The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics.

The lipid bilayer plays a crucial role in gating of mechanosensitive (MS) channels. Hence it is imperative to elucidate the rheological properties of lipid membranes. Herein we introduce a framework to characterize the mechanical properties of lipid bilayers by combining micropipette aspiration (MA) with theoretical modeling. Our results reveal that excised liposome patch fluorometry is superior to traditional cell-attached MA for measuring the intrinsic mechanical properties of lipid bilayers. The computational results also indicate that unlike the uniform bilayer tension estimated by Laplace's law, bilayer tension is not uniform across the membrane patch area. Instead, the highest tension is seen at the apex of the patch and the lowest tension is encountered near the pipette wall. More importantly, there is only a negligible difference between the stress profiles of the outer and inner monolayers in the cellattached configuration, whereas a substantial difference (∼30%) is observed in the excised configuration. Our results have farreaching consequences for the biophysical studies of MS channels and ion channels in general, using the patch-clamp technique, and begin to unravel the difference in activity seen between MS channels in different experimental paradigms.MscL | azolectin | electrophysiology | finite element modeling L iposome reconstitution has been used for both functional and structural studies of bacterial mechanosensitive (MS) channels (MscL and MscS) for many years (1-4) and also more recently for the study of eukaryotic MS channels (5-8). Most frequently azolectin, a crude extract of phospholipids from animal and plant tissue consisting mainly of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) (9), has been used for liposome preparations. Given the frequent use of azolectin bilayers for the study of MS channels and the importance of protein-lipid interactions in MS channel gating it is imperative to consider the best way to accurately calculate and assess its material properties. This is also essential for studies of voltage-and ligand-gated ion channels as the activity of these proteins can also be modulated by membrane tension (6-8, 10-12).The mechanical properties of different types of lipids have been studied extensively using the micropipette aspiration (MA) approach (13-18). These experiments have shown that the mechanical properties of lipids differ considerably and that lipid bilayers exhibit elastic behavior (14,15,18,19). The MA technique is versatile and has the advantage of exerting a wide range of aspiration pressures on a specific portion of a bilayer (20-23).Here we use this technique in combination with finite-element (FE) simulation to examine in depth the mechanical properties of azolectin bilayers in both cell-attached and excised patch configurations (SI Materials and Methods). It is widely known that lipid behavior is vastly different at low (<0.5 mN/m) compared with high tensions, due to thermal shape fluctuations (24). Given that the m...

Mechanosensitive (MS) channels are ubiquitous molecular force sensors that respond to a number of different mechanical stimuli including tensile, compressive and shear stress. MS channels are also proposed to be molecular curvature sensors gating in response to bending in their local environment. One of the main mechanisms to functionally study these channels is the patch clamp technique. However, the patch of membrane surveyed using this methodology is far from physiological. Here we use continuum mechanics to probe the question of how curvature, in a standard patch clamp experiment, at different length scales (global and local) affects a model MS channel. Firstly, to increase the accuracy of the Laplace’s equation in tension estimation in a patch membrane and to be able to more precisely describe the transient phenomena happening during patch clamping, we propose a modified Laplace’s equation. Most importantly, we unambiguously show that the global curvature of a patch, which is visible under the microscope during patch clamp experiments, is of negligible energetic consequence for activation of an MS channel in a model membrane. However, the local curvature (RL < 50) and the direction of bending are able to cause considerable changes in the stress distribution through the thickness of the membrane. Not only does local bending, in the order of physiologically relevant curvatures, cause a substantial change in the pressure profile but it also significantly modifies the stress distribution in response to force application. Understanding these stress variations in regions of high local bending is essential for a complete understanding of the effects of curvature on MS channels.