Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK

Correspondence to: L Iversen, Department of Pharmacology, University of Oxford, Mansfield Rd, Oxford OX1 3QT, UK

More than 20 members have been identified in the neurotransmitter transporter family. These include the cell surface re-uptake mechanisms for monoamine and amino acid neurotransmitters and vesicular transporter mechanisms involved in neurotransmitter storage. The norepinephrine and serotonin re-uptake transporters are key targets for antidepressant drugs. Clinically effective antidepressants include those with selectivity for either NE or serotonin uptake, and compounds with mixed actions. The dopamine transporter plays a key role in mediating the actions of cocaine and the amphetamines and in conferring selectivity on dopamine neurotoxins. The only clinically used compound to come so far from research on amino acid transporters is the antiepileptic drug tiagabine, a GABA uptake inhibitor. Molecular Psychiatry (2000) 5, 357-362.

neurotransmitter transporters; vesicular transporters; antidepressants; serotonin; norepinephrine; dopamine; cocaine; amphetamines

Introduction

The concept that neurotransmitters are inactivated by uptake of the released chemical into the nerve terminal from which it had been released or into adjacent cells is less than 40 years old. Prior to this it was generally assumed that the inactivation of noradrenaline and the other monoamine neurotransmitters after their release from nerves was likely to involve rapid enzymatic breakdown, akin to that seen with acetylcholinesterase. The degradation of monoamines by the enzyme monoamine oxidase was known early on, and in the 1950s a second enzyme catechol-O-methyl transferase (COMT) was discovered and was thought to play a key role in inactivating noradrenaline and other catecholamines.

It was not until high specific activity tritium-labelled radioactive catecholamines became available in the late 1950s, however, that experiments could be performed using quantities of monoamine small enough to mimic the very low concentrations of adrenaline or noradrenaline normally encountered in body fluids. When the first experiments were performed in the Axelrod laboratory at the National Institutes of Health with ³H-adrenaline¹ and later with ³H-noradrenaline² they yielded an unexpected result. Although in laboratory animals most of the injected dose of labelled catecholamine was rapidly metabolised (mainly by COMT), a substantial proportion of the injected monoamine (30-40%) was removed from the circulation by a rapid uptake into tissues, where it remained for some time unchanged. A key observation was that the uptake of ³H-noradrenaline into the heart was virtually eliminated in animals in which the sympathetic innervation had been destroyed by surgical removal of the superior cervical ganglion.³ This led Hertting and Axelrod⁴ to propose that the reuptake of noradrenaline by the same nerves from which it had been released might represent a novel mechanism for inactivating this neurotransmitter.

The discovery of noradrenaline uptake was followed by the finding that similar but distinct transporters were involved in the inactivation of 5-HT and dopamine, and that similar mechanisms existed for the inactivation of the amino acid neurotransmitters GABA, glycine and L-glutamate.^5,6 Much research interest has focused on these mechanisms, including in recent years the identification and cloning of the genes encoding the transporter proteins involved and the development of knock-out strains of genetically engineered mice lacking one or other of these gene products. The family of neurotransmitter transporters has turned out to be far more extensive than previously imagined, with more than 20 different members (for review see Masson et al).⁷

The neurotransmitter transporters have also proved to be very important targets for CNS drug discovery, particularly for antidepressant drugs. Virtually all of the modern generation of antidepressants act as inhibitors of one or other of the monoamine transporters. Fluoxetine and the related serotonin selective reuptake inhibitors (SSRIs) are among the most widely used and commercially valuable CNS drugs of all time, with annual sales of this group of compounds currently in excess of $5 billion. This brief review will attempt to give an overview of what has become a large and rapidly growing area of neuroscience research.

The family of neurotransmitter transporters

The molecular characterisation of the neurotransmitter transporter family began with the cloning of the rat GABA transporter by Radian and Kanner in 1986.⁸ When the norepinephrine transporter was independently cloned by Pacholczyk et al in 1991,⁹ it became apparent that there was a high degree of sequence homology between the GABA and NE transporters, and this led to the recognition that they belonged to larger gene family whose other members were soon identified (Table 1). Separate transporters exist for GABA, GABA/betaine, NE, serotonin, dopamine, glycine, taurine and proline. In addition homology cloning has identified four 'orphan' transporter genes, although the transport functions of these or their substrates remain unknown.⁷ All of these related proteins are dependent on sodium and chloride ions for their function. They use the electrochemical gradient of sodium between the outside and inside surfaces of the cell membrane to provide the thermodynamic energy required to pump neurotransmitters from low concentrations outside the cell to the much higher concentrations inside the cell. Chloride ions accompany the entry of neurotransmitter and sodium, and there is a net movement of positively charged ions into the cell, although not in sufficient amounts to appreciably alter the resting membrane potential of the cell.

A further family of five transporters was later discovered which mediate the uptake of L-glutamate into neurons and glial cells; these proteins require the presence of both sodium and potassium ions for their function, but are not chloride-dependent.⁷

The vesicular neurotransmitter transporters represent another family⁷ whose function is to maintain the very high concentrations of monoamine and amino acid neurotransmitters in storage vesicles. They use the proton gradient that exists across the vesicular membrane as the motive force. The vesicular monoamine transporters (VMAT) recognise serotonin, dopamine, NE, epinephrine and histamine. VMAT-1 is present chiefly in amine-containing endocrine and paracrine cells in peripheral organs, while VMAT-2 is the predominant form found in monaminergic neurons in CNS. It is also expressed in the histamine-containing cells of the stomach, and in the adrenal medulla and in blood cells.

Cholinergic neurons possess their own vesicular acetylcholine transporter, and a fourth vesicular transporter is expressed in GABA and glycine-containing neurons.

The uptake of monoamines into non-neuronal tissues, first described as Uptake₂ is a mechanism that has a particularly high affinity for O-methylated catecholamine metabolites and is sensitive to inhibition by steroids.^5,6 Uptake₂ was at first thought to be mediated by a member of the organic cation carrier family, but it is now known to have its own steroidsensitive transporter molecule.¹⁰

Immunocytochemical and in situ hybridization techniques have been used to study the cellular distribution of the transporters.⁷ Whereas NET, DAT and PROT are expressed exclusively in neurons many members of the family are expressed in non-neural cells and several in both neurons and glia (eg SERT). Among the various GABA transporters GAT1 is localised exclusively to nerve terminals while other members of this group are found in glial cells. Among the L-glutamate transporters EAAT-1 and 2 are expressed exclusively in the brain, while other members of the group are also found in some peripheral tissues. In the brain EAAT-3 and 4 are present only in neurons, while EAAT-1 and 2 are exclusively expressed in glial cells. There are differences in the proportions of the various glutamate transporters found in different brain regions. Overall they are strategically placed to control the extracellular levels of this important excitatory amino acid in the CNS.

The Na⁺/Cl⁺-dependent transporters and the vesicular transporters are membrane proteins consisting of a single polypeptide chain of 5-600 amino acid residues, with 12 -helical membrane-spanning domains.⁷ The Na⁺/K⁺-dependent glutamate transporters are proteins of similar length but the -helical regions are less clearly defined and estimates of the number of membrane-spanning domains range from six to ten.¹¹ The molecular mechanisms underlying the function of the neurotransmitter transporters remain unclear. Unlike flux through an open ion channel, there must be a gating cycle every time solute is transported, but the exact molecular details of this are not understood. No doubt selective mutations of amino acids in the transporter molecules will throw light on these questions in the future.¹²

Neurotransmitter transporters as targets for CNS drugs

Monoamine transporters¾NET and SERT

By far the most important group of CNS drugs that target the NE and serotonin neurotransmitter transporters is the tricyclic antidepressants and their modern counterparts. The discovery by the Axelrod group in 1961¹³ that imipramine potently inhibited the uptake of norepinephrine led to the first understanding of the mechanism of action of the first generation tricyclic antidepressants. Following the discovery of the serotonin uptake system in brain it soon became apparent that the classical tricyclic drugs imipramine and amitriptyline were almost equally potent as inhibitors of NE and 5-HT uptake (Table 2). This reinforced the monoamine hypothesis of depression as a monoamine deficiency state, and stimulated much further research in the pharmaceutical industry to discover new inhibitors of monoamine uptake. The debate as to whether inhibition of NE or 5-HT was the most important in conferring antidepressant efficacy has swung one way and the other over the past 40 years and there is no definitive answer to this question. A fascinating account of the historical development of research in this field is given by David Healy in his monograph 'The Antidepressant Era'.¹⁴ An early effort to improve the selectivity of antidepressants was made in the 1970s by scientists at the CIBA-GEIGY Company in Switzerland, who developed the selective NE uptake inhibitor maprotiline. This proved to be clinically effective as an antidepressant but it was not a great success commercially and had few clear advantages over the classical tricyclics. This idea was also swept away by the wave of enthusiasm for serotonin selective reuptake inhibitors (SSRIs) that started with the success of fluoxetine.¹⁵ Fluoxetine and the related group of SSRIs have proved to be a remarkably important advance in psychopharmacology. Although these drugs have no greater antidepressant efficacy than the classical tricyclics, they have a greatly reduced cardiac toxicity and lack the cholinergic properties of the older drugs. The SSRIs exhibit some adverse side effects, notably in impaired sexual function in both men and women, but because they are relatively safe physicians have been less inhibited about using them. This has extended the clinical recognition and treatment of depression to a much greater number of people than hitherto. At the same time new indications have been demonstrated and officially approved for the SSRIs, including syndromes associated with anxiety and panic, extending still further the wide use of these agents in psychopharmacology (Table 3).

The remarkable success of the SSRIs has prompted the question of whether genetic defects in SERT or in the regulation of its expression might explain the etiology of mood disorders. The gene coding for human SERT is localized on chromosome 17q11.2. It spans over 35 kilobases and is organised in 14 introns. No genetic variations have been found in the coding region of the SERT gene in depressed patients, but a number of studies have found that certain variants in a polymorphic region flanking the 5' region or in the second intron are associated with depressive illness, anxiety-related personality traits or suicidal alcoholism.⁷

Ironically, some of the most recently introduced antidepressants hark back to the non-selective compounds of the earlier era. Thus the new entrants venlafaxine and milnacipran are described as drugs that combine both NE and serotonin re-uptake inhibition¹⁷ (although in vitro binding data show that venlafaxine binds with some 300 times higher affinity to human SER than to NET).¹⁶ The compound sibutramine is also an inhibitor of both NE and serotonin uptake, but it has been approved for use as an anti-obesity agent rather than an antidepressant.¹⁸ At the same time reboxetine is the first antidepressant drug in a new class of NET-selective inhibitors. It is claimed to be particularly suitable for depressed patients who 'feel tired all the time'.¹⁷ Reboxetine is reported to be as effective as the SSRIs or older tricylics, but is not associated with sexual dysfunction.¹⁹ The older antidepressant bupropion, which acts as a weak inhibitor of NE and dopamine uptake, with little effect on serotonin uptake has had only modest success as an antidepressant, but has been approved in the US as an aid to smoking cessation.¹⁷

What are we to make of these twists and turns? How can drugs that are selective NE uptake inhibitors be equally effective as those that selectively target serotonin? In practice it is difficult to know how selective the monoamine uptake inhibitors are in vivo. None of the antidepressants is completely selective for NE or SERT. The SSRIs have some affinity for NET, and some (eg paroxetine) are quite potent inhibitors of NET.¹⁶ In some cases the formation of active metabolites alters the drug selectivity profile. Thus the non-selective compounds imipramine and lofepramine are extensively metabolised to desipramine, a highly potent and selective NE uptake inhibitor. Similarly whereas amitriptyline has little selectivity for NET or SERT, the metabolite nortriptyline is a selective NET inhibitor. It seems likely that both NE-selective and SSRIs exert their effects through some common final pathway in the brain. Perhaps the SSRIs act indirectly to modulate noradrenergic function.²⁰ The original monoamine hypothesis of depression as formulated by Schildkraut in 1965²¹ stated:

'Some, if not all, depressions are associated with an absolute or relative deficiency of catecholamines, particularly norepinephrine, at functionally important adrenergic receptor sites in the brain. Elation conversely may be associated with an excess of such amines'.

Opinion currently seems to be swinging back in support of the view that an up-regulation of noradrenergic function may be the key element underlying the efficacy of antidepressant drugs.²⁰

The molecular mechanisms in the brain that are triggered by the antidepressants, however, remain obscure. The fact that all drugs require a period of several weeks before they become fully effective suggests that they modify gene expression in the brain and that the resulting altered biochemical state takes a long time to become stabilised. Many theories have been proposed, including alterations in the expression of alpha and beta-adrenergic receptors, changes in transcription factors and/or neurotrophic factors, and even morphological alterations in the connectivity of monoaminergic nerves.²⁰

It is possible that antidepressant drugs have other targets in addition to their actions at cell surface monoamine transporters. Al-Damluji and Kopin²² have described a novel amine uptake process in peptide-containing hypothalamic neurons, which they named 'transport-P'. Like the vesicular transporters this process is driven by a proton gradient, but it is distinct from the vesicular transporters in being insensitive to reserpine, but sensitive to a variety of tricyclic antidepressants at micromolar concentrations.²³ It is not clear, however, what role if any transport-P plays in the inactivation of the monoamine neurotransmitters.

Monoamine transporters¾DAT

Some antidepressants, notably mazindol and bupropion, inhibit the dopamine transporter (DAT) as well as NET or SERT. The DAT is best known, however, as one of the principal sites of action of the psychostimulant drug cocaine. Mice that are genetically engineered to knock out the expression of the DAT gene are profoundly hyperactive and fail to show any further stimulation of activity in response to cocaine or d-amphetamine.²⁴ Such animals, nevertheless, will continue to self-administer cocaine,²⁵ suggesting that the rewarding properties of the drug cannot be explained entirely by its ability to inhibit DAT. Cocaine is also a potent inhibitor of both serotonin and NE uptake. It retains some rewarding properties even in SERT knock-out mice²⁶ suggesting that inhibition of NE uptake may also contribute importantly to its pharmacology.

A corollary of the understanding that cocaine owes important parts of its overall CNS profile to mechanisms other than inhibition of DAT is that more selective inhibitors of dopamine uptake might be useful and free of dependence liability. One such compound, brasofensine, is in clinical development for the treatment of Parkinson's disease.²⁷ Other selective DAT inhibitors have been proposed for the treatment of the withdrawal phase of CNS drug abuse.

Another important group of psychopharmaceuticals, the amphetamines, also serve as both substrates and inhibitors of DAT. Amphetamines have the ability to be taken up by dopaminergic neurons and to promote the release of dopamine. d-Amphetamine and methyl phenidate have become widely used in the treatment of children with attention-deficit hyperactivity disorder (ADHD) in recent years.²⁸ The concept of administering psychostimulants to hyperactive children might at first sight appear paradoxical. Recent findings in the DAT knock-out mouse model may offer new insight into the mechanisms involved. The administration of d-amphetamine or methyl phenidate at doses that cause behavioural stimulation in normal mice had a paradoxical calming effect on the hyperactive DAT knockout animals.²⁹ This calming effect appeared to be due to the ability of the amphetamines to cause an increased release of serotonin, as they also have appreciable affinity for both SERT and NET in addition to their affinity for DAT.

Studies of the human gene encoding DAT have suggested an association between polymorphisms in a non-coding region and ADHD.³⁰

The DAT is also important in mediating the actions of the selective dopaminergic neurotoxins 6-hydroxydopamine, and 1-methyl-4-phenylpiperidinium ion (MPP⁺). The selective accumulation of these compounds by DAT in dopaminergic neurons explains their targeted neurotoxicity. One possible explanation for the selective loss of dopaminergic neurons in Parkinson's disease is that patients may have been exposed to some environmental dopaminergic neurotoxin, but the search for such a compound has so far proved fruitless.

Vesicular monoamine transporters

The vesicular monoamine transporters represent the site of action of reserpine and tetrabenazine, which cause a long-lasting impairment of monoamine storage resulting in the depletion of monoamines from CNS and peripheral aminergic neurons. Although reserpine was used briefly during the 1950s as an anti-schizophrenic drug it had several adverse side effects, including the ability to cause profound depression of mood.

The vesicular monoamine transporters are important, however, as they represent a second important target for the amphetamines. The action of these psychostimulants depends on their ability to be taken up into dopaminergic and other aminergic neurons, and by interaction with the vesicular amine transporters to cause a release of dopamine and other endogenous monoamines.

Amino acid neurotransmitter transporters

A number of experimental compounds have been developed that act as selective inhibitors of GABA uptake. As might be expected, such compounds exhibit anticonvulsant properties in animal seizure models, but so far tiagabine is the only compound that has been approved for human use as an anti-epileptic drug.³¹ Tiagabine selectively inhibits the GAT-1 transporter, present in both neurons and astrocytes in the brain. Inhibitors of the other GABA transporters, or the various L-glutamate transporters are not yet available, nor have the other amino acid transporters (Table 1) attracted much pharmacological interest.

Future prospects

The neurotransmitter transporter family has provided many valuable targets for psychopharmacology. There seems every prospect that this will continue. It might seem that the monoamine transporters had already been fully exploited, but the re-emergence of NET-specific antidepressants, and the possible applications of selective inhibitors of DAT suggest that there may still be room for innovation even in such a crowded field.

The amino acid neurotransmitter transporters represent another potentially fruitful field for future CNS drug discovery, with only one useful drug approved for human use so far, the GABA uptake inhibitor tiagabine. Inhibiting the inactivation of L-glutamate after its release in CNS may not be without hazards, as inhibitors of the EAAT family may be liable to provoke seizure activity or to act as pro-convulsants. On the other hand this is a complex family with five distinct transporters. It is possible that the selective inhibition of one or other member of the family could yield beneficial effects (eg cognitive enhancement) with little hazard.

A completely novel target has been discovered in research on the mechanisms involved in the inactivation of the naturally occurring brain cannabinoid substance anandamide. It appears to be inactivated in part by a tissue uptake mechanism, and this could offer an attractive target for drug discovery.³² Inhibitors of anandamide uptake might provide a means of up-regulating endogenous cannabinoid function without the unwanted psychic side effects of direct acting cannabinoid receptor ligands.

The next 50 years of research on neurotransmitter transporters may well provide as many new discoveries and new CNS drugs as the previous half century.

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Figure 1 Cartoon depicting the role of neurotransmitter transporters at the synapse. PNT = plasma membrane neurotransmitter transporter; VNT = vesicular neurotransmitter transporter. Redrawn from Mason et al.⁷

Table 1 Neurotransmitter transporters

Table 2 Antidepressant amine uptake inhibitors

Table 3 Additional indications for SSRI antidepressants