Characterizing the Dynamic Regulation of the K2P ion channel, TRESK
An essential component to better understanding cellular electrical signaling is to address how the resting membrane potential in neurons is established and modified to affect excitability. Two-pore domain potassium (K2P) ion channels are directly involved in this process and comprise a unique protein family that is essential for the maintenance of this resting membrane potential. The TWIK-related spinal cord K+ channel (TRESK) is considered a major contributor to background K+ currents and is expressed abundantly in DRG neurons. This K2P channel is thought to be involved in pain sensation and sensory transduction, where its down-regulation may induce pain disorders such as allodynia and neuropathy, and loss of TRESK channel activity is strongly correlated to hereditary migraines with aura in humans. The goal of my research is to characterize the molecular structure of TRESK to understand the physical mechanism by which this channel operates.This characterization will begin with the expression and purification of TRESK genes from a multitude of species that will be screened via fluorescence size-exclusion chromatography (FSEC). Promising constructs will be scaled up and purified using consecutive metal affinity and size-exclusion chromatography to isolate the TRESK channel from all other molecules in our expression systems. Purified channels will then be reconstituted into artificial bilayers, a reductionist system that allows us to study how TRESK activity changes in response to particular regulatory elements involved in its associated signaling pathways. This is an essential step to characterize the basis of the channels function and to ensure the integrity of our purified components before embarking on structural studies using X-ray crystallography, currently the most powerful technique for determining atomic resolution structures of biological macromolecules.
Message to Sponsor
- Major: MCB Neurobiology
- Mentor: Stephen Brohawn