Exploring the Dynamics of Mechanosensitive Channels Utilizing a Post Translational Modification Approach
type: CreativeWork
description: It is essential for all living organisms to possess the ability to sense and respond to changes in their environment. The ability to detect external and internal mechanical force is very important as well. The detection of mechanical forces underlies the ability to sense touch, gravity, blood pressure, and changes in osmolarity. The bacterial mechanosensitive channel of large conductance (MscL) and the mechanosensitive channel of small conductance (MscS), from E. coli, have been the most studied mechanosensors to date. These channels coordinate a response to rapid changes in osmolarity to serve as emergency release valves during an osmotic downshock (sudden change to a lower osmotic environment). Crystal structures have been solved for homologues of each of these channels. In addition, patch clamp of giant bacterial spheroplasts has allowed for the study of channel activities in native membranes, and both channels have been functionally reconstituted into membranes and studied by electrophysiological single-channel analyses. However, even though well studied, it is still unclear how specific regions of the protein interact with the membrane, and how these interactions change upon channel gating. Here we hypothesize that during channel gating specific residues of MscL change their local environment. We investigate this hypothesis by using a post-translational modification approach to determine how changing hydrophobicity or charge at specific locations influences gating. Briefly, we used MscL channels with specific residues in the linker between the second transmembrane region (TM2) and the cytoplasmic α-helical bundle changed to cysteine. Changing the individual amino acids to cysteine allows us to use Methanethiosulfonate (MTS) sulfhydryl reagents that can form disulfide bonds with cysteines to change the properties (charge or hydrophobicity) of specific residues. We then use the double knockout strain, ∆MscL/∆MscS, which is osmotically fragile and has a reduced vi…
abstract: It is essential for all living organisms to possess the ability to sense and respond to changes in their environment. The ability to detect external and internal mechanical force is very important as well. The detection of mechanical forces underlies the ability to sense touch, gravity, blood pressure, and changes in osmolarity. The bacterial mechanosensitive channel of large conductance (MscL) and the mechanosensitive channel of small conductance (MscS), from E. coli, have been the most studied mechanosensors to date. These channels coordinate a response to rapid changes in osmolarity to serve as emergency release valves during an osmotic downshock (sudden change to a lower osmotic environment). Crystal structures have been solved for homologues of each of these channels. In addition, patch clamp of giant bacterial spheroplasts has allowed for the study of channel activities in native membranes, and both channels have been functionally reconstituted into membranes and studied by electrophysiological single-channel analyses. However, even though well studied, it is still unclear how specific regions of the protein interact with the membrane, and how these interactions change upon channel gating. Here we hypothesize that during channel gating specific residues of MscL change their local environment. We investigate this hypothesis by using a post-translational modification approach to determine how changing hydrophobicity or charge at specific locations influences gating. Briefly, we used MscL channels with specific residues in the linker between the second transmembrane region (TM2) and the cytoplasmic α-helical bundle changed to cysteine. Changing the individual amino acids to cysteine allows us to use Methanethiosulfonate (MTS) sulfhydryl reagents that can form disulfide bonds with cysteines to change the properties (charge or hydrophobicity) of specific residues. We then use the double knockout strain, ∆MscL/∆MscS, which is osmotically fragile and has a reduced vi…
author: Parker, Juandell Latrice
language: en
keywords: Ion Channels, Mechanoreceptors, Protein Processing, Post-Translational
date published: 2014-05-12T19:10:32Z
date modified: 2026-07-24T05:52:31Z
provider: University of Texas Southwestern Medical Center
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Texas Southwestern Medical Center

PropertyValue
property ID: Handle
value: https://hdl.handle.net/2152.5/1412