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CryoEM Studies of Novel Ion Channel Families Involved in Cellular Sensory Processes
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Dissertation
CryoEM Studies of Novel Ion Channel Families Involved in Cellular Sensory Processes
2018
Overview
Every living thing must recognize and respond to changes in its environment. Sensory receptors are the molecules that connect the outside world with the internal environment of the cell. They convert stimuli like light, heat, sound, and physical force into signals that the cell can use like membrane depolarization, protein modification, and changes in gene expression. My work has been on two channels, one involved in sensing physical force (Piezo1) and the other in changes in osmolarity (Volume-Regulated Anion Channel). Here I describe how to isolate these specialized membrane channels as well as structural details about the Volume-Regulated Anion Channel revealed by cryo-electron microscopy. In Chapter I, I describe the methods used to choose conditions for producing pure, stable membrane proteins for structural studies, with specific advice for optimizing these samples for cryo-electron microscopy (cryoEM). Chapter II outlines the molecular biology tools that were used to modify the genome of a cell line used for protein expression in order to simplify the study of a ubiquitously expressed heteromeric channel. Finally, in Chapter III, the cryoEM structure of the homomeric Volume-Regulated Ion Channel composed of SWELL1 is presented revealing its trimer of dimers architecture and providing details about how ions might permeate its pore and changes in ionic strength might alter its conformation. These studies lay the foundation for the study of these important channels and add to the momentum of membrane protein structure solution by cryoEM.
Publisher
ProQuest Dissertations & Theses
Subject
ISBN
9781658495639, 1658495632
