Dataset Information

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.

SUBMITTER: Machiyama H

PROVIDER: S-EPMC2726311 | biostudies-other | 2009 Aug

REPOSITORIES: biostudies-other

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Structural changes in the cytoplasmic domain of the mechanosensitive channel MscS during opening.

Machiyama Hiroaki H Tatsumi Hitoshi H Sokabe Masahiro M

Biophysical journal 20090801 4

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 dur ...[more]

PMID: 19686652

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Project description:Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1-TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1-TM2 shaft and the state of the associated hollow cytoplasmic domain ("cage"). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the ? domain of the cage. We found that mutations destabilizing the TM3b-? interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.

Dataset Information

Structural changes in the cytoplasmic domain of the mechanosensitive channel MscS during opening.

Publications

Structural changes in the cytoplasmic domain of the mechanosensitive channel MscS during opening.

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