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Neurotransmitter Transporters: Diversity of Form and Function
Summary: Susan Amara's laboratory studies the molecular and cellular biology of neurotransmitter transport systems.
Neurotransmitter transporters at the plasma membrane contribute to the clearance and recycling of neurotransmitters and can have a profound impact on the extent of receptor activation that occurs during neuronal signaling. Work in our laboratory has focused on the structure, regulation, and cellular physiology of two families of sodium-dependent transporters: one family includes the biogenic amine neurotransmitters, norepinephrine, dopamine, and serotonin; another family includes the excitatory amino acid neurotransmitters, aspartate and glutamate. The dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT) are well-established targets for addictive drugs, including cocaine and amphetamines, as well as for therapeutic antidepressants. A crucial question is how these different small-molecule drugs bind to the carriers and mediate effects on neurotransmitter clearance that ultimately lead to profound behavioral changes.
We have recently examined the impact of psychostimulant drugs on the signaling properties, physiology, and acute regulation of the DAT in cultured midbrain dopaminergic neurons. Clearance of dopamine by the DAT is surprisingly slow and continues at the same rate despite the rapid voltage changes that occur during neuronal firing. A large number of transporter proteins are required to accomplish dopamine removal, because each carrier takes between one and two seconds to transport a single molecule of dopamine into the cell. Our studies have shown that neurotransmitter transporters can also mediate macroscopic ionic currents that are not directly coupled to substrate movement. Intriguingly, in neurons DAT substrates activate a chloride current associated with the DAT at much lower concentrations than are required for optimal transport. This channel activity has been observed only in dopaminergic neurons and may play an important role in regulating their membrane potential and excitability.
Biogenic Amine Transporters and Drug Action
Drugs that block neurotransmitter uptake have powerful physiological actions. The monoamine carriers in particular have been the focus of extensive research into the development of potentially useful therapeutic drugs. For example, the SERT is potently inhibited by antidepressant drugs such as fluoxetine (Prozac), sertraline, paroxetine, and citalopram, and the NET is selectively blocked by the tricyclic antidepressant desipramine. The affinities of psychostimulants such as cocaine and related drugs for the DAT correlate with their reinforcing properties, supporting the notion that inhibition of dopamine uptake is the primary mechanism mediating their euphoric and addictive actions.
Not all the effects of psychostimulant drugs are explained, however, by a hypothesis based solely on the DAT. Although it is generally believed that the function of each transporter is to accumulate its particular substrate, studies in vitro and in vivo have provided evidence that the transporters are not completely selective. We have shown that mammalian serotonin carriers readily transport dopamine. This aspect of their function has generally been overlooked, because dopamine itself is an extremely poor inhibitor of serotonin transport. Experiments examining the cloned human SERT expressed in Xenopus oocytes demonstrate that dopamine transport by the SERT occurs by a different mode from serotonin transport: dopamine transport is inhibited noncompetitively by serotonin, displays a different ion dependence, and is more potently inhibited by a variety of drugs, including cocaine and fluoxetine. Additional experiments have supported the in vivo relevance of these findings, using voltammetry to show reduced clearance of dopamine when serotonin carriers are blocked and using behavioral assays to show that a serotonin transport inhibitor potentiates dopamine-induced behaviors at doses that produce no effects on its own.
These results not only imply that the SERT can adopt multiple functional states but also support its physiological contribution to the clearance of dopamine, challenging our view about how "serotonin-selective" the serotonin-selective reuptake inhibitors such as Prozac really are. The findings may also shed light on recent data obtained with mice that lack the DAT gene in which cocaine is rewarding even in the absence of DAT protein.
Excitatory Amino Acid Transporters: Carrier and Channel Properties
Excitatory amino acid transporters (EAATs) in the central nervous system maintain extracellular glutamate concentrations below neurotoxic levels and limit the action of glutamate released during neurotransmission. Five different human EAAT subtypes (EAAT1–EAAT5) and their homologs in several species have been identified. Transport of substrates by these carriers is thermodynamically coupled to the cotransport of sodium ions, a proton, and the countertransport of a potassium ion, a process that is electrogenic. These transporters also possess a ligand-gated chloride channel activity that can regulate neuronal excitability and synaptic signaling. The relative proportion of the current generated by ion-coupled substrate transport or the ligand-gated Cl– conductance varies for each of the five different human EAAT subtypes. For example, with EAAT1, EAAT2, and EAAT3 the Cl– flux is a relatively small component of the currents, whereas for the neuronal transporters EAAT4 and EAAT5, the currents elicited by substrates are almost entirely composed of a gated anion flux.
To understand the structural basis of the different properties of these closely related transporters, we are characterizing the functional role of specific sequence domains. Until recently, models for the structure and topology of glutamate transporters were based on predictions from the protein sequence. Using cysteine substitutions together with thiol-modifying reagents, we have evaluated and revised various aspects of this model. Currently we are identifying domains and residues that influence transport and/or the various conductances associated with these proteins. Our studies have revealed a number of domains that appear to influence the kinetics, ion dependence, and substrate selectivity of transport.
Applying electrophysiological methods together with cysteine-scanning approaches, we have shown that transport and chloride channel activities require the different domains of the carrier. In one mutant, for example, modification of a single cysteine residue abolishes uptake activity and generates a transporter that operates exclusively as a substrate-activated chloride channel. The results illustrate, for the first time, the structural separation between glutamate translocation and the gated anion flux and support a model in which substrate binding, but not transport, is required to elicit the anion current.
The National Institute on Drug Abuse and the National Institute of Neurological Disorders and Stroke provided support for some of this work.
Last updated November 09, 2001
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