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PubMed2004

The patch clamp technique in ion channel research.

To understand the pathogenesis of a given ion channel disorder, knowledge of the mutation alone is insufficient, instead, the description of the associated functional defect is decisive. The patch clamp technique enables to achieve this both in native tissue as well as heterologous expression systems. By this technique, structure-function relationships of ion channels were elucidated that not only support the homology already suggested by amino acid alignments of different channel types, but that also pointed to regions important for gating, ion selectivity, or subunit interaction. Currently, effort is being made to develop automation of the technique which will result in a cost-effective, fast, and highly accurate method to test for drug actions on high throughput scales. This review contains an overview of channel structures, channel diseases, and methods to study channel function by the patch clamp technique.

Animals↗

PubMed1990

The structure and dynamics of patch-clamped membranes: a study using differential interference contrast light microscopy.

We have developed techniques for micromanipulation under high power video microscopy. We have used these to study the structure and motion of patch-clamped membranes when driven by pressure steps. Patch-clamped membranes do not consist of just a membrane, but rather a plug of membrane-covered cytoplasm. There are organelles and vesicles within the cytoplasm in the pipette tip of both cell-attached and excised patches. The cytoplasm is capable of active contraction normal to the plane of the membrane. With suction applied before seal formation, vesicles may be swept from the cell surface by shear stress generated from the flow of saline over the cell surface. In this case, patch recordings are made from membrane that was not originally present under the tip. The vesicles may break, or fuse and break, to form the gigasealed patch. Patch membranes adhere strongly to the wall of the pipette so that at zero transmural pressure the membranes tend to be normal to the wall. With transmural pressure gradients, the membranes generally become spherical; the radius of curvature decreasing with increasing pressure. Some patches have nonuniform curvature demonstrating that forces normal to the membrane may be significant. Membranes often do not respond quickly to changes in pipette pressure, probably because viscoelastic cytoplasm reduces the rate of flow through the tip of the pipette. Inside-out patches may be peeled from the walls of the pipette, and even everted (with positive pressure), without losing the seal. This suggests that the gigaseal is a distributed property of the membrane-glass interface.

Animals↗

PubMed1998

Cytochemical and patch-clamp studies of calcium influx through voltage-dependent Ca2+ channels in vestibular supporting cells of guinea pigs.

To clarify whether or not vestibular supporting cells have voltage-dependent Ca2+ channels, cytochemical and patch-clamp studies were performed using cells isolated from the ampullae of the semicircular canal of the guinea pig. Image analysis used fura-2 as a Ca(2+)-sensitive fluorescence dye and showed that the intracellular Ca2+ concentration ([Ca2+]i) increased with bath application of high (150 mM)K+, but was unaffected by 80 mM K+. The increase in [Ca2+]i induced by high K+ was completely blocked by 1 microM nifedipine as an L-type Ca2+ channel antagonist. In the patch-clamp whole-cell recording of the isolated supporting cells, the voltage-dependent inward current was induced by a depolarizing pulse lasting 2 s in a high (50 mM) Ca2+ and tetraethylammonium-containing external solution replaced by choline chloride and a Cs(+)-containing internal solution. The inward current was obtained when the membrane was depolarized to -50 mV and maximum current was observed at -10 to +10 mV. This inward current was completely blocked by 1 microM nifedipine. These findings strongly suggest that voltage-dependent Ca2+ channels exist in the vestibular supporting cells and regulate Ca2+ concentration in the vestibular endolymph.

Animals↗

PubMed2006

Micro-agar salt bridge in patch-clamp electrode holder stabilizes electrode potentials.

Maintaining a stable electrode potential is critical for patch-clamp measurements. The electrode potential of conventional patch electrode-holder assembly, where an Ag/AgCl wire is in direct contact with the patch pipette filling solution, is subject to drift if the pipette solution contains a low concentration of chloride ions (Cl-). We developed an agar bridge of 3 M KCl filled in a polyimide microtubing which forms an electrical connection between an Ag/AgCl wire and the pipette solution. We examined the offset potentials of the micro-agar salt bridge electrode assembly in parallel with a conventional electrode assembly in generic recording conditions (the pipette solution contained 5 mM NaCl). The junction potential between the Ag/AgCl wire and the pipette filling solution in the conventional electrode contributed to most of the offset potential drift observed during the course of 30 min recordings. The drift was up to 27.3 mV after several changes of the glass pipette. In contrast, the micro-agar salt bridge stabilized the electrode potential within typically 2 mV without affecting the patch electrode resistance, capacitance or noise level. Numerical simulations showed that Cl- diffusion from the agar bridge to the tip caused a negligible 0.4 microM Cl- concentration change at the pipette tip within 30 min. This method is easy to implement and provides long-term recording stability. The micro-agar salt bridge can fit in most commercial patch electrode holders and can be conveniently maintained.

Agar↗

PubMed1991

Patch clamping VDAC in liposomes containing whole mitochondrial membranes.

Whole mitochondrial membranes isolated from Neurospora crassa were reconstituted into liposomes and patch clamped. Clear activity characteristic of the mitochondrial channel VDAC was found, namely: open state conductance of 650 pS (in 150 mM KCl, 1 mM CaCl2, 20 mM HEPES, pH 7.2), voltage-dependent closure at both positive and negative potentials, change in conductance upon channel closure of about 450 pS in response to negative and positive potentials, and increased voltage dependence in the presence of König's polyanion. This is the first clear demonstration of VDAC single channels using the patch-clamp technique, even though others used this method before to study whole mitochondrial membranes and liposomes containing mitochondrial proteins. We also found one other channel with a conductance change of about 120 pS.

Electric Conductivity↗

PubMed2001

Screening of ion channel receptor agonists using capillary electrophoresis-patch clamp detection with resensitized detector cells.

Efficient techniques for identifying endogenous and synthetic ligands of ion channels are important in understanding neuronal communication and for screening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection where a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbachol, and (-)-nicotine were fractionated and detected by patch-clamped pheochromcytoma detector cells. The high-conductance state of the nAChr during CE-PC detection was maintained and repetitively resensitized using pulsed-flow superfusion with agonist-free buffer. In this way, each agonist evoked an ensemble of peak currents that reflected the spatiotemporal distribution for the ligand at the cell surface. The technique takes advantage of the intrinsic high selectivity and sensitivity of membrane-expressed receptors and allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.

Acetylcholine↗

PubMed1992

Ca-dependent K channels in smooth muscle cells permeabilized by beta-escin recorded using the cell-attached patch-clamp technique.

Using the cell-attached patch-clamp technique, the activity of single, Ca-dependent K channels was recorded in single smooth muscle cells permeabilized by beta-escin. The conductance and the relationship between the open probability of the channels and pCa recorded in permeabilized cells were very similar to those obtained in excised inside-out patches. At pCa 7, application of 30 microM acetylcholine (ACh) or 0.1 microM substance P (SP) together with 1 mM guanosine 5'-trisphosphate to permeabilized cells elicited transient bursts of channel openings similar to those which occur in intact cells. Transient activation was also observed when 2-30 microM inositol trisphosphate (IP3) was applied to permeabilized cells. This single channel activity was inhibited by pretreatment with low-molecular-weight heparin at 50-100 micrograms/ml. Channel activity at pCa 7.0 was greatly enhanced by 200 microM cyclic adenosine monophosphate. These results provide direct evidence that single Ca-dependent K channel activity is regulated by the transmitters ACh and SP, as well as a second messenger, IP3, via the release of intracellular Ca from intracellular sites which are blocked by heparin. This novel approach is valuable in elucidating second messenger mechanisms involved in the regulation of single channel activity by transmitters and autocoids, since permeabilization by beta-escin preserves the entire system of receptor-operated signal transduction and allows intracellular application of second messengers at fixed concentrations.

Acetylcholine↗

PubMed2000

Evidence for cooperativity between nicotinic acetylcholine receptors in patch clamp records.

It is often assumed that ion channels in cell membrane patches gate independently. However, in the present study nicotinic receptor patch clamp data obtained in cell-attached mode from embryonic chick myotubes suggest that the distribution of steady-state probabilities for conductance multiples arising from concurrent channel openings may not be binomial. In patches where up to four active channels were observed, the probabilities of two or more concurrent openings were greater than expected, suggesting positive cooperativity. For the case of two active channels, we extended the analysis by assuming that 1) individual receptors (not necessarily identical) could be modeled by a five-state (three closed and two open) continuous-time Markov process with equal agonist binding affinity at two recognition sites, and 2) cooperativity between channels could occur through instantaneous changes in specific transition rates in one channel following a change in conductance state of the neighboring channel. This allowed calculation of open and closed sojourn time density functions for either channel conditional on the neighboring channel being open or closed. Simulation studies of two channel systems, with channels being either independent or cooperative, nonidentical or identical, supported the discriminatory power of the optimization algorithm. The experimental results suggested that individual acetylcholine receptors were kinetically identical and that the open state of one channel increased the probability of opening of its neighbor.

Animals↗

PubMed1995

Persistent Na+ conductance in medium-sized neostriatal neurons: characterization using infrared videomicroscopy and whole cell patch-clamp recordings.

1. In the present study we investigate the expression of a persistent Na+ conductance (INaP) in identified medium-sized neostriatal neurons. Nomarski optics and infrared videomicroscopy were used for cell visualization and identification in thick slices (350 microns). Current- and voltage-clamp recordings were obtained utilizing whole cell patch-clamp methodology. 2. Application of depolarizing ramp voltage commands from a holding potential of -70 mV induced a slow, noninactivating inward current that occurred before and independent of the rapidly inactivating sodium current that subserves action potential generation. INaP began to activate at potentials less negative than -70 mV and peaked at -34 +/- 1 (SE) mV. Its average peak amplitude was -100 +/- 17 pA. INaP was abolished by tetrodotoxin (TTX, 0.5-1 microM) or an Na(+)-free solution. In contrast, it was not affected by Ca2+ channel blockers. Depolarizing ramp commands also induced tetraethylammonium-sensitive outward currents. 3. Dopamine (DA) (20-100 microM) produced a significant reduction of INaP. 4. These results demonstrate the existence of a TTX-sensitive persistent Na+ conductance in medium-sized neostriatal neurons. This conductance is modulated by DA and could play a role in the generation of rhythmic oscillations and in supporting repetitive firing.

Animals↗

PubMed1998

In vitro patch-clamp studies in skin fibroblasts.

We have conducted single-channel patch-clamp experiments in skin fibroblasts maintained in culture. Two different cell lines, a mouse 3T3-L1 cell line and a human B17 cell line, were selected for these pilot studies. Recordings were made from both cell-attached and excised inside-out patches at room temperature. In the case of the 3T3-L1 cells, the success rate in obtaining good seals (> 1Gomega) was low, and channel openings in either cell-attached or excised patches were rare. We have, however, identified a channel in a cell-attached configuration with a slope conductance of 39 pS in symmetrical K+ solutions. In the case of the human B17 cells, good quality seals were more readily obtained. One principal type of channel opening was identified. In cell-attached patches, the prevalent type of channel in symmetrical K+ solutions had a conductance of 187 pS. This channel was activated by strong depolarization, and there was usually more than one active channel in the patch. It was blocked by extracellular tetraethylammonium (20 mM), and persisted when external Cl- was replaced by aspartate. In excised inside-out patches bathed in symmetrical K+, this channel was activated by an increase in Ca+ applied to the intracellular face. A large conductance channel (175 pS) was also observed in excised inside-out patches, with a reverse physiological K+ gradient. This channel had a reversal potential > 40 mV and appeared not to be voltage-dependent under these recording conditions (2 mM Ca(2+)i). We conclude that the channel we have identified in these cells belongs to the maxi-K+ channel class.

3T3 Cells↗

PubMed1992

GABA-mediated synaptic transmission in neuroendocrine cells: a patch-clamp study in a pituitary slice preparation.

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline (Kd = 0.1 microM), indicating that they were mediated by gamma-aminobutyric acid A (GABAA) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1-2 ms) rise time and a biexponential decay, with time constants of 21 +/- 4 ms and 58 +/- 14 ms at a holding potential of -60 mV (n = 6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 microM tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1 +/- 2.3 pA (n = 4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA (n = 6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system.

Animals↗

PubMed2001

Patch-clamp analysis of voltage-activated and chemically activated currents in the vomeronasal organ of Sternotherus odoratus (stinkpot/musk turtle).

The electrophysiological basis of chemical communication in the specialized olfactory division of the vomeronasal (VN) organ is poorly understood. In total, 198 patch-clamp recordings were made from 42 animals (Sternotherus odoratus, the stinkpot/musk turtle) to study the electrically and chemically activated properties of VN neurons. The introduction of tetramethylrhodamine-conjugated dextran into the VN orifice permitted good visualization of the vomeronasal neural epithelium prior to dissociating it into single neurons. Basic electrical properties of the neurons were measured (resting potential, -54.5 +/- 2.7 mV, N=11; input resistance, 6.7 +/- 1.4 G Omega, N=25; capacitance, 4.2 +/- 0.3 pF, N=22; means +/- S.E.M.). The voltage-gated K(+) current inactivation rate was significantly slower in VN neurons from males than in those from females, and K(+) currents in males were less sensitive (greater K(i)) to tetraethylammonium. Vomeronasal neurons were held at a holding potential of -60 mV and tested for their response to five natural chemicals, female urine, male urine, female musk, male musk and catfish extract. Of the 90 VN neurons tested, 33 (34 %) responded to at least one of the five compounds. The peak amplitude of chemically evoked currents ranged from 4 to 180 pA, with two-thirds of responses less than 25 pA. Urine-evoked currents were of either polarity, whereas musk and catfish extract always elicited only inward currents. Urine applied to neurons harvested from female animals evoked currents that were 2-3 times larger than those elicited from male neurons. Musk-evoked inward currents were three times the magnitude of urine- or catfish-extract-evoked inward currents. The calculated breadth of responsiveness for neurons presented with this array of five chemicals indicated that the mean response spectrum of the VN neurons is narrow (H metric 0.11). This patch-clamp study indicates that VN neurons exhibit sexual dimorphism in function and specificity in response to complex natural chemicals.iol

Animals↗

PubMed1990

A nonenzymatic preparation of epithelial basolateral membrane for patch clamp.

A preparation has been developed that permits patch clamping of the basolateral membrane of Necturus gallbladder epithelial cells with a high success rate. The epithelium is separated from the underlying tissues mechanically, without enzymatic treatment. Its apical surface is attached to a plastic cover slip, and the basolateral surface, facing up, is cleaned with a suction pipette under microscopic observation. With this cleaning procedure, the success rate in obtaining gigaohm seals increases from less than 1% to approximately 10% of the attempts. The cells appear to retain their structural and functional integrity, as evidenced by electron-microscopic appearance and magnitude of cell membrane voltages. Major advantages of the preparation are that the basolateral membrane domain is preserved and that enzymatic treatment, which could potentially alter membrane proteins, is not necessary.

Animals↗

PubMed2003

Isolation of AtSUC2 promoter-GFP-marked companion cells for patch-clamp studies and expression profiling.

K+ channels control K+ homeostasis and the membrane potential in the sieve element/companion cell complexes. K+ channels from Arabidopsis phloem cells expressing green fluorescent protein (GFP) under the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RT-PCR. Single green fluorescent protoplasts were selected after being isolated enzymatically from vascular strands of rosette leaves. Companion cell protoplasts, which could be recognized by their nucleus, vacuole and chloroplasts, and by their expression of the phloem-specific marker genes SUC2 and AHA3, formed the basis for a cell-specific cDNA library and expressed sequence tag (EST) collection. Although we used primers for all members of the Shaker K+ channel family, we identified only AKT2, KAT1 and KCO6 transcripts. In addition, we also detected transcripts for AtPP2CA, a protein phosphatase, that interacts with AKT2/3. In line with the presence of the K+ channel transcripts, patch-clamp experiments identified distinct K+ channel types. Time-dependent inward rectifying K+ currents were activated upon hyperpolarization and were characterized by a pronounced Ca2+-sensitivity and inhibition by protons. Whole-cell inward currents were carried by single K+-selective channels with a unitary conductance of approximately 4 pS. Outward rectifying K+ channels (approximately 19 pS), with sigmoidal activation kinetics, were elicited upon depolarization. These two dominant phloem K+ channel types provide a versatile mechanism to mediate K+ fluxes required for phloem action and potassium cycling.

Arabidopsis↗

PubMed1992

Whole-cell patch-clamp recordings from respiratory neurons in neonatal rat brainstem in vitro.

Whole-cell recordings were obtained from respiratory neurons by applying patch-clamp techniques in the en bloc medulla of in vitro neonatal rat brainstem-spinal cord preparations. Stable voltage-clamp recordings of excitatory or inhibitory synaptic drive currents and current-clamp recordings of spike discharge of inspiratory and expiratory neurons could be maintained for periods of 1-2 h. Parameters of whole-cell recording, including membrane seal resistances and series resistances, obtained in the en bloc medulla were similar to those obtained in corresponding regions of thin slices where neurons were directly visualized to optimize conditions for whole-cell patch clamp.

Animals↗

PubMed1992

[Ervin Neher and Bert Sakmann, 1991 Nobel Prize laureates for physiology and medicine. Ion channels and the patch clamp technic].

E. Neher and B. Sakmann were awarded the Nobel Prize for achievements in Physiology or Medicine in 1991. They developed the "patch clamp technique" which enables measurement of ionic currents through channels in the plasma membrane of living cells, and characterized their functional properties. An overview of three types of ion channels is presented: voltage gated channels, ligand gated channels and G-protein gated channels. An attempt was made to correlate their function with the structure of the protein complexes that represent ionic channels. The perforated patch clamp technique is mentioned as an alternative that enables recording of membrane currents without washing out the content of the interior of the cells.

Animals↗

PubMed1996

Evidence from incorporation and patch-clamp experiments for a nonselective channel of large conductance at the luminal membrane of rabbit proximal tubule.

The presence of nonselective ionic channels at the apical membrane of rabbit proximal tubule cells was investigated by fusion of proximal tubule brush border membrane vesicles onto a planar bilayer and by means of patch clamp measurements on primary cultured proximal tubule cells. The fusion of brush border membrane vesicles onto a phosphatidylethanolamine-phosphatidylcholine (1:1) planar bilayer led to the identification of a nonselective channel of 115 pS in 200 mM NaCl conditions with an ionic permeability sequence for Na+, K+, and Cl- corresponding to PNa/PK = 0.3 and PNa/PCl = 3, respectively. This channel showed an increased open probability at depolarizing potentials, but its activity was independent of the Ca2+ concentration in both the cis and trans compartments. These findings were subsequently confirmed in inside-out patch clamp experiments carried out on primary cultured proximal tubule cells. This channel was seldom seen in cell-attached recordings on resting cells, but its presence was observed with a higher probability in patch excised experiments performed following superfusion of the cells with a hypotonic solution. It is suggested on the basis of these observations that the apical membrane of rabbit proximal tubule cells contains a voltage-dependent nonselective channel, the activity of which may contribute to the regulation of the membrane potential.

Animals↗

PubMed1995

Patch-clamp analysis of synaptic transmission to cerebellar purkinje cells of prion protein knockout mice.

The prion protein (PrP) plays a pivotal role in transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. Previous experiments have suggested that the normal cellular prion protein (PrPc) is involved in synaptic function in the hippocampus. Here, we utilized the controlled recording conditions of the patch-clamp technique to investigate the synaptic function of prion protein in cerebellar Purkinje cells. By performing whole-cell and outside-out patch-clamp experiments in thin slices, we investigated synaptic transmission in prion protein knockout mice (PrP-null) and control animals. In PrP-null mice, the kinetics of GABA- and glutamate receptor-mediated currents showed no significant deviation from those in control animals. In contrast to previous results in hippocampal neurons, our findings support the view that synaptic transmission is unimpaired in prion protein-deficient mice.

Animals↗