Targeting of the Human Serotonin Transporter

Neurotransmission is terminated by removal of the neurotransmitter from the synaptic cleft. This can be achieved by enzymatic breakdown or by the - more economic - retrieval of the neurotransmitter. The latter task is accomplished by neurotransmitter transporters. A large subfamily thereof (>12 in man) are the Na+ /Cl- -dependent neurotransmitter transporters. They share conserved structural features (e.g. a hydrophobic membrane-embedded core of twelve transmembrane spanning segments) and have several functional properties in common (e.g. they utilise the Na+ -gradient for cotransport of the substrate; substrate flux is associated with an ionic current; transport reversal is seen in the presence of exogenous substrates). In polarised cells, where these transporters are endogenously expressed, transporters are not uniformly distributed over the cell surface but they reside in distinct subcompartments of the cell surface. This is, in particular, true in nerve cells where transporters are targeted to axons and where they are retained on the presynaptic membrane (typically at perisynaptic sites surrounding the very synapse with its active zones). The mechanisms by which individual transporters are sorted and delivered to as well as retained in axons are poorly understood. In the working hypothesis underlying the current grant application, we postulate that the complexity of signal transduction and signal integration in nerve cells can only be understood if the factors that govern the spatial distribution of transporters are known. We propose to focus on the mechanisms by which the human serotonin transporter (SERT) is targeted in neurons. SERT is both, the site of action of widely employed therapeutic drugs (classical tricyclic antidepressants and selective serotonin-reuptake inhibitors etc.) and of drugs that are abused (e.g. ecstasy and other amphetamine congeners). Hence, understanding the regulation SERT is highly relevant to clinical medicine. In neurons, SERT is obviously subject to axonal targeting and retention. Our assumptions are as follows: The spacial segregation of SERT within neurons results from its interaction with distinct proteins. This selective interaction is specified by portions of the transporter protein. These must contain the information governing axonal delivery and presynaptic retention and, perhaps, clustering. The targeted distribution of SERT is subject to regulation; the (presynaptic) site of localisation of SERT is at a large distance from the site of its synthesis; there must be a mechanism that accounts for continuous replenishment. The goals of the current project are to verify these conjectures; specifically, we intend (i) to verify targeting of SERT in a neuron-like, permanent cell line and in nerve cells, (ii) to identify the sequence elements in SERT that specify targeting and the proteins that associate with these portions of the transporter (iii) and to prove that these proteins participate in axonal delivery and/or retention of SERT at specific sites. It is anticipated that the findings that will be obtained are also relevant to other (related) neurotransmitter transporters (e.g. the noradrenaline transporter and the dopamine transporter). Thus, the project aims at the generating insights that contribute to understanding the mechanisms that underlie neuronal signalling and synaptic plasticity. We hope to develop a model that may also serve as a framework to understand the role of SERT targeting in diseases.

Neurons release neurotransmitters and these are retrieved from the synaptic cleft by neurotransmitter transporters. They represent a large family of proteins that share structural, functional and topological properties. In the current work, we focused on sodium-dependent neurotransporter symporters, which are of particular interest, because (i) they represent important targets for pharmacotherapy (antidepressants block the noradrenaline and the serotonin transporter; antiepileptic drugs block the GABA-transporter-1), (ii) they are the site of action of illicit drugs [cocaine, amphetamine, methamphetamine, MDMA (= ecstasy) etc.], (iii) they are delivered to specific sites of neurons (i.e. the presynaptic specialization). It is not clear, how this specific delivery is achieved: the transporter is inserted into the endoplasmic reticulum (= ER) during its synthesis, then shuttled from the ER through additional intracellular compartments into the axon and delivered at the rim of the synapse. The current project aimed at shedding light onto this process. We failed to make any significant progress by employing the experimental strategy that we had originally proposed. However, at the start of this project, we made the exciting and surprising observation that neurotransmitter transporters were homooligomers (i.e. they associated with each other to form a larger complex). We then identified structural elements within the transporter that supported oligomerization. These insights allowed us to introduce mutations which disrupted the oligomer formation. The resulting mutants were retained within the cell, i.e. they did not leave the endoplasmic reticulum. This observation suggested that export of the transporter from the ER was contingent on oligomerization. We outlined a testable model in which we postulated that oligomerization was required because it supported the assembly of the components of the ER-export machinery. This "oligomerization hypothesis" was verified by identifying binding sites for these components in the carboxyl terminus of the transporter. What good are these insights for? There is a disease, referred to as familial postural hypotension, which is caused by a mutation in the norepinephrine transporter. Interestingly, the mutation has a dominant effect; in other words, the transporter encoded by the normal allele is also not functional. Based on our insights (with different transporters), the enigma was solved: The mutated transporter is retained in the ER; because transporters are oligmers, it traps the transporter encoded by the normal allele in an oligomer. Finally, we have shown that the action of amphetamines (which reverse transport direction) is contingent on transporter oligomerization: amphetamines take two to tango..