Mechanosensitive (MS) channels sense and respond to mechanical forces by opening an ion-conducting
pathway. MS channels are found in all kingdoms of life, and in humans play essential roles in a number of
sensory processes, including hearing, the sense of touch, balance and regulation of blood pressure. The first
MS channels likely evolved in early prokaryotes as protection from hypoosmotic stress. Because bacterial MS
channels are ubiquitously expressed in bacteria, but not in humans, and because their uncontrolled opening has
a deleterious and often lethal effect on the bacteria, presumably due to the loss of important metabolites, bacterial
MS channels are intriguing targets for developing novel antibiotics. Bacteria express two types of MS channels,
MS channels of large conductance (MscL) and MS channels of small conductance (MscS). Members of the MscL
family are highly conserved and MscL has become a paradigm for the understanding of MS channels because
of its simplicity and amenability to different experimental approaches. MscS channels are more diverse, and
bacteria often express more than one paralog. Both bacterial MS channels are gated based on the ‘force-from-
lipids’ principle and respond to the transmembrane pressure profile of the surrounding membrane. However,
even though structures are available for MscL and MscS in different functional states, the mechanism by which
membrane tension opens these channels has remained enigmatic. We have recently determined cryo-electron
microscopy (cryo-EM) structures of MscS in different membrane environments, provided by nanodiscs, including
one mimicking a membrane under tension. The structures, complemented by molecular dynamics (MD)
simulations and electrophysiological studies, allowed us to visualize the channel in different functional states and
to deduce what roles lipids associated with MscS play in mechanosensation. We will continue to use a
combination of single-particle cryo-EM, patch-clamp electrophysiology and MD simulations to study the structure
and gating of bacterial MS channels. In Aim 1, we will continue to explore the function of lipids in MscS function,
in particular whether it adopts a defined open conformation in a native lipid environment, how modulators affect
MscS by changing its lipid environment, and whether 16-carbon acyl chains play a specific role in MscS gating.
In Aim 2, we will expand our studies to bacterial cyclic nucleotide-gated (bCNG) channels to elucidate how the
MscS fold was adapted to make the channel respond to cAMP binding rather than membrane tension. Aim 3 will
focus on MscL. We will determine the structure of MscL in a native lipid environment to confirm (or disprove) the
existence of lipid-filled nano-pockets that were suggested to play a critical role in gating. Finally, we will determine
the structure of MscL opened by different effectors to visualize the structure of this channel in the open state and
to test our hypothesis that different effectors result in open conformations with different pore diameters. The
results of these studies will not only provide new insights into the gating mechanism of bacterial MS channels,
but also help in exploiting these channels for biomedical applications.