The full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period, even for Texas A&M users with NetID.
K2P Channel Lipid Binding and Structural Impacts
Abstract
Two-pore potassium channels (K₂ᴘ) are leak channels responsible for maintaining the resting membrane potential. While they are known to be activated by lysophospholipids and particularly sensitive to their lipid environment as mechanosensitive channels, their direct lipid interactions are poorly understood. Found in the nervous system, TREK channels are a subset of K₂ᴘ channels implicated in disease and responsible for pain sensation. Understanding these channels interactions with the surroundings can provide insight into the function of these and in turn the prevention or treatment. High resolution native mass spectrometry allows us detect post translational modifications as well as perform individual lipid binding measurements other techniques cannot detect. Interestingly, TRAAK is found to have two isoforms expressed in humans, each with unique lipid binding preferences, however both isoforms bind POPA avidly. During lipid screening, the protein is able to discriminate lipid tail length, unsaturation as well as the sn-1 linkage to the glycerol backbone resulting in various lipid binding affinities. When the channel is reconstituted in proteoliposomes, TRAAK is activated by POPA more than other PO lipids and can be measured in a dose dependent fashion. These fascinating results led us to perform the lipid studies on another member of the family TREK-2, a highly glycosylated member of the TREK family. After the improving the glycosylation, we were able to perform the lipid titrations as well as the functional assays. Even with a 45% sequence similarity to TRAAK, the lipid binding patterns proved to be very different, showing a higher affinity for most lipids except for a plasmalogen and a PIP lipid. The high affinity lipids most likely have a defined lipid binding spot that can be analyzed using structural studies. The Fab for TRAAK is particularly difficult to work with and binding is not reproducible increasing the difficulty for crystallography. A cryoEM structure of TRAAK in the presence of POPA was able to resolve a lipid binding site near the TM1 and 4. We have also explored alternative Fabs using helical epitope tags with high affinity in an attempt to find more viable options for structural studies.
Subject
TRAAKTREK
K2P
native mass spectrometry
lipids
dissociation constant
xray crystallography
FAB
Citation
Schrecke, Samantha Rose (2022). K2P Channel Lipid Binding and Structural Impacts. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /197756.