An electrophysiological assessment of the glycine transporter 1 and 2

Cells of the brain, called neurons, communicate via chemical signals in a process termed neurotransmission. These chemical signals, called neurotransmitters, are released at a point of contact between two cells, called synapses. Once information has been transmitted, removal of the neurotransmitter from the synapse is required. This job is performed by specialized molecular machines that are called neurotransmitter transporters. These proteins pump the neurotransmitters either back into the cell from which the transmitter was released or into specialized helper cells, called glial cells. One of the important neurotransmitters is glycine, a molecule that belongs to the family of amino acids. The neurotransmitter glycine has two important and, at first sight, opposing roles. On one hand, glycine reduces brain activity by acting on molecules called glycine receptors which inhibit neuronal firing. On the other hand, glycine is also required for the activation of another receptor, the NMDA receptor, which increases brain activity and is important for the formation of new memories. Glycine is removed from the synaptic cleft by the glycine transporters GlyT1 or GlyT2. The two transporters are found in distinct areas of the brain, with GlyT1 being found in glia cells of the brain and GlyT2 being mostly found in the brain stem and the spinal cord. The exact requirements for transport differ between GlyT1 and GlyT2, for example, GlyT1 can pump glycine in both directions but GlyT2 can only pump glycine into the cell. The focus of our study is to understand how the two transporters work. To achieve this goal, we will measure electrical signals that are produced by the transporter while it pumping glycine into the cell. These signals provide important information on the speed and the direction in which glycine is pumped. By comparing these electrical signals under different conditions, we will be able to create a detailed description of how the two transporters work. We will specifically study how ions and voltage influence the transporter and how GlyT1 is able to pump glycine in both directions. Our results will be combined into two mathematical models, one for GlyT1 and one for GlyT2, that will allow us to predict how these transporters will behave in specific situations. Because glycine is required for NMDA receptor activation, this information could be very important for specific diseases of the brain where NMDA receptors do not work, such as Alzheimers disease or Schizophrenia.

Two solute carriers exist that can transport the neurotransmitter glycine across the cellular membrane. By this action, they allow for the uptake of this neurotransmitter from the extracellular space into the interior of the cell. These transporter are called glycine transporter 1 (GlyT1) and glycine transporter 2 (GlyT2). Although both transporters can carry glycine they have different physiological roles as evidenced by them being expressed in different regions of the central nervous system and different cell types (i.e., GlyT1 and GlyT2 are expressed in glial cells and neurons, respectively). In the present project, we addressed the question of whether these two transporters also differ in other properties such as their transport velocity, their dependence on the cellular membrane potential, and their ability to concentrate glycine within the cell. To this end, we used electrophysiological methods because these allow for monitoring the conformational rearrangements within these proteins, which occur in the millisecond time range. Our results show that these two transporter can meet their different physiological obligations because they differ substantially in the way they operate. For instance, GlyT1 cannot only take up glycine from the extracellular space but also release it from the cell interior. This is not possible for GlyT2. The releasing action of GlyT1 is important because receptors exist that are regulated by glycine. Our results have therefore contributed to a better understanding of which tasks these transporters can take on and which they cannot. Moreover, we tackled a problem that in the realm of enzyme kinetics is known as product inhibition. Enzymes fail to further catalyze a reaction when the concentration of the reaction product rises above a certain level. The glycine transporters encounter a similar problem. Accumulated glycine within the cell slows down the transport velocity of the glycine transporters up to a point where they are practically inactive. This is because intracellular glycine can rebind to the transporter. These rebinding events occur more and more frequently as the glycine concentration rises. Above a certain concentration, the transporters cannot get rid of the bound glycine which then stalls their activity entirely. Our analysis revealed how the glycine transporters solve this problem. For this, they bind their substrate (glycine) and their co-substrate (sodium) in a highly cooperative manner. As a consequence, glycine only binds strongly to the transporters if also sodium is present. This is advantageous because normally the sodium concentration within a cell is low. After all, this ion is constantly pumped out of the cell by the sodium-potassium pump. Therefore the affinity of glycine for its transporters is low once it reaches the interior of the cell. For this reason, it does not bind to the transporters, which allows them to work undisturbed.