Understanding the Molecular Mechanism of Ion and Amino Acid Transport in Neurons
Lead Research Organisation:
University of Oxford
Department Name: Interdisciplinary Bioscience DTP
Abstract
In neurons, 2 types of transmembrane proteins are required for function: ion channels and transporters. Two-pore domain potassium channels (K2Ps) are a family of membrane proteins involved in the control of neuronal excitability, while transporters of the solute carrier 1 (SLC1) family are involved in amino acid uptake. The 3-dimensional (3D) structures of K2P channel (TREK-2) have recently been solved, providing initial insights into the molecular mechanisms of ion channel function. Much less is known about the function of SLC1 transporters. The aim of this DPhil is to determine the 3D structure of SLC1A transporters by cryo-electron microscopy and to investigate the channel function using identified modulators (e.g. ions or drugs). Similarly, the molecular mechanism of TREK-2 channel will be elucidated using known modulators and X-ray crystallography. The resulting information may be beneficial in the design of pharmacological K2P and SLC1 modulators through medicinal chemistry. Thus, this project will address the Bioscience for Health Doctoral Training Partnerships (DTP) priority area. This project addresses the BBSRC Systems approaches to the biosciences priority, as the biological questions will be answered through integrating data collection with computational modeling.
BfH, WCUB, ENWW
BfH, WCUB, ENWW
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011224/1 | 01/10/2015 | 31/03/2024 | |||
| 1801228 | Studentship | BB/M011224/1 | 01/10/2016 | 23/12/2020 |
| Description | During my DPhil, I aimed to understand how two membrane proteins, a solute carrier transporter and an ion channel, function in neuronal membranes. I used structural and biophysical methods to identify the molecular elements involved in the substrate selectivity and transport regulation. EAAT3, a member of SLC1 family, is a glutamate transporter. It is expressed ubiquitously in brain but is thought to play distinct roles in different areas of the brain. EAAT3 malfunction is linked to several neurological diseases and disorders, making it a therapeutic target. Due to its complex regulation, EAAT3 activity could be modulated at various levels. The lack of EAAT3-specific inhibitors has hindered our understanding of EAAT3 involvement in cellular processes with the efforts so far focusing on ligand-based approaches. To study the basis of substrate selectivity in the SLC1 family, I solved two structures of EAAT3 at 3.3 Å resolution with bound inhibitors using the cryo-EM method. The two novel inhibitor binding modes expand the current knowledge of the substrate binding site and how small molecules inhibitors interact with this site within the SLC1 family. TREK-2 is a member of the K2P potassium channel family, expressed in the CNS and implicated in neuropathic pain. The polymodal regulation of TREK-2 is not well understood, despite available 3D structures in two conformations. I used functional binders (nanobodies) to investigate the mechanism underlying TREK-2 regulation. I used X-ray crystallography to determine the structures of TREK-2 with bound activatory and inactivatory nanobodies at 2.4 Å and 3.5 Å resolution, respectively. Both nanobodies bind to the channel from the extracellular side but interact with different domains to elicit the opposite effects on the channel conductivity. These structures provide us with new insights into TREK-2 channel regulation. |
| Exploitation Route | The solved structures of EAAT3 provide us with information for subtype-specific drug design. Due to different expression patterns for members of SLC1 family (with EAAT3 selectively expressed in neurons) specific inhibitors are of particular interest. This is needed for conditions such as ischemia when EAAT3 is more likely to run in reverse transport mode, releasing glutamate from the cells and contributing to neuronal damage. Although the substrate binding site is highly conserved, there are subtype-specific differences, highlighted by the EAAT2-specific inhibitor WAY- 213613, which is substrate (L-aspartate) analogue. Allosteric modulators are an attractive alternative that would limit the cross-reactivity. This was successfully demonstrated by the EAAT1-specific inhibitor UCPH101 which binds in a conformation-dependent hydrophobic pocket, locking the transporter in outward-facing state. With the available 3D models of EAAT3, these conformation-dependent pockets can be explored for their druggability. Since EAAT3 plasma membrane expression is highly regulated, with the majority of the protein within intracellular vesicles, modulators that prevent its internalisation by shielding the internalisation motif could be used to increase its apparent activity. To learn more about K2P channel activation and inactivation mechanisms, the 3D structures of TREK- 2 with bound functional nanobodies were solved. The activating nanobody Nb67 was found to bind to the cap domain. To date, the cap domain has not been identified as a modulatory site in TREK-2. To investigate the mechanism of channel activation, mutagenesis studies of residues involved in Nb67 binding to the cap could be performed. This would potentially identify the residues within the cap domain involved in signal sensing that the Nb67 is utilising. The inactivating nanobody Nb61 also binds from the extracellular side but interacts with a TREK-2 loop previously identified essential for channel inactivation. Further mutagenesis studies of residues involved in Nb61 binding would be required to fully understand the underlying mechanism. |
| Sectors | Pharmaceuticals and Medical Biotechnology |