Modulation of mechanosensitive channel activity by plasma membrane lipids

Biological cells are sensitive to physical stimuli, however the mechanisms underlying this sensitivity are still poorly understood. The sensation and conversion of a physical stimulus into electrical and biochemical intracellular signalling, a process termed mechanosensory transduction (mechanotransduction), is mediated by a variety of membrane-bound proteins loosely referred to as mechanoreceptors or mechanotransducers. These encompass all the active and passive membrane components of the mechanotransductory process, including the extracellular matrix, cytoskeleton and crosslinking intermediate filaments, non-conductive membrane proteins, transporters, ion channels, chromatin and lastly, the plasma membrane itself. Biological membranes are essential structural components of living cells which encapsulate and compartmentalize the biochemical processes essential for life and act as a primary host for mechanoreceptors. A specific class of mechanoreceptors, the mechanosensitive (MS) ion channels, is responsible for the fast conversion of physical stimuli into ionic currents. Currently little is known about the role of membrane lipids in regulating the activity of mechanosensitive channels in humans. The following thesis aims to describe how specific membrane lipids can modulate the activity of tension-gated MS channels, focussing specifically on the eukaryotic channel PIEZO1. By employing a combination of patch-clamp electrophysiology and fluorescence microscopy techniques it was possible to characterize the effects of cholesterol and poly-unsaturated fatty acids on the function and localization of PIEZO1 in cultured human cells. Manipulation of membrane lipids produced dramatic effects on the sensitivity and kinetics of PIEZO1, an essential receptor of membrane tension involved in development and pathology. Furthermore, the observed effects could be recapitulated in minimal in vitro liposome systems where the purified PIEZO1 channel was reconstituted. This work demonstrates that altered lipid environments can impact on the functioning of PIEZO1 and can result in phenotypes reminiscent of mutant channel variants involved in pathology. This work furthers our understanding of the general principles behind the membrane dependence of the mechanosensory processes that govern many aspects of cellular life and will provide new insights on how cells regulate essential adaptive processes such as growth, movement and gene expression

Binding of fullerenes and nanotubes to MscL

Multi-drug resistance is becoming an increasing problem in the treatment of bacterial infections and diseases. The mechanosensitive channel of large conductance (MscL) is highly conserved among prokaryotes. Evidence suggests that a pharmacological agent that can affect the gating of, or block the current through, MscL has significant potential as a new class of antimicrobial compound capable of targeting a range of pathogenic bacteria with minimal side-effects to infected patients. Using molecular dynamics we examine the binding of fullerenes and nanotubes to MscL and demonstrate that both are stable within the MscL pore. We predict that fullerenes will attenuate the flow of ions through MscL by reducing the pore volume available to water and ions, but nanotubes will prevent pore closure resulting in a permanently open pore. Moreover, we confirm experimentally that it is possible to attenuate the flow of ions through MscL using a C60-γ 3 cyclodextrin complex

Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-β-cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP’s response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure–response curve, increased channel latency in response to mechanical stimuli, and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM superresolution imaging revealed that, at the nanoscale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of polyunsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane composition and lateral organization of membrane cholesterol domains, which coordinate the activity of clustered PIEZO1 channels

Patch clamp characterization of the effect of cardiolipin on MscS of E. coli

The bacterial mechanosensitive channels MscS and MscL are gated by an increase in membrane tension when the bacterium experiences hypoosmotic shock. It has been well established that membrane lipids modulate the mechanosensitivity and gating behavior of these channels. The focus of this study is a negatively charged phospholipid, cardiolipin, which has been shown to localize at curved regions of the bacterial cell, including the poles and the septum, and to have a strong preference for binding to membrane proteins. Here we characterize the effect of cardiolipin on MscS, the mechanosensitive channel of small conductance, using patch-clamp electrophysiology. We compare the gating kinetics and mechanosensitivity of the channel in both azolectin and mixtures of pure lipids DOPE/DOPC liposomes with and without cardiolipin. In azolectin liposomes, the addition of 10 % cardiolipin abolishes hysteresis of MscS, but MscL remains largely unaffected, indicating that cardiolipin may stabilize the closed state of MscS. On the other hand, mixtures of DOPE/DOPC abolish the hysteresis gating of MscS even in the absence of cardiolipin, and the addition of cardiolipin increases the opening and closing thresholds of both MscS and MscL. In addition, we show that MscS gates more frequently when cardiolipin is present in both the azolectin and pure lipid systems; this dose-dependent effect ultimately destabilizes the open state of MscS and we consider the functional implications of this cardiolipin effect in the bacterial osmotic response. Our results show that cardiolipin modulates the mechanosensitivity and gating characteristics of MscS, indicating its important role in the physiology of bacterial cells

Systematic discovery of the ‘force-from-lipid’ principles

The functioning of bacterial mechanosensitive channels is governed by the lipid environment in which they are embedded. Several physical parameters such as lipid charge, saturation and topography, work in concert during transmission of tension from the bilayer onto the ion channel, thus making it a difficult task to dissect the contribution of each parameter to the channel's mechanosensitivity

Systematic discovery of the ‘force-from-lipid’ principles

The functioning of bacterial mechanosensitive channels is governed by the lipid environment in which they are embedded. Several physical parameters such as lipid charge, saturation and topography, work in concert during transmission of tension from the bilayer onto the ion channel, thus making it a difficult task to dissect the contribution of each parameter to the channel's mechanosensitivity

Lipid-protein interactions: Lessons learned from stress

Full-text article is free to read on the publisher's website Biological membranes are essential for normal function and regulation of cells, forming a physical barrier between extracellular and intracellular space and cellular compartments. These physical barriers are subject to mechanical stresses. As a consequence, nature has developed proteins that are able to transpose mechanical stimuli into meaningful intracellular signals. These proteins, termed Mechanosensitive (MS) proteins provide a variety of roles in response to these stimuli. In prokaryotes these proteins form transmembrane spanning channels that function as osmotically activated nanovalves to prevent cell lysis by hypoosmotic shock. In eukaryotes, the function of MS proteins is more diverse and includes physiological processes such as touch, pain and hearing. The transmembrane portion of these channels is influenced by the physical properties such as charge, shape, thickness and stiffness of the lipid bilayer surrounding it, as well as the bilayer pressure profile. In this review we provide an overview of the progress to date on advances in our understanding of the intimate biophysical and chemical interactions between the lipid bilayer and mechanosensitive membrane channels, focusing on current progress in both eukaryotic and prokaryotic systems. These advances are of importance due to the increasing evidence of the role the MS channels play in disease, such as xerocytosis, muscular dystrophy and cardiac hypertrophy. Moreover, insights gained from lipid-protein interactions of MS channels are likely relevant not only to this class of membrane proteins, but other bilayer embedded proteins as well. This article is part of a Special Issue entitled: Lipid-protein interactions

Functional similarities between heterogeneously and homogenously expressed MscL constructs

The mechanosensitive channel of large conductance MscL is a well-characterized mechanically gated non-selective ion channel, which often serves as a prototype mechanosensitive channel for mechanotransduction studies. However, there are some discrepancies between MscL constructs used in these studies, most notably unintended heterogeneous expression from some MscL expression constructs. In this study we investigate the possible cause of this expression pattern, and compare the original non-homogenously expressing constructs with our new homogeneously expressing one to confirm that there is little functional difference between them. In addition, a new MscL construct has been developed with an improved molar extinction coefficient at 280 nm, enabling more accurate protein quantification

“Force-from-lipids” gating of mechanosensitive channels modulated by PUFAs

The level of fatty acid saturation in phospholipids is a crucial determinant of the biophysical properties of the lipid bilayer. Integral membrane proteins are sensitive to changes of their bilayer environment such that their activities and localization can be profoundly affected. When incorporated into phospholipids of mammalian cells, poly-unsaturated fatty acids (PUFAs) determine the mechanical properties of the bilayer thereby affecting several membrane-associated functions such as endo- and exo-cytosis and ion channel/membrane receptor signalling cascades. In order to understand how membrane tension is propagated through poly-unsaturated bilayers, we characterized the effect of lipid saturation on liposome reconstituted MscS and MscL, the two bacterial mechanosensitive ion channels that have for many years served as models of ion- channel-mediated mechanotransduction. The combination of NMR and patch clamp experiments in this study demonstrate that bilayer thinning is the main responsible factor for the modulation of the MscL threshold of activation while a change in transbilayer pressure profile is indicated as the main factor behind the observed modulation of the MscS kinetics. Together, our data offer a novel insight into how the structural shape differences between the two types of mechanosensitive channels determine their differential modulation by poly-unsaturated phospholipids and thus lay the foundation for future functional studies of eukaryotic ion channels involved in the physiology of mechanosensory transduction processes in mammalian cells. Summary Mechanosensitive channels MscL and MscS are differentially modulated by poly-unsaturated fatty acids in lipid bilayers. MscL becomes sensitized because of increased hydrophobic mismatch while MscS open state is stabilized due to changes in the bilayer lateral pressure profile determined by NMR