Abstract
ABSTRACT Mechanosensitive (MS) ion channels are widespread mechanisms for cellular mechanosensation that can be directly activated by increasing membrane tension. The well-studied MscS family of MS ion channels is found in bacteria, archaea, and plants. MscS-Like (MSL)1 is localized to the inner mitochondrial membrane of Arabidopsis thaliana, where it is required for normal mitochondrial responses to oxidative stress. Like Escherichia coli MscS, MSL1 has a pore-lining helix that is kinked. However, in MSL1 this kink is comprised of two charged pore-lining residues, R326 and D327. Using single-channel patch-clamp electrophysiology in E. coli, we show that altering the size and charge of R326 and D327 leads to dramatic changes in channel kinetics. Modest changes in gating pressure were also observed while no effects on channel rectification or conductance were detected. MSL1 channel variants had differing physiological function in E. coli hypoosmotic shock assays, without clear correlation between function and particular channel characteristics. Taken together, these results demonstrate that altering pore-lining residue charge and size disrupts normal channel state stability and gating transitions, and led us to propose the “sweet spot” model. In this model, the transition to the closed state is facilitated by attraction between R326 and D327 and repulsion between R326 residues of neighboring monomers. In the open state, expansion of the channel reduces inter-monomeric repulsion, rendering open state stability influenced mainly by attractive forces. This work provides insight into how unique charge-charge interactions can be combined with an otherwise conserved structural feature to help modulate MS channel function.
Highlights
Living organisms constantly experience physical force from both internal and external sources and possess a variety of mechanisms for detecting and responding to key mechanical stimuli [1,2,3]
We investigated the roles of R326 and D327 in MSL1 rectification and other hallmarks of MSL1 channel behavior using single-channel patch-clamp electrophysiology and physiological assays in E. coli
The Arabidopsis mitochondrial MS channel MSL1 contains a notable feature midway through its porelining TM5 helix: a kink formed by charged residues R326 and D327
Summary
Living organisms constantly experience physical force from both internal and external sources and possess a variety of mechanisms for detecting and responding to key mechanical stimuli [1,2,3] Among these mechanisms are mechanosensitive (MS) ion channels, which are found in all king doms of life [4,5,6,7]. While MS ion channels are united by their primary gating stimulus rather than a common mechanosensory sequence or structure, individual MS channel families have been identified by the presence of conserved domains One such family is the MscS family, which is defined by similarity to the E. coli Mechanosensitive ion channel of Small conductance (EcMscS) [9,10,11]. Increased membrane tension in turn opens MscS and MscL, allowing for rapid osmoregulation and pre venting cell damage [12,13,14,15,16]
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