An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation

Abstract

Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

“…These data show good agreement with an earlier, electrophysiological, molecular sieving study carried out by Cruickshank et al in 1997 (38) and a 2010 FRET study by Corry et al (31) and suggest that structural plasticity is an inherent property of MscL. The cytoplasmic domain starts at the end of the TM2 helix, where the RKKEE motif in MscL of E. coli (RKKGE in M. tuberculosis), critical for pH sensing and channel function, is located (57,73).…”

“…Similar to the EPR spectroscopic studies (121,123), Corry et al labeled specific MscL residues that had been mutated to cysteines with flurophores for FRET studies (31,33 (31), with the TM2 helix lying closer to the channel pore than in previously suggested models (18,121,141). Furthermore, the FRET studies found that the N-terminus remains anchored at the surface of the membrane, suggesting it can either guide the tilt of or directly translate membrane tension to the TM1 porelining helix.…”

“…The pore closure is exposed on the periplasmic side, while on the cytoplasmic side, the pore is shielded by the five C-terminal helices (24,114). The pore region of MscL lacks any charged residues for imparting an ion selectivity filter function and, by opening a pore of *30 Å in diameter, expands to such a degree that ion selection becomes impossible in the open state, rendering the channel nonselective (31,38,61,121). This lack of ion selectivity in MscL and the large size of its open pore are essential for allowing passage of large organic osmolytes of *1000 in molecular weight and, hence, fulfilling its physiological role as a highly efficient emergency valve to release solutes from bacterial cells under .…”

“…Electron microscopy studies, along with the observed ability of MscL to let molecules as large as insulin pass through the pore, suggest that the C-terminal domain might dissociate and incorporate into the transmembrane domain during channel opening (151, 167). At odds with this, however, are MD simulations and mutational analyses, which suggest that the C-terminal domain remains intact and undergoes only minor dissociation proximal to the TM2 helix (31,40,52,146,162).…”

“…Several functional studies have demonstrated that even small N-terminal deletions or changes to the N-terminal amino-acid sequence are poorly tolerated, resulting in nonfunctional channels or channels which exhibit altered pressure sensitivity (20,59,143). More recent results suggest that the N-terminus contributes to MscL mechanosensitivity by directly sensing membrane tension and transferring it to the pore lining helices, most likely by maintaining its position along the membranecytoplasm interface (31,68). Here, the N-terminal domain functions as an anchor, guiding the TM1 transmembrane helix to a greater tilt during MscL opening (67).…”