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

Rosholm, Kadla R.; Baker, Matthew A. B.; Ridone, Pietro; Nakayama, Yoshitaka; Rohde, Paul R.; Cuello, Luis G.; Lee, Lawrence K.; Martinac, Boris

doi:10.1038/srep45180

Cited by 44 publications

(43 citation statements)

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“…M. Beurg and R. Fettiplace J Physiol 595.23 activated by tensional forces in the bilayer in the absence of any other cellular components (Coste et al 2012;Syeda et al 2016). Direct force sensation, mediated via the lipid bilayer, has also been demonstrated for the bacterial MscL channel (Teng et al 2015;Rosholm et al 2017). However, despite modelling (Kim, 2015), there is no experimental evidence that the hair cell MT channel can be similarly activated.…”

Section: Discussionmentioning

confidence: 99%

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PIEZO2 as the anomalous mechanotransducer channel in auditory hair cells

Beurg

Fettiplace

2017

The Journal of Physiology

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Throughout postnatal maturation of the mouse inner ear, cochlear hair cells display at least two types of mechanically gated ion channel: normal mechanotransducer (MT) channels at the tips of the stereocilia, activated by tension in interciliary tip links, and anomalous mechanosensitive (MS) channels on the top surface of the cells. The anomalous MS channels are responsible for the reverse‐polarity current that appears in mutants in which normal transduction is lost. They are also seen in wild‐type hair cells around birth, appearing 2 days earlier than normal MT channels, and being down‐regulated with the emergence of the normal channels. We review the evidence that the normal and anomalous channels are distinct channel types, which includes differences in localization, susceptibility to pharmacological agents, single‐channel conductance and Ca2+ permeability. The dichotomy is reinforced by the observation that the anomalous current is absent in cochlear cells of Piezo2‐null mice, even though the normal MT current persists. The anomalous current is suppressed by high intracellular Ca2+, suggesting that influx of the divalent ion via more Ca2+‐permeable normal MT channels inhibits the anomalous channels, thus explaining the temporal relationship between the two. Piezo2‐null mice have largely normal hearing, exhibiting up to 20 dB elevation in threshold in the acoustic brainstem response, so raising questions about the significance of PIEZO2 in the cochlea. Since the anomalous current declines with postnatal age, PIEZO2 may contribute to hair cell development, but it does not underlie the normal MT current. Its role in the development of hearing is not understood.

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Section: Discussionmentioning

confidence: 99%

“…; Rosholm et al . ). However, despite modelling (Kim, ), there is no experimental evidence that the hair cell MT channel can be similarly activated.…”

Section: Introductionmentioning

confidence: 97%

PIEZO2 as the anomalous mechanotransducer channel in auditory hair cells

Beurg

Fettiplace

2017

The Journal of Physiology

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“…We used KcsA as a positive control for electrical activity as also previously reported [18]. We verified that at pH 8 there was no observed activity and recorded gating events at pH 4.0 ( Fig.…”

Section: Discussionmentioning

confidence: 55%

“…5) with two current levels indicating the channel activity. The first gating level was at 4 pA, with the second level at 8 pA corresponding to a single channel conductance of ~20 pS [18].…”

Section: Kcsa As a Ph-sensitive Positive Controlmentioning

confidence: 99%

Fluorescence Microscopy of Piezo1 in Droplet Hydrogel Bilayers

Jaggers

Ridone

Martinac

et al. 2018

Preprint

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Mechanosensitive ion channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, mechanosensitive ion channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis (Xerocytosis) and lymphatic dysplasia. Here we utilize an established in-vitro artificial bilayer system to interrogate single Piezo1 channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 ion channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertion and activation of hPiezo1-GFP in bilayers of varying composition. Furthermore, we compare the Piezo1 bilayer reconstitution with measurements of insertion and activation of KcsA channels to reproduce the channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of ion channel gating while visualising the channel proteins using fluorescence.

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“…For example, bilayer tension has been applied by injecting the solution into a droplet. 76,77) 4.1. Detach-Attach of the Bilayer To date, no one has imagined what happens to channel activity when a bilayer is detached and membrane embedded channels are withdrawn from the bilayer.…”

Section: Other Membrane Manipulation Tech-niquesmentioning

confidence: 99%

Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay

Oiki

Iwamoto

2018

Biological & Pharmaceutical Bulletin

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Fluidity and mosaicity are two critical features of biomembranes, by which membrane proteins function through chemical and physical interactions within a bilayer. To understand this complex and dynamic system, artificial lipid bilayer membranes have served as unprecedented tools for experimental examination, in which some aspects of biomembrane features have been extracted, and to which various methodologies have been applied. Among the lipid bilayers involving liposomes, planar lipid bilayers and nanodiscs, recent developments of lipid bilayer methods and the results of our channel studies are reviewed herein. Principles and techniques of bilayer formation are summarized, which have been extended to the current techniques, where a bilayer is formed from lipid-coated water-in-oil droplets (water-in-oil bilayer). In our newly developed method, termed the contact bubble bilayer (CBB) method, a water bubble is blown from a pipette into a bulk oil phase, and monolayer-lined bubbles are docked to form a bilayer through manipulation by pipette. An asymmetric bilayer can be readily formed, and changes in composition in one leaflet were possible. Taking advantage of the topological configuration of the CBB, such that the membrane's hydrophobic interior is contiguous with the surrounding bulk organic phase, oil-dissolved substances such as cholesterol were delivered directly to the bilayer interior to perfuse around the membrane-embedded channels (membrane perfusion), and current recordings in the single-channel allowed detection of immediate changes in the channels' response to cholesterol. Chemical and mechanical manipulation in each monolayer (monolayer technology) allows the examination of dynamic channel-membrane interplay.

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

Cited by 44 publications

References 50 publications

PIEZO2 as the anomalous mechanotransducer channel in auditory hair cells

PIEZO2 as the anomalous mechanotransducer channel in auditory hair cells

Fluorescence Microscopy of Piezo1 in Droplet Hydrogel Bilayers

Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay

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