High Resistance Seal Research Articles

Modeling Ion Channels in the Gigaseal

The ability to form gigaseals is essential for patch-clamp electrophysiology; however, ion channels located in the seal can produce measureable currents. To explore the expected properties of channels in the seal (i.e., rim channels), we created a mathematical model. The seal was a two-dimensional cable filled with saline and bounded on one side by membrane (with resistance and capacitance) and on the other side by glass (nonconductive and noncapacitive). We included ion depletion/accumulation around the channels. The channels were ohmic with a conductance that increased with the concentration of permeant ions. The aqueous seal thickness was set nominally to 1 nm. Imaging with fluorescent dyes in the pipette showed that the hydrophilic dye Alexa 488 is impermeant, but lipophilic FM1-43 labels the seal. The model showed that to obtain high-resistance seals, the conductivity of the seal media has to be <10% that of the bath. Stimulus voltages decreased with distance down the seal. In agreement with results in the literature, channels in the seal can produce currents similar to those in the pipette-spanning dome. The transition times of currents are slower due to membrane capacitance. If channel densities are uniform, patch currents are dominated by channels in the dome.

Lipid Bilayer Mechanics in a Pipette with Glass-Bilayer Adhesion

Electrophysiology is a central tool for measuring how different driving forces (e.g., ligand concentration, transmembrane voltage, or lateral tension) cause a channel protein to gate. Upon formation of the high resistance seal between a lipid bilayer and a glass pipette, the so-called “giga-seal”, channel activity can be recorded electrically. In this article, we explore the implications of giga-seal formation on the mechanical state of a lipid bilayer patch. We use a mechanical model for the free energy of bilayer geometry in the presence of glass-bilayer adhesion to draw three potentially important conclusions. First, we use our adhesion model to derive an explicit relationship between applied pressure and patch shape that is consistent with the Laplace-Young Law, giving an alternative method of calculating patch tension under pressure. With knowledge of the adhesion constant, which we find to be in the range ∼0.4–4 mN/m, and the pipette size, one can precisely calculate the patch tension as a function of pressure, without the difficultly of obtaining an optical measurement of the bilayer radius of curvature. Second, we use data from previous electrophysiological experiments to show that over a wide range of lipids, the resting tension on a electrophysiological patch is highly variable and can be 10–100 times higher than estimates of the tension in a typical cell membrane. This suggests that electrophysiological experiments may be systematically altering channel-gating characteristics and querying the channels under conditions that are not the same as their physiological counterparts. Third, we show that reversible adhesion leads to a predictable change in the population response of gating channels in a bilayer patch.

The feasibility and limitation of patch-clamp recordings from neonatal rat cardiac ventricular slices

Examine the feasibility of whole-cell patch-clamp recordings from the cardiac ventricular slices of newborn (P(3)-P(7)) Sprague-Dawley rats to identify a better substitute for single cardiac myocytes prepared using enzymatic treatment. High resistance seals (>1 GΏ) were obtained from cardiac ventricle tissues prepared without using enzymatic treatment. Thereafter, cell-attached and whole-cell patch-clamp techniques were used on thin cardiac slices (200 μm thick) in 2009 in the Institute of Molecular Medicine of Peking University. An averaged sodium current (n=11 cells) was recorded in the cell-attached mode, and this displayed features similar to those previously reported for isolated rat ventricular myocytes. The outward potassium current, hyperpolarization-activated cation channel or I (f) channel (HCN channel), and action potential were recorded in the whole-cell mode (n=2 cells), and the identical properties were observed from the cardiac slices. The cell-attached mode is stable and reliable for recording the ion current. The resting potential for cardiac slices measured using current-clamp recording in the whole-cell mode was -50 to -70 mV. The resting potential of cardiac slices has properties similar to those of enzyme-prepared cardiomyocytes, with the exception that it is positive. We achieved whole-cell patch-clamp recordings from cardiac slices and affirmed the feasibility and values of both cell-attached and whole-cell recording modes using this technique. Nevertheless, there remain difficulties and limitations associated with the application of whole-cell patch-clamping to cardiac slices, due primarily to the existence of large amounts of connective tissue even in newborn rats.

Dual Micropores in Glass Substrates for Ion Channel and Gap Junction Recording

Despite the commercial success of automated high-throughput patch clamp technologies, instruments on the market typically cannot match the quality of single-channel recordings obtained by traditional patch clamp experiments with glass micropipettes. In automated approaches that use suction to position a cell to a micropore, the inner surface of the pore can be contaminated with proteins in solution prior to seal formation. Traditional patch clamping avoids this problem by applying a positive pressure to the micropipette prior to contacting the cell membrane, which enables significantly higher electrical seal resistances. This research aims to develop a universal platform for high-quality transport assays on glass chips that circumvents the problem of surface contamination and takes advantage of the high seal resistances and low capacitive current noise realized with glass substrates. Femtosecond laser ablation is used to fabricate a pair of micropores in close proximity to each other (distance < 5 µm) in glass, while the access channels to these pores are kept separate. This configuration makes it possible to use one pore for automated positioning of a cell by suction, while the other pore is kept clean by applying positive pressure. Once the cell is pre-positioned onto the first pore, suction may be applied to the second pore to establish a high quality seal that could be used to interrogate individual transmembrane transporter proteins and single ion channel proteins. In addition, this dual micropore configuration may enable the first automated gap junction recordings, bypassing the difficulties of manually positioning two micropipettes.

Formation of Essential Ultrastructural Interface between Cultured Hippocampal Cells and Gold Mushroom-Shaped MEA- Toward ?IN-CELL? Recordings from Vertebrate Neurons

Using cultured Aplysia neurons we recently reported on the development of a novel approach in which an extracellular, non-invasive multi-electrode-array system provides multisite, attenuated, intracellular recordings of subthreshold synaptic potentials, and action potentials (APs), the so called “IN-CELL” recording configuration (to differentiate it from intracellular recordings). Because of its non-invasive nature, the configuration can be used for long term semi intracellular electrophysiological monitoring of APs and synaptic potentials. Three principals converge to generate the IN-CELL configuration: (a) engulfment of approximately 1 μm size gold mushroom-shaped microelectrodes (gMμE) by the neurons, (b) formation of high seal resistance between the cell’s plasma membrane and the engulfed gMμE, and (c), autonomous localized increased conductance of the membrane patch facing the gMμE. Using dissociated rat hippocampal cultures we report here that the necessary morphological and ultrastructural relationships to generate the IN-CELL recording configuration are formed between hippocampal cells and the gMμEs. Interestingly, even <1 μm thin branches expand and engulf the gMμE structures. Recordings of spontaneous electrical activity revealed fast ∼2 ms, 0.04–0.75 mV positive monophasic APs (FPMP). We propose that the FPMP are attenuated APs generated by neurons that engulf gMμEs. Computer simulations of analog electrical circuits depicting the cell–gMμE configuration point out the parameters that should be altered to improve the neuron–gMμE electrical coupling.

Viability Assessment of Primo-node Slices From Organ Surface Primo-vascular Tissues in Rats

Abstract The primo-vascular system is a novel thread-like structure which is recently rediscovered, but its cellular properties are largely unknown. In this study, a slice preparation for primo-nodes was developed to facilitate study of the cellular properties of primo-node cells in vitro. Slices (4–8 slices; 200 μm thick) were sectioned from single primo-nodes collected from the abdominal organ surface of rats and incubated in oxygenated Krebs solution at 25°C or 31°C for up to 7 hours. Trypan blue staining and whole-cell patch-clamp recordings were performed to estimate the viability of cells in the slices. Viability was largely maintained during the first 3 hours, but subsequently decreased (from 80% to 21%, p < 0.001). In addition, the viability of slices incubated at 31°C was higher than those incubated at 25°C (80% vs. 47%, p < 0.001). In whole-cell patch-clamp experiments, high resistance seals readily formed and primo-node cells showed a mean resting membrane potential (-38 mV) comparable to that recorded with sharp electrodes and outwardly-rectifying current-voltage relationships. The results show that the primo-node slices developed in this study maintained viability for up to 4 hours in vitro.

Gating of a G protein-sensitive Mammalian Kir3.1 Prokaryotic Kir Channel Chimera in Planar Lipid Bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip

We present a polymer microchip capable of monitoring neuronal activity with a fidelity never before obtained on a planar patch-clamp device. Cardio-respiratory neurons Left Pedal Dorsal 1 (LPeD1) from mollusc Lymnaea were cultured on the microchip's polyimide surface for 2 to 4 hours. Cultured neurons formed high resistance seals (gigaseals) between the cell membrane and the surface surrounding apertures etched in the polyimide. Gigaseal formation was observed without applying external force, such as suction, on neurons. The formation of gigaseals, as well as the low access resistance and shunt capacitance values of the polymer microchip resulted in high-fidelity recordings. On-chip culture of neurons permitted, for the first time on a polymeric patch-clamp device, the recording of high fidelity physiological action potentials. Microfabrication of the hybrid poly(dimethylsiloxane)-polyimide (PDMS-PI) microchip is discussed, including a two-layer PDMS processing technique resulting in minimized shrinking variations.

Evaluation of the QPatch HT and HTX Systems As Methods for Ion Channel Screening

The introduction of high throughput planar automated electrophysiology systems has brought the potential of ion channels as targets for pharmacological modulation to the forefront. Sophion Biosciences QPatch systems are fully automated electrophysiology platforms generating high resistance seals with resultant ion channel biophysical and pharmacological properties similar to conventional patch clamp recordings. In this study, using a CHO cell line stably expressing the TRPM2 channel, the performance of the QPatch HTX, which records from a population of 10 cells per recording site, was evaluated against the QPatch HT which records from a single cell per recording site.TRPM2 can be challenging to measure, particularly when determining inhibitor potencies, due to poor recording stability characterised by current rundown. A compound set was screened against TRPM2 on the QPatch HT and HTX to assess their relative performances. There was a good correlation between the IC50 values generated on the two systems (r2 = 0.91). The current rundown on the QPatch HTX was considerably reduced compared to the QPatch HT (19.5 ± 1.5% and 37.5 ± 3.0% respectively, mean ± SEM, n = 4 experiments, 767 cells profiled). This resulted in an increase in the number of successful recordings obtained from 51.6 ± 7.1% for HT to 83.1 ± 4.5% for HTX (Mean ± SEM, n = 4 experiments, 912 cells profiled) for a 5 point IC50 assay and from 67.2 ± 8.9% for HT to 98.6 ± 1.4% for HTX (Mean ± SEM, n = 3-4 experiments, 480 cells profiled) for a single concentration assay.The QPatch HT has made a significant impact on the volume of high quality electrophysiology data that can be generated. This preliminary study suggests that the QPatch HTX system can further enhance throughput, particularly with channels where current stability is problematic.

A PDMS microfluidic system with poly(ethylene glycol)/SU-8 based apertures for planar whole cell-patch-clamp recordings

Poly(dimethylsiloxane) (PDMS) has been used as an attractive material for planar patch-clamp system fabrication. However, the hydrophobic property of PDMS prevents the establishment of a high-resistance seal with cell membrane. Here, we describe the first use of poly(ethylene glycol) (PEG)/SU-8 mixture for patch-clamp partition electrode fabrication. This PEG/SU8 mixture with weight ratio of 1:1 has been proved to have better properties compared with PDMS for cell-patch interface, such as improved mechanical property, permanent hydrophilic surface property, and easiness to achieve high-resistance cell sealing. The PEG/SU-8 partitions with 2 μm apertures were successfully fabricated by employing a two-step process of soft lithography for PDMS mold (thermal curing) and PEG/SU-8 chip (UV light curing). The partitions could be reversibly sealed to the PDMS microfluidic system including electrode solution inlet/outlet, cell manipulation inlet/outlet, cell reservoir, microchannels and microfabricated Ag/AgCl electrodes. The integrated chip was successfully used to trap Hela cells onto 2 μm aperture by a negative pressure. We demonstrated that a planar PDMS patch-clamp system with PEG/SU-8 based cell-patch interface for successful patch-clamp recording on Hela cells.

POSH Stimulates the Ubiquitination and the Clathrin-independent Endocytosis of ROMK1 Channels

POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation of lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels in HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-alpha, beta, and gamma subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Delta373-378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHDeltaRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHDeltaRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK ubiquitination. We conclude that POSH inhibits ROMK channels by enhancing dynamin-dependent and clathrin-independent endocytosis and by stimulating ubiquitination of ROMK channels.

Automated Patch-Clamp Technique: Increased Throughput in Functional Characterization and in Pharmacological Screening of Small-Conductance Ca2+ Release-Activated Ca2+ Channels

The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release-activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (I( CRAC)) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 +/- 226 MOmega (n = 44). CRAC channels were activated by a number of methods that raise intracellular calcium concentration, including EGTA, ionomycin, Ins(1,4,5)P3, and thapsigargin. I(CRAC) whole-cell currents ranged from 30 to 120 pA with rise times of 40 to 150 s. An initial delay in current activation was observed in particular when I(CRAC) was activated by passive store depletion using EGTA. Apparent rundown of I(CRAC) was commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. I(CRAC) was blocked by SKF-96365 and 2-APB with IC50 values of 4.7 +/- 1.1 microM (n = 9) and 7.5 +/- 0.7 (n = 9) microM, respectively. The potencies of these blockers were similar to values reported for I(CRAC) in similar conventional patch-clamp experiments. The study demonstrates that CRAC channels can be rapidly and efficiently targeted with automated patch-clamp techniques for characterization of physiological and pharmacological properties.

In situ recording from gut pacemaker cells

Interstitial cells of Cajal (ICC) associated with the myenteric plexus of the small intestine are crucial players in gut physiology performing pacemaker functions and directing peristalsis and segmentation. ICC have been studied after chemical isolation and under culture conditions, but concerns that these methods affect the intrinsic properties have hindered progress in our understanding of ICC. To overcome this problem, we have developed a method to obtain electrophysiological recordings from ICC in situ. The critical feature is the ability to make high resistance seals onto cells that are embedded within tissue to obtain patch clamp recordings. Our first results show a prominent presence of a chloride channel, one of the proposed ICC pacemaker channels. The developed method can be applied to auxiliary cells of the enteric nervous system such as glial cells or fibroblasts and will be ideal for the study of cell-cell communication in tissue.

Ionic Requirements for Membrane-Glass Adhesion and Giga Seal Formation in Patch-Clamp Recording

Patch-clamp recording has revolutionized the study of ion channels, transporters, and the electrical activity of small cells. Vital to this method is formation of a tight seal between glass recording pipette and cell membrane. To better understand seal formation and improve practical application of this technique, we examine the effects of divalent ions, protons, ionic strength, and membrane proteins on adhesion of membrane to glass and on seal resistance using both patch-clamp recording and atomic force microscopy. We find that H +, Ca 2+, and Mg 2+ increase adhesion force between glass and membrane (lipid and cellular), decrease the time required to form a tight seal, and increase seal resistance. In the absence of H + (10 −10 M) and divalent cations (<10 −8 M), adhesion forces are greatly reduced and tight seals are not formed. H + (10 −7 M) promotes seal formation in the absence of divalent cations. A positive correlation between adhesion force and seal formation indicates that high resistance seals are associated with increased adhesion between membrane and glass. A similar ionic dependence of the adhesion of lipid membranes and cell membranes to glass indicates that lipid membranes without proteins are sufficient for the action of ions on adhesion.

The patch clamp technique: Principles and technical considerations

The development of techniques that allow the high fidelity measurement of small scale ionic currents ushered in a new era of investigation into the role of ion channels in the physiologic and pathophysiologic function of excitable tissue. Based upon the formation of a high resistance (gigaohm) seal with the membrane of the cell being studied, these “patch clamp” techniques have improved our understanding of a wide variety of cardiac disease states with respect to diagnosis, treatment and prognosis. This review outlines the basic principles underlying the patch clamp technique, including the properties of biological membranes and ion channels, and provides an elementary summary of its application to the recording of cardiac ionic currents, with a particular focus on issues related to myocyte isolation, electrode manufacturing and the voltage clamp configuration.

Molecular Identification and Physiological Roles of Parotid Acinar Cell Maxi-K Channels

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca(2+)-activated Cl(-) and K(+) channels. Parotid acinar cells express two types of Ca(2+)-activated K(+) channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the K(Ca)3.1 gene, and the K(Ca)1.1 gene is a likely candidate for the maxi-K channel. To confirm the genetic identity of the maxi-K channel and to probe its specific roles, we studied parotid glands in mice with the K(Ca)1.1 gene ablated. Parotid acinar cells from these animals lacked maxi-K channels, confirming their genetic identity. The stimulated parotid gland fluid secretion rate was normal, but the sodium and potassium content of the secreted fluid was altered. In addition, we found that the regulatory volume decrease in acinar cells was substantially impaired in K(Ca)1.1-null animals. We examined fluid secretion from animals with both K(+) channel genes deleted. The secretion rate was severely reduced, and the ion content of the secreted fluid was significantly changed. We measured the membrane potentials of acinar cells from wild-type mice and from animals with either or both K(+) channel genes ablated. They revealed that the observed functional effects on fluid secretion reflected alterations in cell membrane voltage. Our findings show that the maxi-K channels are critical for the regulatory volume decrease in these cells and that they play an important role in the sodium uptake and potassium secretion process in the ducts of these fluid-secreting salivary glands.

A single cell electrophysiological analysis device with embedded electrode

A single cell electrophysiological analysis device with a sub-micrometer opening on embedded recording electrode has been demonstrated using a combined micro- and nano-machining process. This technique can reduce the series pipette resistance while keeping high seal resistance for electrophysiological measurements. In the prototype demonstration, the electrolyte pipette resistance is reduced to 200 kΩ using the microfabricated electrophysiological system with opening size of 500 nm in diameter. A 6 GΩ seal resistance is achieved on a HeLa cell, a cervical cancer cell line with size between 10 and 20 μm, on the same device. In a second experiment, the measured electrolyte pipette resistance is 1 MΩ using different pipette solution on a device with 800 nm opening and a 500 MΩ seal resistance is achieved on a vacuolar yeast cell of 3–5 μm in size.

Design and Fabrication of a Micromachined Planar Patch-Clamp Substrate With Integrated Microfluidics for Single-Cell Measurements

We have designed, fabricated, tested, and integrated microfabricated planar patch-clamp substrates and poly(dimethylsiloxane) (PDMS) microfluidic components. Substrates with cell-patch-site aperture diameters ranging from 300nm to 12 /spl mu/m were produced using standard MEMS-fabrication techniques. The resistance of the cell-patch sites and substrate capacitance were measured using impedance spectroscopy. The resistance of the microfabricated apertures ranged from 200 k/spl Omega/ to 47 M/spl Omega/ for apertures ranging from 12 /spl mu/m to 750 nm, respectively. The substrate capacitance was 17.2 pF per mm/sup 2/ of fluid contact area for substrates with a 2-/spl mu/m-thick layer of silicon dioxide. In addition, the ability of the planar patch-clamp substrates to form high-resistance seals in excess of 1 G/spl Omega/ has been confirmed using Chinese hamster ovary cells (CHO-K1). Testing shows that the microfluidic components are appropriate for driving human embryonic kidney cells (HEK 293) to patch apertures, for trapping cells on patch apertures, and for exchanging the extracellular fluid environment.

A high-performance elastomeric patch clamp chip

Ion channels play key roles in cell physiology and underlie a broad spectrum of disorders. To this day, the gold standard for studying ion channels is the patch clamp technique. Patch clamping involves careful positioning of a fine-tipped glass micropipette onto the surface of the cell to form a high-resistance (>1 Gohms) seal ("gigaseal"), a procedure that is laborious, vibration-sensitive, and not easily amenable to automation. In addition, the solution inside the pipette cannot be easily exchanged. Recently reported patch clamp chips offer the potential of increased throughput, but to date the overall per-cell performance of most designs has been very low when compared to pipettes, and/or the fabrication process is prohibitively expensive. Here we demonstrate a replica-molded elastomeric patch clamp chip incorporating nanofabricated constrictions, which delivers high-stability gigaseals, with success rates comparable to those of pipettes, using rat basophilic leukemia (RBL) cells. The high stability enables exchanges of both the extracellular and intracellular solution during whole-cell recordings. In a sample of 103 experiments, 66 cells (64%) were successfully immobilized at the patch aperture; 38 cells (58% of immobilized cells, 37% of all cells) were successfully gigasealed; and 25 cells (65% of gigasealed cells, 34% of immobilized cells, 24% of all cells) were successfully perforated for whole-cell access. In the last group of 27 experiments, 79% of the cells could be immobilized, of which 68% could be gigasealed and 46% perforated for whole-cell access, indicating that dexterity is important.

G Protein-induced Trafficking of Voltage-dependent Calcium Channels

Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.