Biophysical implications of lipid bilayer rheometry for mechanosensitive channels

Bavi, Navid; Nakayama, Yoshitaka; Bavi, Omid; Cox, Charles D.; Qin, Qing Hua; Martinac, Boris

doi:10.1073/pnas.1409011111

Cited by 63 publications

(72 citation statements)

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“…Nevertheless, direct functional studies of membrane curvature and leaflet asymmetry are experimentally challenging (6,47), particularly with eukaryotic MS channels. Traditionally, pipettebased electrophysiological recordings are used in conjunction with the application of either positive or negative pressure to stretch the membrane; however, numerous factors arising from the Ω shape of the membrane within the pipette and preferential adhesion of the outer leaflet to the glass complicate the interpretation of such experiments (6,18,48), and might have contributed to the differences in the reported effects of negative vs. positive pressure on K2P channels (23,24,43). Therefore, we chose a more controlled approach and examined the mechanosensitivity of purified TREK-2 proteins reconstituted in a lipid bilayer system with a defined symmetrical composition and less-complex geometry.…”

Section: Resultsmentioning

confidence: 99%

“…Interactions with the cytoskeleton and other proteins will undoubtedly influence the response (1), but a fundamental understanding of their intrinsic response to changes in bilayer tension is still required. Previous computational and theoretical studies of membrane tension have emphasized how stretch-induced changes in the lateral pressure profile might not be symmetrical between the two leaflets (i.e., inner and outer) of the lipid bilayer and explored how this may influence gating (16)(17)(18)(19)(20), but these studies were restricted mostly to prokaryotic MS channels. More tractable and better-defined experimental systems for the study of eukaryotic MS channels are needed.…”

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confidence: 99%

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Asymmetric mechanosensitivity in a eukaryotic ion channel

Clausen

Jarerattanachat

Carpenter

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian twopore domain (K2P) K + channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli.any forms of sensory perception in higher organisms depend on the rapid conversion of mechanical and physical stimuli into the electrochemical language of nerve and muscle cells (1). These transduction mechanisms often involve "mechanosensitive" (MS) ion channels that are able to rapidly convert physicomechanical stimuli into the electrical signals required for complex physiological responses (e.g., touch, pain, hearing, proprioception), as well those mechanical signals that play key roles in development and cell-cell communication (2). The enormous complexity and diversity of these sensory responses suggests that a variety of molecular mechanisms are likely responsible; for example, while some channels are regulated via physical coupling to proteins of the cytoskeleton and/or cell matrix, others respond directly to changes in lipid membrane tension (3). The precise structural and physical mechanisms underlying these processes remain poorly understood, however (4-6).Our current understanding of how membrane tension directly regulates ion channel gating is largely derived from studies of prokaryotic MS ion channels involved in the control of bacterial turgor pressure (7,8). The open state of these MS channels has a larger cross-sectional area than the closed state within the membrane, and stretch-induced changes in the forces within the bilayer stabilize the expanded open-state conformation. This is commonly referred to as the force-from-lipid principle of mechanosensitivity (9). Similar physical principles are thought to regulate many eukaryotic MS channels (5, 10).Typically, the functional activity of MS ion channels is examined in pipette-based patch-clamp recordings in which negative or positive pressures are used to alter bilayer tension. These pressure changes stretch the membrane to alter the lateral pressure profile, the complex profile of forces that vary with location acro...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Asymmetric mechanosensitivity in a eukaryotic ion channel

Clausen

Jarerattanachat

Carpenter

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…For comparison we also quantified the tension sensitivity of MscL-G22S in DPhPC bilayers using patch fluorometry as previously described2930. MscL-G22S was incorporated into DPhPC liposomes labeled with 0.1% fluorescent lipid (rhodamine-PE), which were subsequently used in the patch clamp experiments.…”

Section: Resultsmentioning

confidence: 99%

“…S8b). Following a previously established analysis method29 and using Laplace’s equation, we quantified bilayer tension from the patch curvature (Supplementary Fig. S8a) allowing us to plot the open probability, P o , of MscL-G22S as a function of bilayer tension (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers

Rosholm

Baker

Ridone

et al. 2017

Sci Rep

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The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform.

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“…The lipid bilayer was assumed to behave as a linear elastic plate 27,28 , and the secondary structural elements making the MscL protein were treated as elastic cylinders/rods, as described 6,29,30 . The Young's modulus of the bilayer was assumed to be 4.3 MPa as previously determined using excised patch fluorometry 28 and the Young's moduli of the MscL N-terminal (0.35 GPa), TM1 (2.6 GPa), TM2 (3.4 GPa) and C-terminal (7.7 GPa) α-helices were determined using steered molecular dynamics (MD) simulations 31 . The dimensions of the rods were: radius r = 2.5 Å, the lengths, N-terminus = 18.65 Å, TM1 = 47.33 Å, TM2 = 42.51 Å, C-terminus = 36.06 Å.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

Structural Dynamics of the MSCL C-Terminal Domain

Martinac

Bavi

Cortes

et al. 2017

Biophysical Journal

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The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.The bacterial mechanosensitive channel of large conductance (MscL), has become a prototype molecular system in the study of mechanotransduction and its evolutionary origins 1,2 . The three-dimensional (3D) structure of MscL from Mycobacterium tuberculosis (MtMscL) in its closed conformation was resolved at 3.5 Å 3 . The channel is a homopentamer consisting of N-and C-terminal domains facing the cytoplasm and TM1 and TM2 transmembrane helices connected by a periplasmic loop [3][4][5][6] . The closed channel structure was also determined in the lipid bilayer by site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy 7 . Moreover, the open channel structure was also determined by SDSL EPR spectroscopy 8 allowing for estimation of the size of the open channel pore in a very good agreement with electrophysiological sieving studies 9 . Later on, an improved open E. coli MscL (EcMscL) 3D structure was determined using both ensemble and single molecule site-directed fluorofore labelling (SDFL) FRET spectroscopy in combination with MD simulations 6,10,11 in agreement with the X-ray structure of an expanded form of an archaeal MscL homolog from Methanosarcina acetivorans 12 . While the overall gating-related structural changes in MscL have largely been established 8,11,13,14 the structural dynamics and physiological role of the C-terminal domain has thus far been controversial. Several studies have suggested that the C-terminus should remain intact during gating and could function as a molecular sieve 15,16 , while others suggested that this helical bundle should actively participate in gating and that opening of the MscL channel was accompanied by complete dissociation of the bundle 13,17 . More recently, the X-ray structure of the EcMscL C-terminus domain was obtained at 1. 45 Å 18 , suggesting that even in the absence ...

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Biophysical implications of lipid bilayer rheometry for mechanosensitive channels

Cited by 63 publications

References 50 publications

Asymmetric mechanosensitivity in a eukaryotic ion channel

Asymmetric mechanosensitivity in a eukaryotic ion channel

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers

Structural Dynamics of the MSCL C-Terminal Domain

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