Andrew R. Battle

Differential effects of lipids and lyso-lipids on the mechanosensitivity of the mechanosensitive channels MscL and MscS

Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

Glutamate efflux mediated by Corynebacterium glutamicum MscCG, Escherichia coli MscS, and their derivatives.

Corynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, in particular glutamate. The mechanism of glutamate excretion, however, is not yet fully understood. Recently, evidence was provided that the NCgl1221 gene product from C. glutamicum ATCC 13869, a MscS-type mechanosensitive efflux channel, is responsible for glutamate efflux [1]. The major difference of NCgl1221 and the homologous protein MscCG of C. glutamicum ATCC 13032 from Escherichia coli MscS and most other MscS-type proteins is the presence of an additional, 247 amino acid long C-terminal domain. By topology analysis, we show that this domain in MscCG carries a transmembrane segment. We have generated selected C-terminal truncations of MscCG, gain-of-function and loss-of-function constructs of both E. coli MscS and C. glutamicum MscCG, as well as fusion constructs of the two proteins. These mutant proteins were investigated for mechanosensitive efflux, MS channel activity, glutamate excretion and their impact on membrane potential. We provide evidence that the channel domain of MscCG mediates glutamate efflux in response to penicillin treatment, and that the E. coli MscS channel is to some extent able to function in a similar manner. We further show that the C-terminal domain of MscCG has a significant impact for function and/or regulation of MscCG. Significantly, a positive effect on glutamate efflux of the C-terminal extension of MscCG from C. glutamicum was also observed when fused to the E. coli MscS channel.

Mechano-regulation of the beating heart at the cellular level – Mechanosensitive channels in normal and diseased heart

The heart as a contractile hollow organ finely tunes mechanical parameters such as stroke volume, stroke pressure and cardiac output according to filling volumes, filling pressures via intrinsic and neuronal routes. At the cellular level, cardiomyocytes in beating hearts are exposed to large mechanical stress during successive heart beats. Although the mechanisms of excitation-contraction coupling are well established in mammalian heart cells, the putative contribution of mechanosensitive channels to Ca²⁺ homeostasis, Ca²⁺ signaling and force generation has been primarily investigated in relation to heart disease states. For instance, transient receptor potential channels (TRPs) are up-regulated in animal models of congestive heart failure or hypertension models and seem to play a vital role in pathological Ca²⁺ overload to cardiomyocytes, thus aggravating the pathology of disease at the cellular level. Apart from that, the contribution of mechanosensitive channels (MsC) in the normal beating heart to the downstream force activation cascade has not been addressed. We present an overview of the current literature and concepts of mechanosensitive channel involvement in failing hearts and cardiomyopathies and novel data showing a likely contribution of Ca²⁺ influx via mechanosensitive channels in beating normal cardiomyocytes during systolic shortening.

Lipid–protein interactions: Lessons learned from stress

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.

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 10xa0% 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.

“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.nnnSUMMARYnMechanosensitive 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.

Biophysics of Mechanotransduction

(Singer and Nicolson 1972). As a physical barrier it presents a major target of mechanical forces stretching, compressing, bending or even breaking it, if an excessive force is acting on it. As a composite of a large variety of lipid molecules such as phospholipids, sphingolipids, cholesterol and many others, the lipid bilayer of the cell membrane presents, mechanistically speaking, a supporting matrix for a great variety of membrane-associated proteins. To strengthen its mechanical properties, the lipid bilayer can in different types of cells be supported by a cell wall as in bacterial and plant cells or can associate with extracellular (EC) matrix and cytoskeletal (CSK) proteins as in animal and human cells (Hamill and Martinac 2001). Membrane proteins embedded in the lipid bilayer of the cell membrane are functional units performing a range of functions enabling the survival of biological cells. A class of membrane proteins functioning as biological force-sensing systems are mechanosensitive (MS) ion channels, which together with cytoskeleton and muscles represent firmly established biological mechanosensors (Martinac 2014). MS channels have over the last two decades entered into the focus of the mechanobiology research area and are, together with the lipid bilayer, also the focus of this issue of the European Biophysics Journal. This special issue on the biophysics of mechanotransduction assembles nine papers from a number of leading scientists interested in mechanical properties of cell membrane and its components, which play a role in mechanosensory transduction in living cells. The papers in this issue are based on several presentations given at the Mechanotransduction Satellite Symposium, which took place last year in Broadbeach on the Gold Coast in conjunction with the 2014 IUPAB International Biophysics Congress in Brisbane, Australia. Mechanosensory transduction is ancient, paralleling the appearance of the first primordial cells on the surface of the Earth some 3.8 billion years ago. These primal organisms experienced osmotic pressure as the first likely mechanical stimulus resulting from the inherent role that water plays in the existence of all life forms. Mechanical stimuli acting on the variety of living organisms existing today include, for example, sound and direct contact, for which these organisms developed specialized mechanoreceptors serving as transducers of these mechanical stimuli into senses of hearing and touch, respectively. Other forms of mechanotransduction range from turgor pressure regulation in microorganisms such as bacteria and yeasts to gravitropism in plants or blood flow regulation in humans (Hamill and Martinac 2001). Central to mechanotransduction is the cellular membrane surrounding every living cell. The cell membrane provides a separation between the extracellular and intracellular compartments and serves as a highly dynamic functional barrier composed of membrane proteins and lipid bilayer, controlling the traffic of ions, water and nutrients between these compartments

Force from Lipids: A Multidisciplinary Approach to Study Bacterial Mechanosensitive Ion Channels

Since their discovery 25 years ago mechanosensitive (MS) ion channels have been the topic of intensive scientific research. Much of what we know about structure and function of these channels derives from prokaryotic sources, and from patch clamp electrophysiology techniques and X-ray crystallography. But as technology develops so too do the methods employed to investigate the structure and function of these channels become more diverse. Techniques such as electron paramagnetic resonance (EPR) spectroscopy, Forster resonance energy transfer (FRET) imaging and nuclear magnetic resonance (NMR) spectroscopy have all been used to increase our understandings of these channels. But with every new technique come new challenges in sample preparation. Just what is the best way to prepare a MS ion channel for FRET imaging? Which mutants need to be generated? Can we incorporate a MS channel protein into an artificial lipid or will we have to perform the experiments in vivo? To help answer these questions we have documented some of the most common techniques that have been used to investigate MS ion channels to date. We discuss what these techniques have been able to tell us about MS ion channel structure, their molecular dynamics, and just how these channels are capable of responding to mechanical forces exerted from their surrounding lipid bilayer.