An electrophysiological approach to the multiple states of the serotonin transporter

The transporters for the monoamines dopamine, serotonin and noradrenalin are import targets for the treatment of depression but also for illicit drugs such cocaine and amphetamines. The monoamine transporters are Na+-symporters that belong to the SLC6 (solute carrier) gene family. They are involved in the clearance of extracellular monoamines after their exocytotic release and in the replenishment of vesicular neurotransmitter pools. Several polymorphisms and de novo mutations in the coding and regulatory regions of the respective genes have been linked to increased susceptibility to various psychiatric disorders and neurological diseases such as depression and Parkinsons disease. Monoamine transporters likely function by an alternating access type mechanism. The alternating access model predicts that solutes (i.e. Na+, Cl- and substrate) bind to extracellular sites on the transporter and then - following conformational rearrangements- dissociate into the intracellular space of the cell. The exact mechanism that governs these events is poorly understood. Clinically relevant defects in the function of monoamine transporters that are caused by mutations can be subtle and difficult to diagnose. Therefore, there is a clear need for diagnostic tools that are able to detect changes in function also in case they are small. Electrophysiological techniques are very suitable because they can assess transporter function in real time by measurements of the currents associated with the transport of substrate. However, in order to interpret the results from such measurements a model is required that can faithfully relate the measured currents to transport function. The main aim of this proposal is to develop such a model for the serotonin transporter (SERT). The transport cycle of SERT is complex because it is contingent on multiple ions such as Na+, Cl-, and K+. Ambiguities exist in regard to the action of Cl- and K+. There is evidence suggesting that Cl- is not symported and moreover there is also evidence in conflict with the idea that K+ is antiported. Thus SERT might function differently as currently believed. Accordingly this proposal intends to clarify how Cl- and K+ act on SERT. When clarity on this issue is obtained, partial reactions of SERT transport cycle will be determined and this information will be used to build a kinetic model of SERT function- for the first time based on realistic time constants. This model will then be employed to understand how mutant transporters that are relevant to diseases differ from transporters that are functionally intact.

The most important results of the project are (i) that we were able to determine the rate of the partial reactions that the serotonin transporter must undergo to support uptake of its cognate substrate into presynaptic nerve terminals, (ii) that we could show that it is possible to accelerate dopamine uptake via the dopamine transporter by a pharmacological intervention and (iii) that cells expressing the serotonin transporter are a suitable model system to demonstrate that it is feasible to monitor ligand binding to a membrane protein with high temporal precision utilizing electrophysiological tools. Serotonin and dopamine are neurotransmitters, which are released into the synaptic cleft. The serotonin and the dopamine transporter (SERT and DAT) are tasked with re-uptake of these molecules into the nerve cells from which they were released. SERT and DAT are important drug targets for the treatment of diseases such as major depression and anxiety disorders. However, all licensed drugs acting on these transporters are either inhibitors or synthetic substrates. Compounds, which bind to so-called allosteric sites (remote from the substrate binding site) may affect these transporters in ways that are novel. This can open new therapeutic possibilities. Understanding, which drug actions are feasible, requires detailed knowledge on how these transporters operate. The aim of this project was therefore to identify the states that SERT adopts during serotonin transport and to measure the transition rates between these states. To achieve this we relied on electrophysiological recordings as these provided the necessary temporal resolution. With the obtained information we build a kinetic model, which was able to fully explain the function of SERT. We were able to show that it is possible to accelerate dopamine transport by DAT pharmacologically (i.e. by the application of Zinc). It was previously unclear whether a ligand that binds to a transporter can achieve this. Our results provide proof of concept. Thus, in the future small molecules might be identified that affect a given transporter in the way Zinc affects DAT. Activation of DAT by a drug, for instance, might become a treatment option for addiction and for hyperdopaminergic diseases such as schizophrenia. Our electrophysiological analysis of SERT led to the discovery of a new electrophysiological phenomenon. Charged ligands can change the potential at the surface of the membrane upon binding to membrane proteins. Based on this phenomenon we established a new method to measure ligand binding in real time and in a label free manner. Although this method was developed using SERT expressing cells it can in principle be applied to any other membrane protein. Possible candidates are membrane receptors for which there is a need of methods that can measure ligand binding with high temporal precision.