Energy of Liposome Patch Adhesion to the Pipet Glass Determined by Confocal Fluorescence Microscopy

Abstract

The formation of the gigaseal in the patch clamp technique is dependent on the adhesion between the cell or liposome membrane and the glass pipet. The adhesion results in a capillary force causing creep of the patch membrane up the pipet. The membrane can be immobilized by counteracting the capillary force by positive pressure applied to the patch pipet. We use this phenomenon to develop a method for static measurement of the adhesion free energy of the lipid bilayer to the glass. Confocal fluorescent microscopy is used to track the bilayer creep inside the pipet and measure the immobilization pressure at various salt concentrations and pH. The adhesion energy is simply related to this pressure. For the studied phospholipid bilayers, its values were in the 0.3-0.7 mJ/m range, increased with salt concentration, and had a maximum as a function of pH. This method offers a way to measure bilayer-glass adhesion energy in patch clamp experiments that is more precise than dynamic methods.

PIEZO1-Mediated Currents Are Modulated by Substrate Mechanics

“…The model predicts that, in the pillar array assay, a deflection of 100 nm on substrate R 3 results in a maximum stress of 0.9 MPa in the cytoskeletal structures, whereas membrane stress is minimal at only 0.15 MPa (∼0.3 mN/m). In comparison, the maximal membrane stress arising from the application of −45 mmHg (∼6 kPa) pressure through a micropipette is predicted to be 1.2 MPa (∼5.6 mN/m); however, the resting membrane tension within a patch is variable and influenced by an additional adhesion energy between the membrane and the pipet glass (predicted to range from 0.5 to 4 mN/m). − Indentation with a blunt glass probe is predicted to create 1.5 MPa (∼6.7 mN/m) maximum stress (assuming membrane thickness of 5 nm). In comparison, our simulation predicts that activation of PIEZO1 by pillar deflection occurs at membrane tensions of <0.4 mN/m.…”

“…To empirically test whether the changes in membrane tension generated using pillar deflection could activate a second, well-characterized, stretch-activated channel, we investigated whether TREK-1 currents could be evoked by pillar deflections. The TREK-1 channel is a stretch-activated K2P channel that is gated at low membrane tensions using pressure applied to membrane patches. − In fact, TREK-1 is so sensitive to changes in membrane tension that the channel is activated by the tension that arises from the formation of a high resistance seal between the membrane and glass pipet (estimated to be <4 mN/m). , TREK-1 was overexpressed in HEK-293T cells lacking PIEZO1 (HEK-293T P1KO); deflection stimuli were applied to the cells, and TREK-1 activation was monitored using a whole-cell patch-clamp. Over a wide range of pillar deflections (10 nm–1000 nm), we did not observe any TREK-1 activity in the HEK-293T P1KO cells ( n = 7 cells) (Figure ).…”

“…31−33 In fact, TREK-1 is so sensitive to changes in membrane tension that the channel is activated by the tension that arises from the formation of a high resistance seal between the membrane and glass pipet (estimated to be <4 mN/m). 29,34 TREK-1 was overexpressed in HEK-293T cells lacking PIEZO1 (HEK-293T P1KO); 22 deflection stimuli were applied to the cells, and TREK-1 activation was monitored using a whole-cell patch-clamp. Over a wide range of pillar deflections (10 nm−1000 nm), we did not observe any TREK-1 activity in the HEK-293T P1KO cells (n = 7 cells) (Figure 7).…”

Transduction of Repetitive Mechanical Stimuli by Piezo1 and Piezo2 Ion Channels

“…Specifically, we designed a protocol in which a four second sinusoidal test pulse oscillated between +5 mmHg, a pressure we previously demonstrated to minimize membrane curvature, resting membrane tension and Piezo1 open probability by allowing Piezo1 channels to recover from inactivation, and −50 mmHg, a pressure driving open probability to near saturation, while minimizing stress on the membrane patch as well as membrane creep (Figure 1C) (Lewis and Grandl, 2015; Nakayama et al, 2016). Each test pulse was framed between two 300 ms standard step pulses (‘ step 1 ’ and ‘ step 2 ’), which were used to assess initial current density, monitor patch integrity, and quantify channel rundown (Figure S1).…”

From stretch to deflection: the importance of context in the activation of mammalian, mechanically activated ion channels

“…However, HSPC experiments can be combined with imaging of the patch deformation (largely in excised inside-out patches). This approach has allowed the modelling of membrane tension changes within the stimulated patch and the prediction of tension thresholds required for channel gating [22][23][24]. As such, HSPC analysis of MA channel activity is a powerful approach for the study of channels activated by stretch or changes in membrane tension (Figure 1).…”