Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Abstract

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...

Membrane-based biomolecular smart materials

“…This general form of bimolecular material system can be considered a smart material due to the stimuli-responsive properties of the network. Through previous work and our research it has been demonstrated that such a network can be made responsive to the application of a wide range of external stimuli, such as mechanical stress [37,38], optical inputs [27], and thermal inputs [39,40]. Likewise, it has been demonstrated that the control of collective transport across artificial membranes can produce a macroscopic pumping action and material swelling that mimics the properties of certain plant models [28,35].…”

Mechanosensitive channel properties and membrane mechanics in mouse dystrophic myotubes

“…This behaviour is similar to that observed previously in myotubes (Franco‐Obregon & Lansman, 2002), where resting MSC activity increased in mdx patches over a period of several minutes, while in wild‐type patches it decreased. Resting MSC activity in mdx patches was also sensitive to previous suction and pressure stimulation (Franco‐Obregon & Lansman, 2002) and depolariziation (Gil et al 1999).…”

Mechanosensitivity of Nav1.5, a voltage-sensitive sodium channel

“…An Axopatch 200A amplifier, Digidata 1322A, and Clampex 9.0 software (Molecular Devices, Sunnyvale, CA, USA) were used for voltage‐clamp and data acquisition. Previous work showed that large transmembrane voltages (100+ mV) applied over seconds produced patch creep, which caused activation of mechanosensitive channels in Xenopus oocytes (Gil et al 1999). To prevent this creep, we kept the holding potential at 0 mV.…”