Membrane-Pipette Interactions Underlie Delayed Voltage Activation of Mechanosensitive Channels in Xenopus Oocytes

Gil, Ziv; Magleby, Karl L.; Silberberg, Shai D.

doi:10.1016/s0006-3495(99)77463-1

Cited by 18 publications

(16 citation statements)

References 36 publications

(62 reference statements)

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“…While these pressures are larger than reported elsewhere [1], they are consistent with our previous reports [2]. We believe the lower pressures required by other labs are related to differences in the glass or saline compositions used that can affect the resting tension in the patch dome and the patch mechanical properties during stimulation [35], [51].…”

Section: Resultssupporting

confidence: 90%

Caveolae Regulation of Mechanosensitive Channel Function in Myotubes

et al. 2013

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Mutations that lead to muscular dystrophy often create deficiencies in cytoskeletal support of the muscle sarcolemma causing hyperactive mechanosensitive cation channel (MSC) activity and elevated intracellular Ca2+. Caveolae are cholesterol-rich microdomains that form mechanically deformable invaginations of the sarcolemma. Mutations to caveolin-3, the main scaffolding protein of caveolae in muscle, cause Limbe-Girdle muscular dystrophy. Using genetic and acute chemical perturbations of developing myotubes we investigated whether caveolae are functionally linked to MSCs. MSC sensitivity was assayed using suction application to patches and probe-induced indentation during whole-cell recordings. Membrane mechanical stress in patches was monitored using patch capacitance/impedance. Cholesterol depletion disrupted caveolae and caused a large increase in MSC current. It also decreased the membrane mechanical relaxation time, likely reflecting cytoskeleton dissociation from the bilayer. Reduction of Cav3 expression with miRNA also increased MSC current and decreased patch relaxation time. In contrast Cav3 overexpression produced a small decrease in MSC currents. To acutely and specifically inhibit Cav3 interactions, we made a chimeric peptide containing the antennapedia membrane translocation domain and the Cav3 scaffolding domain (A-CSD3). A-CSD3 action was time dependent initially producing a mild Ca2+ leak and increased MSC current, while longer exposures decreased MSC currents coinciding with increased patch stiffening. Images of GFP labeled Cav3 in patches showed that Cav3 doesn’t enter the pipette, showing patch composition differed from the cell surface. However, disruption via cholesterol depletion caused Cav3 to become uniformly distributed over the sarcolemma and Cav3 appearance in the patch dome. The whole-cell indentation currents elicited under the different caveolae modifying conditions mirror the patch response supporting the role of caveolae in MSC function. These studies show that normal expression levels of Cav3 are mechanoprotective to the sarcolemma through multiple mechanisms, and Cav3 upregulation observed in some dystrophies may compensate for other mechanical deficiencies.

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Section: Resultssupporting

confidence: 90%

Caveolae Regulation of Mechanosensitive Channel Function in Myotubes

et al. 2013

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“…Gil et al (10) hypothesize that voltage across the patch influences membrane tension and gates SA channels. Because depolarization activation did not occur with soft glass pipettes and was absent in outside-out patches, they concluded that voltage dependence was an artifact resulting from specific interactions between the glass pipette and membrane patch in Xenopus oocytes.…”

Section: Discussionmentioning

confidence: 99%

A mechanogated nonselective cation channel in proximal tubule that is ATP sensitive

Hurwitz¹,

Hu²,

Segal

2002

American Journal of Physiology-Renal Physiology

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Ion channels that are gated in response to membrane deformation or “stretch” are empirically designated stretch-activated channels. Here we describe a stretch-activated nonselective cation channel in the basolateral membrane (BLM) of the proximal tubule (PT) that is nucleotide sensitive. Single channels were studied in cell-intact and cell-free patches from the BLM of PT cells that maintain their epithelial polarity. The limiting inward Cs+ conductance is ∼28 pS, and channel activity persists after excision into a Ca2+- and ATP-free bath. The stretch-dose response is sigmoidal, with half-maximal activation of about −19 mmHg at −40 mV, and the channel is activated by depolarization. The inward conductance sequence is: NH[Formula: see text] ∼ Cs+ ∼ Rb+> K+ ∼ Na+ ∼ Li+ > Ca2+ ∼ Ba2+> N-methyl-d-glucamine ∼ tetraethylammonium. The venom of the common Chilean tarantula, Grammostola spatulata, completely blocks channel activity in cell-attached patches. Hypotonic swelling reversibly activates the channel. Intracellular ATP concentration ([ATP]i) reversibly blocks the channel (inhibitory constant ∼0.48 mM), suggesting that channel function is coupled to the metabolic state of the cell. We conclude that this channel may function as a Ca2+ entry pathway and/or be involved in regulation of cell volume. We speculate this channel may be important when [ATP]i is depleted, as occurs during periods of increased transepithelial transport or with ischemic injury.

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“…The methods used for single-channel recording from patches of membrane from oocytes obtained from Xenopus laevis have been described (6,7). The neurons used for recording were cultured from the hippocampal region of embryonic (e15) rat brain as described (20).…”

Section: Methodsmentioning

confidence: 99%

“…Two different experimental approaches were used to test whether preventing movement of the membrane also prevented activation of MS channels. The first approach was based on an observation made in a previous study: depolarization-induced delayed activation of MS channels no longer occurs when the tips of the borosilicate glass pipettes are coated with soft glass (7). Perhaps this lack of activation with soft glass arose from a lack of movement of the membrane patch, because soft glass has a different composition and charge density that may interact with the membrane differently than hard glass (24).…”

Section: Depolarization-induced Membrane Displacement and Ms Channelmentioning

confidence: 99%

“…We have shown recently that the delayed activation of the MS channels is not observed in outside-out patches, in membrane outside of the patch pipette in whole-cell recordings from Xenopus oocytes, or when the patch pipettes are fabricated from or coated with soft glass (7). These observations suggest that the delayed cooperative activation of MS channels by voltage is not an intrinsic property of the channels but requires interactions between the patch of membrane and the patch pipette (7).…”

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confidence: 99%

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Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Gil

Silberberg

Magleby

1999

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

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The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, Ϸ1 m with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage. M echanosensitive (MS) ion channels can act as transducers for the senses of touch, hearing, balance, and proprioception and also seem to be involved in the regulation of cell volume (1-3). MS channels in patches of membrane in the tips of patch pipettes are readily activated when the membrane is stretched by the application of negative pressure to the shank of the patch pipette (4). The stretching of the membrane patch by negative pressure is thought to activate the channels by increased tension in the membrane (5). When the pressure is released, the channels then readily deactivate. In addition to stretch activation, some MS channels can also be activated by voltage (1). In this regard, depolarization of membrane patches from Xenopus oocytes activates endogenous MS channels in the patch after prolonged delays of typically 1-20 s (6). After stepping the voltage back to negative potentials, the MS channels then deactivate over several seconds. The delayed activation is observed for both on-cell and excised patches and typically occurs in a cooperative manner, with all channels activating over a short period of time compared with the duration of the delay to activation.We have shown recently that the delayed activation of the MS channels is not observed in outside-out patches, in membrane outside of the patc...

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Membrane-Pipette Interactions Underlie Delayed Voltage Activation of Mechanosensitive Channels in Xenopus Oocytes

Cited by 18 publications

References 36 publications

Caveolae Regulation of Mechanosensitive Channel Function in Myotubes

Caveolae Regulation of Mechanosensitive Channel Function in Myotubes

A mechanogated nonselective cation channel in proximal tubule that is ATP sensitive

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

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