The Structural Basis for Function of the Escherichia coli Mechanosensitive Channel of Small Conductance, MscS

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The 'small' mechanosensitive channel, MscS, resides in cytoplasmic membranes of most free-living bacteria. MscS is gated directly by membrane tension and functions as an osmolyte release valve in bacterial turgor regulation. In contrast to previously studied MscL, which is a strictly prokaryotic molecule, MscS homologs are found in eukaryotes increasing the value of this channel as a general model for gating by membrane stretch.

Presented here are the results of three studies aimed at characterizing the structural basis for function of Escherichia coli MscS. In study one, we provide the first electrophysiological characterization of the wild-type channel in its native membrane free of other mechanosensitive channels. It is, to date, the most complete description the gating cycle specifying the kinetic scheme and dependencies of major rates on tension and voltage. Study two represents a collaborative effort to probe the strength of intersubunit contacts in the homo-heptameric MscS channel. In patch-clamp experiments we show that the dissociating effects of TFE alter MscS gating in a manner that provides significant insight into the mechanics of channel inactivation. In the final study our research group utilized a novel extrapolated motion technique to explore the conformational pathways of the MscS functional cycle. Guided by these new models, channel mutants were generated to alter helical propensity along the pore lining TM3 helix. Patch-clamp analysis revealed a vivid picture of the functioning MscS in which these TM3 domains provide a structural frame for the open channel. Dynamic collapse of these 'struts' at flexible points along TM3 modulates transitions from the open state to the inactivated and closed states.

My contributions to these studies have allowed for (1) refinement of the MscS functional cycle including identification of a new desensitized state; (2) determination of the physical parameters and spatial scales of channel opening, closing and inactivation; and (3) identification of key hinge elements, residing in TM3, that along with membrane tension serve to modulate the functional cycle of MscS. These findings have led to a better understanding of the biophysical principles that underlie mechanotransduction and provide insights into the larger family of mechanically activated phenomena.