Mechanosensitive channels (MSCs) are critical for cells to sense and respond to mechanical stimuli. MSCs are widely expressed, and defects in mechano-transduction are associated with numerous diseases. MSCs is a ubiquitous turgor-regulating bacterial MSC that is used as a model system for the entire family of channels in both prokaryotes and eukaryotes. The existing structures of E. coli MSCs fall into two categories: (1) splayed non-conductive structures with a decoupled gate, typically observed in the presence of regular-chain lipids, and (2) expanded, semi-conductive structures, observed in either delipidated samples or in the presence of short-chain lipids. The G168D mutant of MSCs does not exhibit tension-dependent inactivation in patch-clamp electrophysiology, has dramatically decreased osmotic viability, and does not adopt the splayed, non-conductive conformation associated with the inactivated state under the conditions used in our cryo-EM studies. Glycine 168 is situated at the tightly packed interface between the TM3b helix, which runs parallel to the membrane, and the β domain forming the upper part of the cytoplasmic cage. The destabilization of the inactivated state caused by a bulkier, charged aspartate at this site suggests that these contacts may not be present in the closed state. Our structure of G168D in mixed micelles shows a semi-open state with partially straightened TM3 helices and a loose TM3b-β contact. Interestingly, the particles were divided between regular heptamers and a smaller subset of octamers. The inter-subunit contacts in both stoichiometries appeared similar, and patch-clamp shows no deviation from the standard 1.1 nS unitary channel conductance. Based on the low fidelity in complex assembly of this non-inactivating mutant, we hypothesize that the inactivated state may be integral to the initial assembly stage of newly synthesized MSCs channels from protomers.
Moller et al. (Sun,) studied this question.