Studying Mechanosensitive Ion Channels Using Liposomes

Martinac, Boris; Rohde, Paul R.; Battle, Andrew R.; Petrov, Evgeny; Pal, Prithwish; Foo, Alexander; Vásquez, Valeria; Huynh, Thuan; Kloda, Anna

doi:10.1007/978-1-60761-447-0_4

Cited by 38 publications

(26 citation statements)

References 22 publications

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“…Reconstitution in GUVs was performed according to a technique adapted from Martinac et al (28) starting from proteo-small unilamellar vesicles (SUVs) to achieve large protein densities. 2 mg of 1-pamitoyl-2-oleoyl-sn-glycero-3-phosphocholine was dissolved in trichloromethane in a glass test tube and dried under nitrogen for 30 min followed for 3 h under vacuum.…”

Section: Methodsmentioning

confidence: 99%

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Carnarius

Kreir

Krick

et al. 2012

Journal of Biological Chemistry

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Background: Connexin 43 hemichannels participate in many cellular processes. To elucidate their location and function within living cells, they were labeled with GFP. Results: Recombinantly expressed Cx43 and Cx43-GFP form conducting hemichannels in reconstituted planar membranes. Their conductance states and voltage dependence differ. Conclusion: Fusion of GFP to Cx43 significantly affects the electrophysiological behavior of Cx43 hemichannels. Significance: GFP can significantly alter channel activities.

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Section: Methodsmentioning

confidence: 99%

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Carnarius

Kreir

Krick

et al. 2012

Journal of Biological Chemistry

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“…Cell Physiol Biochem 2011;28:1051-1060 Nanion technologies GmbH (München, Germany; http:// www.nanion.de/products/port-a-patch/port-a-patchdata.html) [45]. Besides giving very good reconstitution efficiencies for both MscL and MscS, the sucrose method also offers significant time-savings over the D/R method with recordings of functional MscL and MscS channel activity being reported in 3 hours [46].…”

Section: Bacterial Ms Channelsmentioning

confidence: 99%

Bacterial Mechanosensitive Channels as a Paradigm for Mechanosensory Transduction

Martinac¹

2011

Cell Physiol Biochem

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Research on bacterial mechanosensitive (MS) channels has since their discovery been at the forefront of the MS channel field due to extensive studies of the structure and function of MscL and MscS, two of the several different types of MS channels found in bacteria. Just a few years after these two MS channels were cloned their 3D structure was solved by X-ray crystallography. Today, the repertoire of multidisciplinary approaches used in experimental and theoretical studies following the cloning and crystallographic determination of the MscL and MscS structure has expanded by including electronparamagnetic resonance (EPR) and Förster resonance energy transfer (FRET) spectroscopy aided by computational modelling employing molecular dynamics as well as Brownian dynamics simulations, which significantly advanced the understanding of structural determinants of the gating and conduction properties of these two MS channels. These extensive multidisciplinary studies of MscL and MscS have greatly contributed to elucidation of the basic physical principles of MS channel gating by mechanical force. This review summarizes briefly the major experimental and conceptual advancements, which helped in establishing MscL and MscS as a major paradigm of mechanosensory transduction in living cells.

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“…Subsequently, bacterial MS channel reconstitution into liposomes has become the gold standard for patch-clamp electrophysiology experiments, especially for MscL, where channel activity can be recorded in liposomes at protein/lipid ratios as low as 1:10,000 w/w (Fig. 5C) (97). Other prokaryotic MS channel proteins also successfully incorporated into liposomes using this method include those of the Archaeon Haloferax volcanii (85), the MscMJ and MscMJLR channels from Methanococcus jannashii (74,75), as well as the MscSP channel from Silicibacter pomeroyi (124).…”

Section: Reconstitution Of Mscl and Mscs Channels Into Liposomesmentioning

confidence: 99%

“…Only MscL of E. coli has been incorporated into liposomes using electroformation, and functional characterization was demonstrated by Martinac and co-workers (37). An automated electroformation method (Nanion Technologies GmbH, Mü nchen, Germany) instead uses a solvent-free approach to produce the MscL-loaded giant liposomes (97).…”

Section: Reconstitution Of Mscl and Mscs Channels Into Liposomesmentioning

confidence: 99%

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

Martinac

Nomura

Chi

et al. 2014

Antioxidants & Redox Signaling

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Significance: Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. Recent Advances: As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. Critical Issues: In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. Future Directions: A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology. Antioxid. Redox Signal. 00, 000-000.

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Studying Mechanosensitive Ion Channels Using Liposomes

Cited by 38 publications

References 22 publications

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Bacterial Mechanosensitive Channels as a Paradigm for Mechanosensory Transduction

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

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