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PubMed2004

Angiotensin II excites paraventricular nucleus neurons that innervate the rostral ventrolateral medulla: an in vitro patch-clamp study in brain slices.

Neurons of the hypothalamic paraventricular nucleus (PVN) are key controllers of sympathetic nerve activity and receive input from angiotensin II (ANG II)-containing neurons in the forebrain. This study determined the effect of ANG II on PVN neurons that innervate in the rostral ventrolateral medulla (RVLM)-a brain stem site critical for maintaining sympathetic outflow and arterial pressure. Using an in vitro brain slice preparation, whole cell patch-clamp recordings were made from PVN neurons retrogradely labeled from the ipsilateral RVLM of rats. Of 71 neurons tested, 62 (87%) responded to ANG II. In current-clamp mode, bath-applied ANG II (2 muM) significantly (P < 0.05) depolarized membrane potential from -58.5 +/- 2.5 to -54.5 +/- 2.0 mV and increased the frequency of action potential discharge from 0.7 +/- 0.3 to 2.8 +/- 0.8 Hz (n = 4). Local application of ANG II by low-pressure ejection from a glass pipette (2 pmol, 0.4 nl, 5 s) also elicited rapid and reproducible excitation in 17 of 20 cells. In this group, membrane potential depolarization averaged 21.5 +/- 4.1 mV, and spike activity increased from 0.7 +/- 0.4 to 21.3 +/- 3.3 Hz. In voltage-clamp mode, 41 of 47 neurons responded to pressure-ejected ANG II with a dose-dependent inward current that averaged -54.7 +/- 3.9 pA at a maximally effective dose of 2.0 pmol. Blockade of ANG II AT1 receptors significantly reduced discharge (P < 0.001, n = 5), depolarization (P < 0.05, n = 3), and inward current (P < 0.01, n = 11) responses to locally applied ANG II. In six of six cells tested, membrane input conductance increased (P < 0.001) during local application of ANG II (2 pmol), suggesting influx of cations. The ANG II current reversed polarity at +2.2 +/- 2.2 mV (n = 9) and was blocked (P < 0.01) by bath perfusion with gadolinium (Gd(3+), 100 muM, n = 8), suggesting that ANG II activates membrane channels that are nonselectively permeable to cations. These findings indicate that ANG II excites PVN neurons that innervate the ipsilateral RVLM by a mechanism that depends on activation of AT1 receptors and gating of one or more classes of ion channels that result in a mixed cation current.

Anesthetics, Local↗

PubMed1996

Permeability increase induced by Escherichia coli hemolysin A in human macrophages is due to the formation of ionic pores: a patch clamp characterization.

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K+ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches.

Bacterial Proteins↗

PubMed1998

Patch-clamp, ion-sensing, and glutamate-sensing techniques to study glutamate transport in isolated retinal glial cells.

We have described how a combination of electrical, ion-sensing, and glutamate-sensing techniques has advanced our understanding of glutamate uptake into isolated salamander retinal glial cells. The next steps in understanding glutamate transport will inevitably depend strongly on molecular biological methods, as described elsewhere in this book, but will also require more detailed study of transporters in their normal environment, perhaps by using patch-clamping or imaging techniques to study cells in situ.

ATP-Binding Cassette Transporters↗

PubMed2004

Ca(2+) channel properties in neuroendocrine tumor cell cultures investigated by whole-cell patch-clamp technique.

Neuroendocrine tumor (NET) cells of the gastroenteropancreatic system express a variety of voltage-operated Ca(2+) channels (VOCCs). In addition, NET cells release hormones and biogenic amines in distinct patterns, leading to typical hypersecretion syndromes. Interestingly, these patterns depend on the primary location of the NET. The aim of this study was to investigate a possible link between clinically distinct entities of NETs and specific properties of VOCCs. Using whole-cell patch-clamp technique, electrophysiological data were obtained from permanent NET cell cultures as well as from primary cultivated NET cells from foregut and midgut tumors. Definite Ca(2+) channel characteristics were analyzed by a color-coding method. As a result, we could demonstrate specific Ca(2+) channel characteristics in NET cells from midgut tumors that were not found in NET cells from a foregut location. This may be important functionally with respect to different cell biological functions of NET cells. In summary, clear differences exist in the VOCC pattern in NET cells from foregut versus midgut. This may be functionally relevant in the hypersecretion syndrome predominantly found in foregut NETs. Therefore, these data on the characteristics of VOCCs could be useful in the development of a novel therapy for NET disease; for example, specific VOCC subtype modulators may help.

Calcium Channels↗

PubMed2001

Plasma membrane Ca2+ release-activated Ca2+ channels with a high selectivity for Ca2+ identified by patch-clamp recording in rat liver cells.

Repetitive waves of increased cytoplasmic Ca2+ concentration play a central role in the process by which hormones regulate liver function. Maintenance of these Ca2+ waves requires Ca2+ inflow through store-operated Ca2+ channels. The properties and mechanism(s) of activation of these channels are not well understood. Store-operated Ca2+ channels (SOCs) in the H4-IIE rat liver cell line were studied by whole-cell patch clamping. Depletion of Ca2+ in intracellular stores by intracellular perfusion with either inositol 1,4,5-trisphosphate (InsP(3)) or thapsigargin in the presence of 10 mmol/L ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA), or with 10 mmol/L EGTA alone, activated an inward current that reversed at a membrane potential above +40 mV. In physiologic extracellular medium, this inward current was carried exclusively by Ca2+ and was blocked by a variety of di- and trivalent cations. In the absence of extracellular Ca2+ and Mg2+, the inward current was carried by monovalent cations. This current was 10 to 30 times larger than that observed in the presence of extracellular Ca2+, and permitted the detection of single-channel events that corresponded to a single-channel conductance of about 40 pS. Both the Ca2+ and Na+ inward currents were blocked by the calmodulin antagonist, N-(6-amino hexyl)-5-chloro-1-naphthalenesulphonamide (W7), but not by calmidazolium or calmodulin-dependent protein kinase II fragment 290-309. It is concluded that liver cells possess plasma membrane Ca2+ channels that have a high selectivity for Ca2+, are activated by a decrease in the concentration of Ca2+ in intracellular stores through a mechanism that is unlikely to involve calmodulin, and are involved in re-filling intracellular Ca2+ stores during Ca2+ signaling.

Animals↗

PubMed1989

Calcium-dependent potassium channel in Paramecium studied under patch clamp.

We have studied a class of Ca2+i-dependent K channels in inside-out excised membrane patches from Paramecium under patch clamp. single channels had a conductance of 72 +/- 9.0 pS in a solution containing 100 mM K+. The channels were selective for K+ over Rb+ with the permeability ratio of 1: 0.56, and over Na+, Cs+ or NH+4 with a ratio 1: less than 0.1. The channel activity was dependent on Ca2+i, which was applied to the cytoplasmic side; the Ca2+i concentration for the half maximal activation was 2 microM. The Hill coefficient for the Ca2+i dependence of the channel activity was 2.58, indicating that more than two Ca2+i bindings are necessary for full activation. Unlike most Ca2+i-dependent K channels in other organisms, the channels in Paramecium were slightly more active upon hyperpolarization than upon depolarization. The voltage dependence was fitted to a Boltzmann curve with 41.2 mV per e-fold change in channel activity. While a high Ca2+i concentration activated the channels, it also irreversibly reduced the channel activity over time. The decay of channel activity occurred faster at higher Ca2+i concentrations. Quaternary ammonium ions suppressed ion passage through the channel; more highly alkylated quaternary ammonium ions were more efficient in blocking. Ba2+i and Ca2+i were relatively ineffective in blockage. it was concluded that these Ca2+i-dependent K channels in Paramecium are different from the previously described Ca2+i-dependent K channels, and are perhaps of a novel class.

Animals↗

PubMed1987

Ca2+ channels from brain microsomal membranes reconstituted in patch-clamped bilayers.

Single Ca2+ channels from brain microsomal membranes were reconstituted in bilayers made at the tips of patch-clamp micropipettes. The single-channel conductance was defined to be 107 pS in 50 mM Ca2+. The channel activity was stimulated by nucleotides and inositol 1,4,5-trisphosphate (Ins-P3), and was inhibited by ruthenium red. Na+ added asymmetrically to the membrane bilayer induced an increase in the Ca2+-channel activity. The described characteristics of these Ca2+ channels suggest that they may be responsible for the Ca2+ transport across the membranes of the endoplasmic reticulum system triggering and modulating various neurosecretory and excitatory processes in nerve cells.

Adenosine Triphosphate↗

PubMed1993

The calcium signal in human neutrophils and its relation to exocytosis investigated by patch-clamp capacitance and Fura-2 measurements.

Intracellular calcium ([Ca2+]i) and exocytosis of human neutrophils were investigated with patch-clamp capacitance and Fura-2 fluorescence measurements. Intracellular application of GTP gamma S induces a calcium transient and exocytosis. The onset of degranulation occurs at the time where the maximal [Ca2+]i is reached. Despite the close correlation in time, buffering [Ca2+]i at the resting level or at approximately 2 microM leaves the extent and the time course of degranulation unchanged. The decay of the calcium transient is due to diffusional equilibration between the cytosol and the pipette volume. GTP gamma S activates no cellular mechanisms for Ca2+ reuptake or extrusion. The endogenous calcium buffer capacity can be estimated to be as low as that of approximately 90 microM Fura-2. Stimulation with fMLP also induces degranulation and a calcium transient. The decay of fMLP-induced calcium transients is much faster than that of GTP gamma S-induced transients and is independent of diffusion indicating that fMLP also induces rapid reuptake or extrusion of Ca2+. Degranulation but not the calcium transient requires the presence of intracellular GTP. Different signalling pathways appear to be involved in GTP gamma S- and fMLP-stimulated calcium signals. The intracellular calcium release is not an essential signal to initiate exocytosis in neutrophils.

Calcium↗

PubMed1998

Neuromuscular blockade by IgG antibodies from patients with Guillain-Barré syndrome: a macro-patch-clamp study.

Guillain-Barré syndrome (GBS) is often associated with serum antibodies to glycoconjugates such as GM1 and GQ1b. The pathogenic role of these antibodies and other serum factors has not yet been clarified. We have investigated the effect of serum, plasma filtrate, and highly purified IgG and IgM from 10 patients with typical GBS on motor nerve terminals in the mouse hemidiaphragm. Quantal endplate currents were recorded by means of a perfused macro-patch-clamp electrode. The plasma filtrate of all GBS patients led to a 5- to 20-fold reduction of evoked quantal release within 7 to 15 minutes of continuous superfusion. In 4 patients, the amplitudes of single quanta were clearly reduced (by 10-66% of control values), indicating an additional postsynaptic action. Blocking effects could be reversed to a variable degree within 15 to 18 minutes after washout. Purified IgG was as effective as native serum, whereas a purified GBS IgM fraction did not block transmission. Sera from convalescent patients and IgG from healthy subjects were without blocking effect. The effects were complement independent and there was no link to the presence (in 6 patients) or absence (in 4 patients) of detectable antibodies to GM1 or GQ1b. In GBS, antibodies to an undetermined antigen depress the presynaptic transmitter release and, in some cases, the activation of postsynaptic channels. We suggest that weakness in the acute stage of GBS may be caused in part by circulating antibodies.

Adult↗

PubMed2002

Patch-clamp recording of Müller glial cells after cryopreservation.

Human and other primate retinal Müller cells display dominating K(+) currents as well as other membrane conductances that may change in cases of retinal pathology. Because the use of human and primate tissue is limited by reasons of availability, a method for long-term storage of these cells is desirable. We describe a cryopreservation method in which isolated Müller cells are stored in liquid nitrogen. After thawing, the cells can be used for patch-clamp experiments immediately, without culturing. We show that the main electrophysiological properties are not altered by this method and that voltage- and ligand-gated currents can be recorded from cryopreserved cells even after 2-years storage.

Adenosine Triphosphate↗

PubMed2000

Patch-clamp analysis of anesthetic interactions with recombinant SK2 subtype neuronal calcium-activated potassium channels.

UNLABELLED: Small conductance calcium-activated potassium channels (SK) mediate spike frequency adaptation and underlie the slow afterhyperpolarization in central neurons. We tested the actions of several anesthetics on the SK2 subtype of recombinant SK channels, cloned from rat brain and functionally expressed in a mammalian cell line. Butanol, ethanol, ketamine, lidocaine, and methohexital blocked recombinant SK2 channel currents, measured in the whole-cell patch clamp recording mode. The block was reversible, dose-dependent, and of variable efficacy. The inhaled anesthetics chloroform, desflurane, enflurane, halothane, isoflurane, and sevoflurane produced little or no block when applied at 1 minimum alveolar anesthetic concentration; varying degrees of modulation were observed at very large concentrations (10 minimum alveolar concentration). The extent of block by inhaled anesthetics did not appear to depend on concentration or membrane voltage. IMPLICATIONS: We describe differential effects of anesthetics on cloned small conductance calcium-activated potassium channels from brain that may play a role in generating the effects or side effects of anesthetics.

Anesthetics↗

PubMed2000

Patch clamp studies of the thr1313met mutant sodium channel causing paramyotonia congenita.

Paramyotonia congenita (PC) is an autosomal-dominant disorder due to a point mutation in the adult skeletal muscle Na channel gene. Muscle fibers from PC patients have normal membrane properties at 32 degrees C. At 27 degrees C, they are inexcitable, have increased Na conductance, and have a reduced resting membrane potential of -40 mV. To define the biophysical basis for the muscle membrane abnormalities, we performed patch clamp whole-cell and outside-out single Na channel studies at 22 degrees C on cultured human muscle cells from 4 control patients and 2 sisters with PC and the thr1313met mutant Na channel. The whole-cell studies showed no difference in window currents. Unlike cells transfected with the thr1313met mutant Na channel, the inactivation time constant, tau(h), for PC cells was similar to control cells. For PC recordings containing long-duration single Na channel openings, mean open time was prolonged at -60, -40, and -20 mV. The long-duration Na channel openings occurred randomly with no evidence of modal gating. The number of channel openings, occurrence of late openings, and the prolonged mean open time resulted in a sustained inward Na current at -40 mV. We suggest that the biophysical marker of the thr1313met mutant Na channel is a voltage- and temperature-dependent abnormality in mutant single Na channel behavior.

Adult↗

PubMed1998

Removing non-random artifacts from patch clamp traces.

Analysis and interpretation of current records from cellular membranes or lipid bilayers is complicated by perturbations. These perturbations can originate from random noise or deterministic artifacts as capacitive currents caused by step depolarization's, drift, 50 Hz pick up or microphonics. Accurate removal of deterministic perturbations is a prerequisite for further analysis of ion channel kinetics. In this report we present two methods developed for parameter estimation of such artifacts in order to remove these perturbations from single channel patch clamp traces. For both methods artifact removal does not require sweeps lacking channel activity. The first method was designed to extract artifacts from sweeps showing moderate channel activity. Within a certain number of sweeps the artifacts should remain rather constant. The second method allows for artifact removal from one individual sweep with channel activity. With the second method, correction for drift, pick up and microphonics is possible without long periods of minor channel activity. To evaluate the correctness of artifact removal a specific test was set up. The methods were carefully examined using simulated records for a wide range of parameters found in single channel experiments. Further, the developed algorithms were applied to original records obtained from cardiac and recombinant L-type Ca2+ channels.

Algorithms↗

PubMed2005

Patch-clamp analysis of the "new permeability pathways" in malaria-infected erythrocytes.

The intraerythrocytic amplification of the malaria parasite Plasmodium falciparum induces new pathways of solute permeability in the host cell's membrane. These pathways play a pivotal role in parasite development by supplying the parasite with nutrients, disposing of the parasite's metabolic waste and organic osmolytes, and adapting the host's electrolyte composition to the parasite's needs. The "new permeability pathways" allow the fast electrogenic diffusion of ions and thus can be analyzed by patch-clamp single-channel or whole-cell recording. By employing these techniques, several ion-channel types with different electrophysiological profiles have been identified in P. falciparum-infected erythrocytes; they have also been identified in noninfected cells. This review discusses a possible contribution of these channels to the new permeability pathways on the one hand and their supposed functions in noninfected erythrocytes on the other.

Adenosine Triphosphate↗

PubMed2003

Xenopus embryonic spinal neurons recorded in situ with patch-clamp electrodes--conditional oscillators after all?

The central pattern generator for swimming Xenopus embryo is organized as two half-centres linked by reciprocal inhibition. Microelectrode recordings suggest that Xenopus neurons are poorly excitable, necessitating a key role for postinhibitory rebound in the operation of the central pattern generator. However the Xenopus central pattern generator seems unusual in that the component neurons apparently have no intrinsic or conditional rhythmogenic properties. We have re-examined the firing properties of Xenopus embryo spinal neurons by making patch-clamp recordings in situ from intact spinal cord. Recordings made from 99 neurons were divided into three groups. Central pattern generator neurons overwhelmingly (44/51) fired trains of action potentials in response to current injection. Just over half of the sensory interneurons (13/22) also fired trains of action potentials. Neurons that received no synaptic inputs during swimming mostly fired just one or two action potentials (22/26). Thirty-four neurons were identified morphologically. Commissural (8/12) and descending (6/6) interneurons, key components of the spinal central pattern generator, fired repetitive trains of action potentials during current injection. Neurons that were not part of the central pattern generator did not demonstrate this preponderance for repetitive firing. Analysis of the interspike intervals during current injection revealed that the majority of central pattern generators, descending and commissural interneurons, could readily fire at frequencies up to twice that of swimming. We suggest that Xenopus neurons can be considered as conditional oscillators: in the presence of unpatterned excitation they exhibit an ability to fire rhythmically. This property makes the Xenopus embryonic central pattern generator more similar to other model central pattern generators than has hitherto been appreciated.

Action Potentials↗

PubMed1990

Giant liposomes: a model system in which to obtain patch-clamp recordings of ionic channels.

Cell-size, giant liposomes have been formed by submitting a mixture of asolectin lipid vesicles and native membranes from Torpedo, highly enriched in acetylcholine receptor (AcChR), to a partial dehydration/rehydration cycle [Criado, M., & Keller, B. U. (1987) FEBS Lett. 224, 172-176]. Giant liposomes can be prepared in bulk quantities, in the absence of potentially damaging detergents or organic solvents, and their formation is mediated by membrane fusion phenomena. In fact, fluorescence microscopy and freeze-fracture data indicate that protein and lipid components of the initial membranes and lipid vesicles are homogenously distributed in the resulting liposomes. Giant liposomes containing AcChR have been used as a model to evaluate whether this system can be used to monitor the activity of ionic channels by using high-resolution, patch-clamp techniques. Excised liposome patches in an "inside-out" configuration have been used in this work. We find that the most frequent pattern of electrical activity in response to the presence of acetylcholine in the patch pipet corresponds to a cation-specific channel exhibiting a dominant conductance level and a sublevel of approximately 78 and 25 pS, respectively. Such channel activity exhibits the pharmacological specificity, ion channel activation, ion selectivity, and desensitization properties expected from native Torpedo AcChR. Thus, it appears that the giant liposome technique offers a distinct advantage over other reconstitution procedures in that it provides a unique opportunity to undertake simultaneous biochemical, morphological, and electrophysiological studies of the incorporated ionic channel proteins.

Animals↗

PubMed2005

In vivo and in vitro patch-clamp recording analysis of the process of sensory transmission in the spinal cord and sensory cortex.

Following the integration and modification of the sensory inputs in the spinal cord, the information is transmitted to the primary sensory cortex where the integrated information is further processed and perceived. Processing of the sensory information in the spinal cord has been intensively investigated. However, the mechanisms of how the inputs are processed in the cortex are still unclear. To know the correlation of the sensory processing in the dorsal horn and cortex, in vivo and in vitro patch-clamp recordings were made from rat dorsal horn and sensory cortex. Although dorsal horn neurons showed spontaneous and evoked EPSCs by noxious and non-noxious stimuli, most somatosensory neurons located at 100 to 1000 microm from the surface of the cortex exhibited an oscillatory activity and received synaptic inputs from non-noxious but not noxious receptors. These observations suggest that the synaptic responses in cortical neurons are processed in a more complex manner; and this may be due to the reciprocal synaptic connection between thalamus and cortex.

Animals↗

PubMed1998

Patch clamp studies on ion pumps of the cytoplasmic membrane of Escherichia coli. Formation, preparation, and utilization of giant vacuole-like structures consisting of everted cytoplasmic membrane.

Formation of giant protoplasts from normal Escherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H+ because of respiration and to ATP hydrolysis by the FoF1-ATPase. Such current was inhibited by inhibitors of the respiratory chain or FoF1-ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.

Cytoplasm↗