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PubMed2003

Characterization of potassium channel modulators with QPatch automated patch-clamp technology: system characteristics and performance.

Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.

Animals↗

PubMed2000

[Single channel and whole cell recordings using patch clamp technique].

Action of ion channel on membrane is the key events of messenger transduction for excitable cells, which can be detected by the patch clamp technique. The developments of patch clamp technique have brought a revolution of life science research. The theory, work modes, single channel and whole cell recording techniques in single cells are described here in detail.

Cells, Cultured↗

PubMed1995

Design of the EPC-9, a computer-controlled patch-clamp amplifier. 1. Hardware.

The EPC-9 patch-clamp amplifier is a digitally controlled analog device for recording currents in membrane patches and in small cells. It has neither front-panel controls nor internal trim adjustments; instead all gain, range-changing, transient cancellation, calibration and other functions are computer controlled. Novel aspects of the circuit design of this instrument are discussed, with special reference to the issues of allowing computer control of all functions.

Computer Storage Devices↗

PubMedDate not supplied

Patch-clamp studies of voltage-gated currents in identified neurons of the rat cerebral cortex.

In the cerebral cortex, neurons can be classified into 2 broad morphological classes, referred to as pyramidal and nonpyramidal (stellate) cells, which correspond to functional classes of projection neurons and local circuit interneurons, respectively. In this study, we demonstrate that specific morphological, immunohistochemical, and physiological features, that allow class distinction of neurons in situ, are retained in acutely isolated neocortical neurons. Furthermore, voltage-clamp analysis with patch-clamp techniques indicate the differences in functional properties in adult neurons, reflect cell-specific, developmental changes in the density and type of specific classes of Na+, K+ and Ca2+ channels expressed. The differences in channel properties contribute to the different input-output relations of neocortical neurons, which enable inhibitory neurons to follow excitatory inputs faithfully and projection neurons to have more integrative roles.

Action Potentials↗

PubMed1994

Patch-clamp recordings from subpopulations of autonomic and afferent neurons identified by axonal tracing techniques.

This study determined whether axonal tracing methods can be used in combination with patch-clamp techniques to examine the electrical properties of identified populations of autonomic and afferent neurons in the adult rat. Fluorescent dyes (Fast Blue, FB and Fluoro-Gold, FG) were injected into the wall of the urinary bladder or colon and into various somatic structures to label postganglionic neurons in the major pelvic ganglia (MPG) as well as visceral and somatic afferent neurons in the lumbosacral dorsal root ganglia (DRG) and trigeminal ganglia (TG). One to 3 weeks after dye injection, neurons were isolated from ganglia by enzymatic dissociation. Following dissociation, single neurons labelled with FB were identified in the three types of ganglion preparations; however FG was only identified consistently in TG neurons. FB was retained in neurons during short-term culture (1-5 days). Following 10 to 20 s exposure to UV light which was required for identification of the cells, whole-cell patch-clamp recordings revealed that the electrophysiological properties of FB-labelled cells did not differ from those of unlabelled cells. However, a more prolonged exposure (1-5 min) of the neurons to UV light produced irreversible damage to the cells which was evident as changes in the action potential, sodium current and resting membrane potential. These results indicate that patch-clamp recording in combination with axonal tracing is a useful approach for studying the electrical properties of identified populations of autonomic and afferent neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed2001

Patch-clamp recording in brain slices with improved slicer technology.

The use of advanced patch-clamp recording techniques in brain slices, such as simultaneous recording from multiple neurons and recording from dendrites or presynaptic terminals, demands slices of the highest quality. In this context the mechanics of the tissue slicer are an important factor. Ideally, a tissue slicer should generate large-amplitude and high-frequency movements of the cutting blade in a horizontal axis, with minimal vibrations in the vertical axis. We developed a vibroslicer that fulfils these in part conflicting requirements. The oscillator is a permanent-magnet-coil-leaf-spring system. Using an auto-resonant mechano-electrical feedback circuit, large horizontal oscillations (up to 3 mm peak-to-peak) with high frequency ( approximately 90 Hz) are generated. To minimize vertical vibrations, an adjustment mechanism was employed that allowed alignment of the cutting edge of the blade with the major axis of the oscillation. A vibroprobe device was used to monitor vertical vibrations during adjustment. The system is based on the shading of the light path between a light-emitting diode (LED) and a photodiode. Vibroprobe monitoring revealed that the vibroslicer, after appropriate adjustment, generated vertical vibrations of <1 microm, significantly less than many commercial tissue slicers. Light- and electron-microscopic analysis of surface layers of slices cut with the vibroslicer showed that cellular elements, dendritic processes and presynaptic terminals are well preserved under these conditions, as required for patch-clamp recording from these structures.

Animals↗

PubMed2002

Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact ganglia.

Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of -47 +/- 6 mV and input resistances (R(in)) of 713 +/- 49 MOmega at voltages ranging from -90 to -40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inward-rectifier current (I(Kir)) decreased R(in) to 103 +/- 10 MOmega. AH neurones had resting potentials of -57 +/- 4 mV and R(in) was 502 +/- 27 MOmega. R(in) fell to 194 +/- 16 MOmega upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, I(h), and to I(Kir). Resting potential and R(in) exhibited a low sensitivity to changes in [K(+)](o) in both AH and S neurones. This indicates that both cells have a low background K(+) permeability. The cationic current, I(h), contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of -72 +/- 2 mV, and a voltage sensitivity of 8.2 +/- 0.7 mV per e-fold change. I(h) has relatively fast, voltage-dependent kinetics, with on and off time constants in the range of 50-350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium-dependent current that was poorly sensitive to TTX. In AH neurones, the post-action-potential slow hyperpolarizing current, I(AHP), displayed large variation from cell to cell. I(AHP) appeared to be highly Ca(2+) sensitive, since its activation with either membrane depolarization or caffeine (1 mM) was not prevented by perfusing the cell with 10 mM BAPTA. We determined the identity of the Ca(2+) channels linked to I(AHP). Action potentials of AH neurones that were elongated by TEA (10 mM) were similarly shortened and I(AHP) was suppressed with each of the three omega-conotoxins GVIA, MVIIA and MVIIC (0.3-0.5 microM), but not with omega-agatoxin IVA (0.2 microM). There was no additivity between the effects of the three conotoxins, which indicates the presence of N- but not of P/Q-type Ca(2+) channels. A residual Ca(2+) current, resistant to all toxins, but blocked by 0.5 mM Cd(2+), could not generate I(AHP). This patch-clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea-pig duodenum are under the control of four major currents, I(AHP), I(h), an N-type Ca(2+) current and a slowly inactivating Na(+) current.

Action Potentials↗

PubMed1990

A pipette holder for use in patch-clamp measurements.

A novel design for a pipette holder for use in patch-clamp experiments is presented. The holder is designed for use in patch-clamp amplifiers equipped with female BNC-type connectors on the headstage. In this design the glass micropipette cannot contact the Ag/AgCl electrode, thus avoiding deterioration and subsequent offset voltages and baseline current drifts of the Ag/AgCl electrode, induced by frequent replacement of micropipettes.

Electrophysiology↗

PubMed1990

Na current in membrane blebs: implications for channel mobility and patch clamp recording.

When suction was applied to loose patch clamp pipettes while recording from enzymatically dissociated muscle fibers, large membrane blebs formed within the pipettes. We initiated a study of these suction-induced blebs because ion channels in the blebs would complicate or possibly invalidate loose patch voltage clamp measurements of membrane current density. The low lateral mobility (Stühmer and Almers, 1982) and steep gradients of Na channels at the end-plate and tendon (Caldwell et al., 1986) imply tight binding of Na channels to cytoskeletal elements and led us to expect few, if any, Na channels in the blebs. Bleb formation produced an increase in membrane capacitance, as expected from the increase in membrane area. Bleb formation also increased the Na current, indicating that the blebs contained Na channels. Assuming that the increased capacitance and Na current were due to lipid and Na channels moving from membrane outside the pipette, ejection of the bleb from the pipette was expected to bring the capacitance and Na current back to their original values. Capacitance did return to its original value, but Na current was lower than expected. The decrease in Na current is explained by Na channels moving from the patch membrane into the bleb. Normalization of bleb and patch Na current to their respective capacitances revealed that bleb membrane had a Na channel density almost 50% that of normal surface membrane. Thus, bleb membrane is neither devoid of proteins nor truly representative of the normal surface membrane from which it arose. It is enriched in membrane lipids and is relatively protein poor. Two conclusions can be drawn.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

[Analysis of molecular basis of neuronal properties using the patch-clamp RT-PCR method].

The AMPA-type glutamate receptor mediates fast neurotransmission in most of the excitatory synapses in the brain. Expression studies of the AMPA receptor subunits, GluR1-GluR4, have shown that the functional properties of AMPA receptors, such as rectification and Ca2+ permeability, are determined by their subunit composition. To analyze the molecular basis of functional properties of AMPA receptors at the single neuron level, we have combined the whole-cell patch clamp recording with the reverse transcription-polymerase chain reaction (patch-clamp RT-PCR method). Using this method, we have demonstrated that expression of the GluR2 subunit determines rectification properties and Ca2+ permeability of AMPA receptors in different types of neurons. The patch-clamp RT-PCR technique will be useful for a wide range of physiological studies to elucidate the molecular basis of functional properties at the single cell level.

Animals↗

PubMed1991

Ba2+ release from soda glass modifies single maxi K+ channel activity in patch clamp experiments.

Glasses used to fabricate patch pipettes may release components which affect ion channels (Cota, G., and C.M. Armstrong. 1988. Biophys. J. 53:107-109; Furman, R.E., and J.C. Tanaka. 1988. Biophys. J. 53:287-292; Rojas, L., and C. Zuazaga. 1988. Neurosci. Lett. 88:39-44). The gating properties of maxi K+ channels from Necturus gallbladder epithelium depend on whether borosilicate glass (BG) or blue tip hematocrit glass (SG) is used to construct the patch pipettes. The data are consistent with solubilization from SG of a component which exerts voltage-dependent, cytosolic-side specific block, closely resembling "slow block" by Ba2+ ions. Ringer's solution preincubated with SG, but not with BG, blocked inside-out maxi K+ channels when used as bathing solution. Mass spectrometry revealed that Ba2+ is released by the glass from fast and slow-release compartments (SG contains 3% wt/wt BaO), and is the only ion found in the solution at concentrations consistent with the observed channel block. Additionally, SG released O2-, Na+, Ca2+, and Mg2+, all to micromolar concentrations. These elements do not interfere with maxi K+ channels but they could in principle alter the properties of other ion channels. Thus, screening for channel-modifying substances released by the glass may be necessary for the adequate interpretation of patch-clamp results.

Animals↗

PubMed1988

Ionic channels in smooth muscle studied with patch-clamp methods.

Application of the patch-clamp technique on smooth muscle has started only a few years ago, but many publications are already emerging. The Ca and K channels so far found in smooth muscles seem to be fundamentally similar to those in other tissues. However, it is likely that Ca channel in smooth muscle is not controlled by cyclic AMP, in contrast to the cardiac muscle. There are at least two different ("fast" and "slow") types of Ca channel, but their relative distribution in different smooth muscles and their physiological significance are not yet fully analyzed. Furthermore, analysis of the receptor-operated Ca channels is still very limited, although these channels are very important for the smooth muscle function. Further careful studies are necessary to examine regulatory mechanisms involved in different types of Ca channel and to clarify the receptor-operated channel. There are also different (voltage- and Ca-activated) types of K channel. The Ca-activated K channel seems predominant in many smooth muscles ad regulating their excitability. However, K channels responsible for the resting potential or for spontaneous activity should be defined and also contribution of K (and other ionic) channels to receptor-mediated responses remains to be investigated.

Animals↗

PubMed1996

Cell-attached patch clamping of the intact rabbit ciliary epithelium.

Following thorough removal of adhering aqueous humor, we have succeeded in patch clamping the intact rabbit ciliary epithelium in the cell-attached and inside-out excised-patch modes. Rapidly fluctuating currents ("chatter activity') were observed during recordings conducted for as long as 1 h. Chatter activity did not reflect seal instability since interconversion was noted between chatter activity and transitions between stable open and closed states, excision of patches into the bath was associated with substantial shifts in the reversal potential, and chatter activity could be triggered by sustained hyperpolarization, but was insensitive to stretch. The chatter channel was identified as cation-nonselective from the reversal potentials both in the cell-attached and excised-patch modes. The channel's kinetics were similar to those of the cGMP-activated phototransduction channel. The results of PCR amplifications of fragments in cDNA libraries from both human ciliary body and human nonpigmented ciliary epithelial (NPE) cells indicated that human ciliary epithelial cells transcribe message for the retinal phototransduction channel. The possible role of the phototransduction channel in expressing chatter activity was further explored by perfusing preparations with a known activator of that channel (cGMP) and with a known inhibitor (L-cis-diltiazem). Neither agent significantly affected chatter behavior. We conclude that: (1) this is the first demonstration of the feasibility of patch-clamping the intact ciliary epithelium; (2) the NPE cells display chatter activity arising from rapidly fluctuating transitions of a cation-nonselective channel; (3) NPE cells can transcribe message for the cation-nonselective phototransduction channel; and (4) if the observed chatter activity is from a homologue of the photo-transduction channel, the homologue is pharmacologically distinct.

Animals↗

PubMed2004

Beta-escin diminishes voltage-gated calcium current rundown in perforated patch-clamp recordings from rat primary afferent neurons.

Perforated patch recordings of neuronal calcium currents (I(Ca)) with amphotericin B or nystatin reduce dialysis of intracellular constituents and current rundown, but can be difficult and frequently unsuccessful. We investigated the saponin beta-escin as a putative ionophore for perforated patch I(Ca) recordings in acutely dissociated, rat dorsal root ganglion neurons. I(Ca) was recorded in time-course studies after including either beta-escin (50 microM), or amphotericin B (240 microg/ml) as perforating ionophores in the internal pipette solution, in comparison to standard ruptured-patch technique, using suction. Perforated patches were allowed to take place spontaneously. The percentage loss of I(Ca) per min (within the first 20 min) was significantly less after beta-escin (0.0518%) (n = 18), versus either amphotericin (1.82%) (n = 12) or standard patch (4.52%) (n = 7), (P < 0.001). The slope of the rundown after linear fit was also less after beta-escin (P < 0.001). Minimal "steady-state" access resistance (R(a)) of 6.6 +/- 1.6 MOmega was achieved within 7.1 +/- 9.3 min following perforation with beta-escin, 7.9 +/- 3.5 MOmega within 44 =/- 14 min after amphotericin B, and 6.8 +/- 1.9 MOmega with standard patch (P < 0.05 for R(a), and P < 0.01 for permeabilization time, respectively). Success rates were 59% with beta-escin versus 27% with amphotericin. Leak >10% of peak I(Ca) was present in 25% of cells after beta-escin versus 20% after amphotericin, and 12% after standard technique. Perforated patches using beta-escin were stable for 15-60 min. We conclude that beta-escin may be used as an alternative ionophore for perforated patch-clamp studies in neurons, and results in minimal rundown that can facilitate long-term recordings of I(Ca). Limited rundown may be due to better preservation of cytosolic ATP content.

Afferent Pathways↗

PubMed2006

Hourglass SiO2 coating increases the performance of planar patch-clamp.

Obtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines.

Animals↗

PubMed2003

Flip the tip: an automated, high quality, cost-effective patch clamp screen.

The race for creating an automated patch clamp has begun. Here, we present a novel technology to produce true gigaseals and whole cell preparations at a high rate. Suspended cells are flushed toward the tip of glass micropipettes. Seal, whole-cell break-in, and pipette/liquid handling are fully automated. Extremely stable seals and access resistance guarantee high recording quality. Data obtained from different cell types sealed inside pipettes show long-term stability, voltage clamp and seal quality, as well as block by compounds in the pM range. A flexible array of independent electrode positions minimizes consumables consumption at maximal throughput. Pulled micropipettes guarantee a proven gigaseal substrate with ultra clean and smooth surface at low cost.

Animals↗

PubMed2002

[Patch-clamp technique in investigations of potassium channel activation in T lymphocytes].

This review focuses on results of patch-clamp studies on modulation of T lymphocyte potassium channel activity by physiologically relevant factors. In the preface the patch-clamp technique is briefly presented and basic properties of potassium channels in T lymphocytes are characterised. The paper contains an overview of the data on modulatory effects of extracellular and intracellular pH, temperature, extracellular potassium, extracellular divalent and trivalent metal cations, channel phosphorylation processes and membrane lipid metabolities on potassium channel activity. Some still unresolved problems in that area are indicated.

Animals↗

PubMed2001

Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings.

1. Simultaneous dendritic and somatic patch-clamp recordings were made from Purkinje cells in cerebellar slices from 12- to 21-day-old rats. Voltage responses to current impulses injected via either the dendritic or the somatic pipette were obtained in the presence of the selective I(h) blocker ZD 7288 and blockers of spontaneous synaptic input. Neurons were filled with biocytin for subsequent morphological reconstruction. 2. Four neurons were reconstructed and converted into detailed compartmental models. The specific membrane capacitance (C(m)), specific membrane resistance (R(m)) and intracellular resistivity (R(i)) were optimized by direct fitting of the model responses to the electrophysiological data from the same cell. Mean values were: C(m), 0.77 +/- 0.17 microF cm(-2) (mean +/- S.D.; range, 0.64-1.00 microF cm(-2)), R(m), 122 +/- 18 kOmega cm(2) (98-141 kOmega cm(2)) and R(i), 115 +/- 20 Omega cm (93-142 Omega cm). 3. The steady-state electrotonic architecture of these cells was compact under the experimental conditions used. However, somatic voltage-clamp recordings of parallel fibre and climbing fibre synaptic currents were substantially filtered and attenuated. 4. The detailed models were compared with a two-compartment model of Purkinje cells. The range of synaptic current kinetics that can be faithfully recorded using somatic voltage clamp is predicted fairly well by the two-compartment model, even though some of its underlying assumptions are violated. 5. A model of I(h) was constructed based on voltage-clamp data, and inserted into the passive compartmental models. Somatic EPSP amplitude was substantially attenuated compared to the amplitude of dendritic EPSPs at their site of generation. However, synaptic efficacy of the same quantal synaptic conductance, as measured by the somatic EPSP amplitude, was only weakly dependent on synaptic location on spiny branchlets. 6. The passive electrotonic structure of Purkinje cells is unusual in that the steady-state architecture is very compact, while voltage transients such as synaptic potentials and action potentials are heavily filtered.

4-Aminopyridine↗