Energy coupling in the serotonin transporter

Neurons are cells specialized in information processing, storing and signaling. A neuron can have an extension that reaches a meter in length. Neurons use very fast traveling electrical signals called action potentials for communication. Communication between neurons requires specialized structures named synapses. Otto Loewi was awarded the Nobel prize in 1936 for his discovery that interneuron communication is carried by small diffusing molecules called neurotransmitters instead of action potentials. These neurotransmitters are packed in small vesicles, and are released upon excitation by an action potential. This sharp increase in neurotransmitter concentrations can trigger new action potential in the second neuron. Neuronal functioning requires efficient remove of these neurotransmitters by dedicated transporters. Abnormal synaptic function is associated with a number of diseases such as depression, attention deficit hyperactivity disorder, autism and bipolar disorder. This research proposal focuses on the transporter of the neurotransmitter serotonin (hSERT), which is targeted by several drugs approved for the patients treatment. During transport, hSERT alternates between two conformations, whereby access to the serotonin binding site changes between exposure to the extracellular and the intracellular side of the membrane. Transport is energized by the transmembrane electrochemical gradient of co-transported ions through a process called energy coupling. The aim of this research project is to answer the following questions: How is energy converted from the ionic gradient into conformational changes? How can energy coupling energize the uphill transport of serotonin? To achieve this goal, we will study energy coupling through quantification of the essential elements of serotonin transport, which are conformations, dynamics, interactions and thermodynamics. The hypothesis put forward states that hSERT builds on imperfect serotonin and ion binding to its initial binding conformation. The energy provided by ion and serotonin binding is than used by hSERT to change its conformation to become oriented to the intracellular side. This enigmatic energy harvesting will be uncovered by the integration of a plethora of methods, which range from hole cell transport assays to extensive computer simulations of substrate transport at the atomic level. Pharmacological industry has reduced in the last years its efforts in developing new drugs for the treatment of neurological diseases, because of difficulties to identify novel and more effective medications. This research proposal does not directly address the drug design challenge, but through uncovering the mechanism of energy conversion and the structural details of transport, its long term prospects are to provide the fertile grounds for innovative drug developments and the discovery of better medications for patients.

Neurons are cells specialized in information processing and transmitting. Information exchange between neurons requires dedicated structures called synapses. The signalling molecules (neurotransmitters) are stored in small vesicles. Neurotransmitters release results in a sharp concentration increase that triggers new action potentials. Neural function requires their efficient removal, while abnormal neuronal function is associated with conditions such as depression, attention and hyperactivity disorder, autism, or bipolar disorder. The subject of this research project is substrat transport by the neurotransmitter serotonin (hSERT). The substrate binding site (S1) is located in the centre of the transporter in the middle of the membrane. The transport cycle model predicts that the 5HT binding to the open transporter starts the transport cycle, followed by SERT occlusion and opening of the inner gate, which allows the release of 5HT into the cytosol of the pre-synaptic neuron. In this research project, we were able to confirm the assumption that SERT is open to the outside in the resting state. Our studies have shown that binding of the co-transport sodium ion is necessary to stabilize this state. We were able to show that the structural transition to the occluded state is very complex and includes several intermediate steps. Using extensive simulations and combination with experiments, we were able to identify the individual steps on the path to SERT occlusion. The first step is recognition of the substrate by aspartate 98, which is part of helix TM1B. This ligand sensor initiates the process: first the movement of TM1B, followed by concerted movements of the bundle domain and the completion of occlusion by movements of TM6A. As a key predictor of these movements, we identified the density of water in the entrance to the substrate binding site, which has a low density immediately before and during the movements. We found that the interface between protein domains sliding relative to each other exhibits properties that allow for seemingly frictionless motion, enabled by conformationally very flexible amino acid side chains. A central result of this project is characterization of conformational coupling between ligand and transporter and quantification of the associated free energy. By characterizing the interaction of 5HT, a congeneric series of 5HT derivatives, and several psychoactive substances, we demonstrated that the ligand triggers SERT occlusion. This means that the ligand initiates its own transport through a few key interactions, which requires that the substrate matches the energetic and geometric requirements of the transporter. The free energy profile clearly shows the molecular origin of these properties. The essential interactions for triggering and driving the individual steps are interactions of the carboxyl group of aspartate 98 and the helical dipole of helix TM6A, while the aliphatic nitrogen is the essential factor in the substrate.