Structural Changes in the Cytoplasmic Domain of the Mechanosensitive Channel MscS During Opening

Abstract

The bacterial mechanosensitive channel MscS forms a homoheptamer of subunits composed of a transmembrane (TM) domain and a large cytoplasmic (CP) domain. Recent studies suggest that a lateral expansion of the TM domain, structural change in the CP domain, and TM-CP interactions are essential to open the channel. However, it has not been examined whether the CP domain undergoes structural changes during channel opening. The aim of this study was to estimate structural changes in the CP domain during channel opening using fluorescence resonance energy transfer (FRET) spectroscopy. To monitor changes in the horizontal diameter of the CP domain, four point mutants (A132C, F178C, L246C, and R259C), all of which had channel activity, were created and labeled with Alexa488 and Alexa568 for FRET analysis. The FRET efficiency of these mutants decreased when lysophosphatidylcholine was applied to open the channel, suggesting that the CP domain swells up when the channel opens. The degree of the decease in FRET efficiency after lysophosphatidylcholine treatment was smaller in the D62N/F178C mutant, which was deficient in the TM-CP interactions, than in the F178C mutant. These findings provide the first, to our knowledge, experimental evidence that the CP domain swells up during channel opening, and the swelling is mediated by the TM-CP interactions.

Genetic Screen for Potassium Leaky Small Mechanosensitive Channels (MscS) in Escherichia coli

“…It is their positions that force the TM3 gate to keep shut. Our model of TM3-␤ domain interaction parallels previous papers that suggested involvement of the cytoplasmic domain in gating (17,19,21,22). Binding/unbinding of ␤ domain to TM3b could influence activity of the channel but could also be the mechanism by which structural changes in the transmembrane domain are transmitted to changes in the cytoplasmic domain.…”

“…A few experimental tests have been conducted to support the model; however, the conclusions drawn from them were restricted to the transmembrane part of the channel. The MscS large cytoplasmic chamber was previously shown to change its conformation upon gating (19,(21)(22)(23); however, it is not understood nor is it modeled how this cage-like shaped domain is involved in gating. To obtain a comprehensive understanding of MscS gating, an approach that provides a broader search in a model-independent manner is required.…”

Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration

“…The retroviral expression vectors for GFP-CasmPR and GFP-Cas15YF/mPR were constructed by site-directed mutagenesis using the vectors for GFP-CasWT and GFP-Cas15YF expression as templates, following a procedure described previously (Machiyama et al, 2009). All Cas mutants used in this study were cloned to the retroviral expression vector pBabe-hygromycin together with the coding sequence for mEGFP or mCherry.…”

Structure and molecular mechanism of an anion-selective mechanosensitive channel of small conductance

“…A structural comparison of EcMscS in the closed state with the EcMscS A106V mutant revealed striking conformational changes in the TM domain but not in the cytoplasmic region (11). However, subsequent physiological (15) and biophysical (16,17) studies suggest that the cytoplasmic region also undergoes a large conformational change when the channel is open.…”