Biophysics and Structure of the Patch and the Gigaseal

Abstract

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 microm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E(a)), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer ( approximately 10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E(a) seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E(a) and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E(a) and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of approximately 7 gigohm/microm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E(a) and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.

One-channel Cell-attached Patch-clamp Recording

“…9. To form a seal, pick up the syringe and apply slight negative pressure through the lateral tubing by pulling the syringe plunger, which pulls the cellular membrane into the tip of the recording pipette and initiates the formation of a GΩ resistance seal 10 . Take note of the test signal waveform on the oscilloscope, in which seal formation is indicated by its complete flattening, with only capacitive transients visible ( Figure 2B).…”

“…A flat tip ensures that when the pipette approaches the cell it will touch and press onto the cell membrane with the entire tip surface at once, helping to seal uniformly onto the cell. A pipette with a tip that is too wide will be less likely to result in one-channel patches; in contrast, upon pulling a pipette with a tip that is too narrow will result in an omega-shaped membrane loop that may seal at the base and clog the pipette 10 .…”

Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle

“…This could result from movement of the channel into an electrically isolated space such as a vesicle/membrane compartment or movement within the region where the cell membrane comes in contact with the recording electrode. 36 Alternatively, changes in membrane tension within the patch in the mutant may promote an inactivation or desensitization process, which occurs in the absence of an applied pressure stimulus. recording from the mdx fiber.…”

“…It is possible that the level of MS channel activity fluctuates over long time periods, either due to random variations in membrane mechanical properties or, perhaps, non-random forces generated by contractile elements or patch adhesion to the electrode. 36,37 Alternatively, MS channels may diffuse within the patch itself to the region where the cell membrane forms a seal with the electrode glass. 36 In the first set of experiments, I tested whether MS channel activity represents a simple stochastic process that is more or less constant over time using time series analysis of channel gating parameters.…”

“…36,37 Alternatively, MS channels may diffuse within the patch itself to the region where the cell membrane forms a seal with the electrode glass. 36 In the first set of experiments, I tested whether MS channel activity represents a simple stochastic process that is more or less constant over time using time series analysis of channel gating parameters. Figure 1c shows measurements of MS channel open probability as a function of time during the experiment.…”