Unidirectional incorporation of a bacterial mechanosensitiv…

Abstract

The bacterial mechanosensitive channel of small conductance (MscS) plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. The functional characteristics of MscS have been extensively studied using liposomal reconstitution. This is a widely used experimental paradigm and is particularly important for mechanosensitive channels as channel activity can be probed free from cytoskeletal influence. A perpetual issue encountered using this paradigm is unknown channel orientation. Here we examine the orientation of MscS in liposomes formed using 2 ion channel reconstitution methods employing the powerful combination of patch clamp electrophysiology, confocal microscopy, and continuum mechanics simulation. Using the previously determined electrophysiological and pharmacological properties of MscS, we were able to determine that in liposomes, independent of lipid composition, MscS adopts the same orientation seen in native membranes. These results strongly support the idea that these specific methods result in uniform incorporation of membrane ion channels and caution against making assumptions about mechanosensitive channel orientation using the stimulus type alone.

Keywords

MscS electrophysiology finite element simulation patch clamp fluorometry reconstitution

MeSH Terms

Escherichia coli/metabolism,physiology Escherichia coli Proteins/chemistry,metabolism,physiology Ion Channel Gating/drug effects,physiology Ion Channels/chemistry,metabolism,physiology Kinetics Lipid Bilayers/chemistry,metabolism Liposomes/chemistry,metabolism Mechanotransduction, Cellular/drug effects,physiology Membrane Potentials/drug effects Microscopy, Confocal Patch-Clamp Techniques Spheroplasts/drug effects,metabolism,physiology Time Factors Trifluoroethanol/pharmacology

Chemicals

Escherichia coli Proteins Ion Channels Lipid Bilayers Liposomes MscS protein, E coli Trifluoroethanol

Authors & Affiliations

5 authors, click to expand affiliations / ORCID

Nomura Takeshi

*Molecular Cardiology and Biophysics Division/Mechanosensory Biophysics Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia; Department of Physical Therapy, Faculty of Rehabilitation, Graduate School of Health Sciences, Kyushu Nutrition Welfare University, Kitakyushu, Japan; St. Vincent's Clinical School, The University of New South Wales, Sydney, New South Wales, Australia; and Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Cox Charles D

*Molecular Cardiology and Biophysics Division/Mechanosensory Biophysics Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia; Department of Physical Therapy, Faculty of Rehabilitation, Graduate School of Health Sciences, Kyushu Nutrition Welfare University, Kitakyushu, Japan; St. Vincent's Clinical School, The University of New South Wales, Sydney, New South Wales, Australia; and Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Bavi Navid

*Molecular Cardiology and Biophysics Division/Mechanosensory Biophysics Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia; Department of Physical Therapy, Faculty of Rehabilitation, Graduate School of Health Sciences, Kyushu Nutrition Welfare University, Kitakyushu, Japan; St. Vincent's Clinical School, The University of New South Wales, Sydney, New South Wales, Australia; and Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Sokabe Masahiro

*Molecular Cardiology and Biophysics Division/Mechanosensory Biophysics Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia; Department of Physical Therapy, Faculty of Rehabilitation, Graduate School of Health Sciences, Kyushu Nutrition Welfare University, Kitakyushu, Japan; St. Vincent's Clinical School, The University of New South Wales, Sydney, New South Wales, Australia; and Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Martinac Boris

*Molecular Cardiology and Biophysics Division/Mechanosensory Biophysics Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia; Department of Physical Therapy, Faculty of Rehabilitation, Graduate School of Health Sciences, Kyushu Nutrition Welfare University, Kitakyushu, Japan; St. Vincent's Clinical School, The University of New South Wales, Sydney, New South Wales, Australia; and Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya, Japan b.martinac@victorchang.edu.au.

Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes.

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