2+) permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.

Alternative Splicing↗
PubMed2004

Transporter structure and mechanism.

During the past several decades, lac permease has assumed almost tutelary proportions as a model for cotransporters. This archetypical membrane protein now exerts its influence by the most dramatic means possible: its structure is solved. This article describes the configuration and implied transport mechanism of the bacterial lactose transporter and compares its structure and function with those of other transporters and those of ion channels. This juxtaposition of transporters and channels is likely to be helpful because LacY is the first cotransporter with known structure and exclusive carrier properties, and it shares topology with neurotransmitter transporters with unknown structure and channel properties.

Animals↗
PubMed2003

Ionic currents in the human serotonin transporter reveal inconsistencies in the alternating access hypothesis.

We have investigated the conduction states of human serotonin transporter (hSERT) using the voltage clamp, cut-open frog oocyte method under different internal and external ionic conditions. Our data indicate discrepancies in the alternating access model of cotransport, which cannot consistently explain substrate transport and electrophysiological data. We are able simultaneously to isolate distinct external and internal binding sites for substrate, which exert different effects upon currents conducted by hSERT, in contradiction to the alternating access model. External binding sites of coupled Na ions are likewise simultaneously accessible from the internal and external face. Although Na and Cl are putatively cotransported, they have opposite effects on the internal face of the transporter. Finally, the internal K ion does not compete with internal 5-hydroxytryptamine for empty transporters. These data can be explained more readily in the language of ion channels, rather than carrier models distinguished by alternating access mechanisms: in a channel model of coupled transport, the currents represent different states of the same permeation path through hSERT and coupling occurs in a common pore.

Animals↗
PubMed2002

Binding and transport in norepinephrine transporters. Real-time, spatially resolved analysis in single cells using a fluorescent substrate.

Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP(+) (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP(+) accumulation is Na(+)-, Cl(-)-, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP(+) competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na(+) and Cl(-), binding is independent of Na(+) and actually increases in low Cl(-). We further demonstrate that ASP(+) interacts with transporters not only in transfected cells but in cultured neurons. ASP(+) is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery.

Adrenergic Uptake Inhibitors↗
PubMed2002

Targeting cell surface receptors with ligand-conjugated nanocrystals.

To explore the potential for use of ligand-conjugated nanocrystals to target cell surface receptors, ion channels, and transporters, we explored the ability of serotonin-labeled CdSe nanocrystals (SNACs) to interact with antidepressant-sensitive, human and Drosophila serotonin transporters (hSERT, dSERT) expressed in HeLa and HEK-293 cells. Unlike unconjugated nanocrystals, SNACs were found to dose-dependently inhibit transport of radiolabeled serotonin by hSERT and dSERT, with an estimated half-maximal activity (EC(50)) of 33 (dSERT) and 99 microM (hSERT). When serotonin was conjugated to the nanocrystal through a linker arm (LSNACs), the EC(50) for hSERT was determined to be 115 microM. Electrophysiology measurements indicated that LSNACs did not elicit currents from the serotonin-3 (5HT(3)) receptor but did produce currents when exposed to the transporter, which are similar to those elicited by antagonists. Moreover, fluorescent LSNACs were found to label SERT-transfected cells but did not label either nontransfected cells or transfected cells coincubated with the high-affinity SERT antagonist paroxetine. These findings support further consideration of ligand-conjugated nanocrystals as versatile probes of membrane proteins in living cells.

Animals↗
PubMed2002

Serotonin transporter function and pharmacology are sensitive to expression level: evidence for an endogenous regulatory factor.

We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires approximately 5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity.

Amphetamine↗
PubMed2002

Flux coupling in the human serotonin transporter.

The serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) catalyzes the movement of 5HT across cellular membranes. In the brain, SERT clears 5HT from extracellular spaces, modulating the strength and duration of serotonergic signaling. SERT is also an important pharmacological target for antidepressants and drugs of abuse. We have studied the flux of radio-labeled 5HT through the transporter stably expressed in HEK-293 cells. Analysis of the time course of net transport, the equilibrium 5HT gradient sustained, and the ratio of the unidirectional influx to efflux of 5HT indicate that mechanistically, human SERT functions as a 5HT channel rather than a classical carrier. This is especially apparent at relatively high [5HT](out) (> or =10 microM), but is not restricted to this regime of external 5HT.

Carrier Proteins↗
PubMed2002

Characterization of cocaine and antidepressant-sensitive norepinephrine transporters in rat placental trophoblasts.

1. This paper reports on a primary cell culture system that predominantly expresses native norepinephrine (NE) transporters (NETs), and is amenable to biophysical as well as biochemical analyses. 2. Previous research has identified human and rat placentas as rich sources of NET. We have exploited this to develop primary cultures of rat placental trophoblasts. NE uptake in these cultures is about 10 times higher when compared to 5HT uptake. The presence of NET protein is revealed by immunoblot analysis, while there is no detectable SERT protein. 3. NE transport in rat trophoblasts is sensitive to NET-specific antagonists, desipramine (DS) and nisoxetine (NX), but not to the dopamine-transporter (DAT) specific antagonist, GBR12909 or to the serotonin (5HT) transporter (SERT) specific antagonist paroxetine (PX). Drugs of abuse such as cocaine and amphetamine also inhibit NE transport in these cells. Together these results suggest that rat placental trophoblasts predominantly express NET over other monoamine transporters. 4. Patch-clamp analysis reveals that NETs in intact rat trophoblasts are electrogenic. Comparison of NE uptake with NE-induced currents suggests that these two modes of transporter activity are differentially regulated.

Animals↗