ORCID
https://orcid.org/0009-0005-6130-2779
Date of Award
12-4-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
All living organisms experience exogenous and endogenous mechanical stress that are fundamental to their growth, survival, and development. A prominent strategy to manage these stresses and ensure efficient physiological responses is through mechanosensitive (MS) ion channels by transforming mechanical forces into electrochemical signaling. However, given their structural diversity and the inherent challenges of applying membrane tension during structural study, how MS channels sense mechanical force and transit into different functional states remain unclear. MSL channels in plants, members of the mechanosensitive ion channel of small conductance (MscS) superfamily, provide a compelling avenue for studying the structural basis for gating of MS ion channels, owing to their diversity in cellular localization, physiological functions, domain arrangement and membrane topology. This dissertation specifically focuses on MSL1, MSL8 and MSL10 channels in Arabidopsis thaliana because these channels have been functionally characterized. In this work, we observed the bowl-shaped transmembrane domain (TMD) of the wildtype AtMSL1 channel. With the combination of mutagenesis and electrophysiology, a gain-ofxi function mutant, AtMSL1 A320V, was identified. Structural studies of this mutant revealed a pronounced conformational change at the TMD upon channel opening. The peripheral TM helices moved as a rigid body and pulled the pore-lining helix to open this channel. Notably, the curved TMD got flattened, resulting in large in-plane expansion in the membrane, while the cytoplasmic domain (CTD) remained stationary. Additionally, we determined the cryo-EM structure of the wild-type AtMSL10 channel. Unprecedently, without applied membrane tension, this structure represented an open conformation of AtMSL10. Moreover, unlike AtMSL1 or EcMscS, AtMSL10 adopted a nondomain swapped architecture, suggesting a distinct gating mechanism. Further structural analysis of a non-conductive mutant, G556V, implied that the reorientation of the phenylalanine side chains at the narrowest site at the pore might cause the channel closure. This local distortion in AtMSL10, in contrast to the dramatic structural rearrangements in AtMSL1 or EcMscS, would align with its rapid opening and closing kinetics required for wind sensing. Furthermore, we performed structural and functional analysis of AtMSL8. Departing from the heptameric channel assemblies in the all other known MscS homologs, the wild-type AtMSL8 channel forms a hexamer in detergent micelles, amphiphilic polymers, or lipid environments. Structural comparison with a close homolog, the AtMSL10 channel, led us to postulate that the C-terminal β-barrel of AtMSL8 might be the determinant factor for its unique oligomerization. In addition, a potential gain-of-function mutant, L707A, was identified through pollen phenotype screening for future studies of the gating mechanism of AtMSL8. Taken together, this thesis provides important structural and functional insights into MS channel gating and advance our understanding of mechanotransduction.
Language
English (en)
Chair and Committee
Jianmin Cui
Recommended Citation
Zhang, Jingying, "Structural Basis of Gating in MSL Channels" (2023). Arts & Sciences Electronic Theses and Dissertations. 3197.
https://openscholarship.wustl.edu/art_sci_etds/3197