SEARCH · PubMed Health

Results for “patch clamp”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources

PubMed1998

A distinct membrane current in rat lens fiber cells isolated under calcium-free conditions.

PURPOSE: To investigate whether lens fiber cells isolated and maintained under calcium-free conditions exhibit distinct membrane currents. METHODS: Fiber cells were isolated from the cortical portion of neonate rat lenses using a trypsin digestion protocol and were maintained in EDTA-buffered Ringer's solutions. Membrane currents were recorded from fiber bundles using the whole-cell patch-clamp technique. RESULTS: Cortical fiber cells of up to 600-microm length were viable and amenable to whole-cell patch-clamp recording. The major current recorded under these conditions was a slowly activating, voltage-dependent current that was markedly increased on membrane depolarization. This current appeared to be fiber cell specific and had similar properties to currents elicited by gap junction hemichannels previously recorded by others in Xenopus oocytes. CONCLUSIONS: This is the first report of whole-cell patch-clamp recordings from intact elongated fiber cells. Fiber cells kept in calcium-free bath medium appear to be electrically "leaky" and exhibit a distinct membrane current that has not been described previously for lens cells. This current is unlikely to be active in the normal lens but may play a role in the depolarized cataractogenic lens.

Animals↗

PubMed2000

R-Type Ca2+ channels are coupled to the rapid component of secretion in mouse adrenal slice chromaffin cells.

Patch-clamp measurements of Ca(2+) currents and membrane capacitance were performed on slices of mouse adrenal glands, using the perforated-patch configuration of the patch-clamp technique. These recording conditions are much closer to the in vivo situation than those used so far in most electrophysiological studies in adrenal chromaffin cells (isolated cells maintained in culture and whole-cell configuration). We observed profound discrepancies in the quantities of Ca(2+) channel subtypes (P-, Q-, N-, and L-type Ca(2+) channels) described for isolated mouse chromaffin cells maintained in culture. Differences with respect to previous studies may be attributable not only to culture conditions, but also to the patch-clamp configuration used. Our experiments revealed the presence of a Ca(2+) channel subtype never before described in chromaffin cells, a toxin and dihydropyridine-resistant Ca(2+) channel with fast inactivation kinetics, similar to the R-type Ca(2+) channel described in neurons. This channel contributes 22% to the total Ca(2+) current and controls 55% of the rapid secretory response evoked by short depolarizing pulses. Our results indicate that R-type Ca(2+) channels are in close proximity with the exocytotic machinery to rapidly regulate the secretory process.

Adrenal Glands↗

PubMed1998

Diverse signal transduction pathways mediated by endogenous P2 receptors in cultured rat cerebral cortical neurons.

The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca2+ concentration ([Ca2+]i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) but not by the Gi/o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca2+ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca2+ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTP gamma S induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein beta gamma subunits and increased [Ca2+]i by a mechanism independent of phospholipase C stimulation and IP3 production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca2+ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P2 receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P2 receptors.

Adenosine Triphosphate↗

PubMed2003

Potassium channels in human umbilical artery cells.

OBJECTIVE: To identify K+ channels of smooth muscle of human umbilical artery using the patch-clamp technique and to study their effect on resting tone of umbilical artery rings. METHODS: Whole-cell and single-channel patch-clamp recordings in enzymatically isolated smooth muscle cells were made. Measurements of developed isometric force were performed on intact tissue. RESULTS: Delayed rectifier K+ channels (KDR) and large-conductance Ca2+-activated K+ channels (BKCa) contribute to the whole-cell voltage- and time-dependent outward K+ current, as it was specifically inhibited by 5 mM 4-aminopyridine (4-AP; KDR blocker) (92 +/- 4% at 0 mV, n = 7), by 1 mM tetraethylammonium (TEA; BKCa blocker) (71 +/- 4% at +60 mV, n = 4), and by 200 nM iberiotoxin (BKCa blocker) (64 +/- 7% at +60 mV, n = 4). In outside-out patches, BKCa channels had a single-channel conductance of 132 +/- 4 pS (n = 24) in asymmetric K+ conditions and 216 +/- 4 pS (n = 4) in a symmetric K+ gradient. The activity of the BKCa channels was significantly augmented by 1 microM Ca2+ in the inside-out configuration. 4-AP had no effect on resting tone of intact arterial rings. TEA produced contraction of arterial rings whereas phloretin, an activator of BKCa, relaxed them, which means that BKCa channels are functional in intact tissue and are involved in the maintenance of resting tone in this human vessel. CONCLUSION: The identities of K+ channels in the human umbilical artery were shown using the patch-clamp technique, and the physiologic effect of K+ channels on resting tone was documented.

4-Aminopyridine↗

PubMed2003

Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells.

myo-Inositol hexakisphosphate (InsP6) is the most abundant inositol phosphate in cells, yet it remains the most enigmatic of this class of signaling molecule. InsP6 plays a role in the processes by which the drought stress hormone abscisic acid (ABA) induces stomatal closure, conserving water and ensuring plant survival. Previous work has shown that InsP6 levels in guard cells are elevated in response to ABA, and InsP6 inactivates the plasma membrane inward K+ conductance (IK,in) in a cytosolic calcium-dependent manner. The use of laser-scanning confocal microscopy in dye-loaded patch-clamped guard cell protoplasts shows that release of InsP6 from a caged precursor mobilizes calcium. Measurement of calcium (barium) currents ICa in patch-clamped protoplasts in whole cell mode shows that InsP6 has no effect on the calcium-permeable channels in the plasma membrane activated by ABA. The InsP6-mediated inhibition of IK,in can also be observed in the absence of external calcium. Thus the InsP6-induced increase in cytoplasmic calcium does not result from calcium influx but must arise from InsP6-triggered release of calcium from endomembrane stores. Measurements of vacuolar currents in patch-clamped isolated vacuoles in whole-vacuole mode showed that InsP6 activates both the fast and slow conductances of the guard cell vacuole. These data define InsP6 as an endomembrane-acting calcium-release signal in guard cells; the vacuole may contribute to InsP6-triggered Ca2+ release, but other endomembranes may also be involved.

Abscisic Acid↗

PubMed2005

Characterization of a hERG screen using the IonWorks HT: comparison to a hERG rubidium efflux screen.

The introduction of parallel patch clamp instruments offers the promise of moderate-throughput, high-fidelity voltage clamp for drug screening assays. One such device, the IonWorks HT (Molecular Devices, Sunnyvale, CA), was evaluated and compared to conventional human ethera- go-go-related gene (hERG) patch clamp data and an alternative functional screen based on rubidium flux. Data generated by the IonWorks HT and rubidium assays were compared to determine if either offered superior predictive value compared to conventional patch clamp. Concentration-effect curves for a panel of known hERG blockers were shifted to higher concentrations on the IonWorks HT compared to conventional voltage clamp determinations. The magnitude of the potency shifts was compound-specific and ranged from no shift (e.g., quinidine) to over 200-fold (astemizole). When the extreme value for astemizole was disregarded, the potency shift for 13 other known reference standards was 12-fold or less, with an average shift of fivefold. The same subset of compounds in the rubidium efflux assay exhibited an average potency shift of 12-fold. To provide a simulation of how the IonWorks HT assay might perform in a single concentration screening mode, a panel of test compounds was evaluated. The IonWorks HT screen did not outperform the rubidium efflux screen in predicting conventional voltage clamp measurements. The most likely explanation appears to rest with variable and compound-specific potency shifts in the IonWorks HT assay. The variable potency shifts make it difficult to select a screening concentration that meets the criterion of a high positive predictive value while avoiding false-positives.

Animals↗

PubMed2000

Effects of nitroprusside, 3-morpholino-sydnonimine, and spermine on calcium-sensitive potassium currents in gastric antral circular myocytes of guinea pig.

AIM: To determine the effect of nitric oxide (NO) on calcium-activated potassium currents in antral circular myocytes of the guinea pig stomach. METHODS: The whole-cell patch-clamp techniques were used, and the cells were isolated by collagenase. Sodium nitroprusside (SNP), spermine, and 3-morpholino-sydnonimine (SIN-1) were used as nitric oxide donors. RESULTS: Outward potassium currents were remarkably inhibited by tetraethylammonium (TEA) 1 mmol.L-1. Charybdotoxin 200 nmol.L-1, a specific inhibitor of KCa channel, also greatly inhibited IK(Ca). NO donors remarkably increased IK(Ca) in guinea pig gastric antral circular myocytes with perforated patch-clamp techniques but not conventional whole-cell patch-clamp techniques. Spermine 100 mumol.L-1 increased IK(Ca) by 14% +/- 10% (at 60 mV, P < 0.01), SIN-1 200 mumol.L-1 increased IK(Ca) by 19% +/- 14% (at 60 mV, P < 0.01), and SNP 100 mumol.L-1 increased IK(Ca) by 24% +/- 13% (at 60 mV, P < 0.01) respectively. SIN-1- and SNP-induced increase of IK(Ca) was blocked by methylene blue 1 mumol.L-1. CONCLUSION: NO increases calcium-activated potassium currents in gastric antral circular myocytes of guinea pig, and the effect of NO on IK(Ca) may be mediated by cyclic GMP.

Animals↗

PubMed1997

Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells.

1. The aim of this study was to investigate the effect of the Na+/K+-ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague-Dawley SPF-rats. 2. Experiments were performed at 22-26 degrees C in the tight-seal whole-cell configuration of the patch-clamp technique by use of Sylgard-coated patch pipettes (3-6 M[omega]). High-resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier controlled by the 'E9SCREEN' software. In addition, a charting programme on another computer synchronously recorded at low resolution (2 Hz) membrane potential and holding current (low-pass filtered at 500 Hz). 3. Na+/K+-ATPase activity was measured as the ouabain-sensitive change in the zero-current potential. The zero-current potential in rat peritoneal mast cells measured 2 min after obtaining whole-cell configuration amounted to 1.7 +/- 2.5 mV (n = 21). Ouabain (5 mM), a Na+/K+-ATPase-inhibitor, had only a very minor effect upon the membrane potential under resting conditions (n = 3). 4. When mast cells were superfused with nominal calcium-free external solution, the cells hyperpolarized (delta mV: 20.2 +/- 3.8 mV (n = 5)). In addition, when the mast cells were preincubated in nominal calcium-free external solution for 12 +/- 1.6 min before whole-cell configuration, the membrane potential amounted to -53.7 +/- 9.8 mV (n = 8). A subsequent superfusion with ouabain (5 mM) depolarized the membrane potential (ouabain-sensitive hyperpolarization (delta mV): 23.0 +/- 8.4 mV (n = 8)). 5. A high intracellular concentration of Na+ ([Na+]i) (26.6 mM) also resulted in hyperpolarization (delta mV: 20.2 +/- 9.1 mV (n = 7)), but only when ATP was present. A subsequent superfusion with ouabain (5 mM) repolarized these cells to -1.2 +/- 14 mV (ouabain-sensitive hyperpolarization (delta mV): 19.7 +/- 7.7 mV (n = 7)). 6. The size of the [Na+]i-dependent hyperpolarization was dose-dependent. Low [Na+]i (1 mM) had no effect on membrane potential and these cells were unaffected by superfusion with calcium-free external solution. 7. These data thus directly confirm that the stimulant effect of calcium-free external solutions on the ouabain-sensitive changes in the zero-current potential, and hence the Na+/K+-ATPase, is mediated through [Na+]i and that the activity of the Na+/K+-ATPase can have an important influence on the resting membrane potential in rat peritoneal mast cells.

Animals↗

PubMed1985

Effects of L-glutamate on the anomalous rectifier potassium current in horizontal cells of Carassius auratus retina.

The effects of externally applied L-glutamate (Glu) on K currents through the anomalous rectifier were studied in solitary horizontal cells dissociated from goldfish retinae under whole-cell voltage-clamp or cell-attached patch-clamp conditions using 'giga-seal' suction pipettes. In the whole-cell clamp experiments, hyperpolarization of the membrane below the resting potential (ca. -57 mV) induced a large voltage-dependent inward current which has been identified as the K current through the anomalous rectifier (Ianomal.). Application of Glu to the external medium reduced Ianomal.. Reduction of the inward current was not seen in preparations in which Ianomal. has been blocked by an application of Cs or Ba ions to the external medium. Single-channel currents through the anomalous rectifier were recorded under cell-attached patch-clamp conditions. The current showed an inward rectification; its amplitude increased with hyperpolarization of the patch membrane, and became below the noise level near the equilibrium potential of K ions (EK). No polarity reversal was observed even by a strong membrane depolarization. The patch membrane potential at which the current amplitude became undetectable shifted in parallel to the shift of EK. The open probability changed little with polarization of the patch membrane. When Glu (greater than 100 microM) was applied to the outside of the patch membrane, the number of available Ianomal. channels was decreased, but neither the single-channel conductance, open or closed time constants, nor the open probability changed significantly. Removal of Glu produced the opposite sequence; i.e. the number of available Ianomal. channels increased with time. It was concluded that the reduction of the Glu-induced current at hyperpolarized potentials in the whole-cell recording configuration is due to the blocking action of Glu on Ianomal..

Action Potentials↗

PubMed2001

Effects of a Lasiodora spider venom on Ca2+ and Na+ channels.

The venom of a Brazilian spider, Lasiodora sp (Mygalomorphae, Theraphosidae), was screened for activity against ion channels using Ca2+ imaging and whole-cell patch clamp in GH3 cells. When tetrodotoxin (TTX) was present to block Na+ channels, the venom abolished the Ca2+ oscillations that are normally present in these cells and reduced the basal level of intracellular Ca2+. Under patch clamp, the venom reduced the L-type Ca2+ channel conductance and caused a positive shift in its voltage dependence of activation. In addition to these effects, when applied without TTX, the venom also caused a slow and noisy increase in intracellular Ca2+. The sensitivity of this second effect to TTX suggested an effect on Na+ channels, which was tested using patch clamp. Control Na+ currents inactivated completely as a single exponential. Treatment with the venom did not affect the amplitude of I(Na), but caused it to divide in two slower exponential components plus a sustained component, all of which were suppressed by TTX. The venom also caused a negative shift in the voltage dependence of activation and steady-state inactivation of I(Na). The observed effects of this venom on whole-cell currents explain the changes it causes in intracellular Ca2+ in GH3 cells and demonstrate that the venom of this spider is a source of toxins active against ion channels.

Algorithms↗

PubMed2001

Discontinuous single electrode voltage-clamp measurements: assessment of clamp accuracy in Vicia faba guard cells.

The use of a discontinuous single electrode voltage-clamp (dSEVC) offers an attractive alternative to the patch-clamp technique, since whole-cell measurements can be performed with a single sharp electrode. Comparison of current-voltage relations, however, revealed a weaker voltage dependence of channels measured with the dSEVC compared to patch clamp. The accuracy of the dSEVC was tested on Vicia faba guard cells impaled with double-barrelled electrodes. The actual clamp potential was measured independently of the dSEVC, at the second barrel. The weaker voltage dependence of ion channels appeared to be due to an overestimation of the clamp potential by the dSEVC. The deviation between the intended and actual clamp potential showed a linear relationship with the injected current; on average a 126 mV deviation was found for a clamp current of 1 nA. The deviation was probably caused by a slow settling capacity at the electrode, not compensated by the dSEVC amplifier. It is concluded that the dSEVC method in its current state is only suited for the study of small ion conductances in plant cells.

Cell Membrane↗

PubMed1993

Calcium currents in pyramidal neurons acutely dissociated from the rat frontal cortex: a study by the nystatin perforated patch technique.

We studied electrical and pharmacological properties of voltage-activated Ca2+ currents (ICa) in freshly dissociated rat frontal cortical pyramidal neurons by using nystatin perforated patch and conventional patch-clamp techniques. With either nystatin perforated patch technique or conventional patch-clamp technique, low voltage-activated ICa (LVA ICa) and high voltage-activated ICa (HVA ICa) were recorded. However, in conventional whole-cell recording, HVA ICa ran down within 5-20 min, whereas the current remained stable for 50 min at least in nystatin perforated patch recording. The HVA ICa recorded by nystatin perforated patch technique was further classified to L-type, N-type and a current component resistant to both nicardipine and omega-conotoxin by their current kinetics, voltage- and drug-sensitivities. Present results suggest that the perforated patch technique is a useful method for studying the properties of high voltage-activated Ca2+ channels.

Animals↗

PubMed2001

Subfornical organ neurons projecting to paraventricular nucleus: whole-cell properties.

The subfornical organ (SFO) has been repeatedly identified as a CNS site that plays a critical role in sensing multiple physiological variables of the "milieu interieur" and, through efferent projections to other CNS sites, initiating physiological responses to change. Many recent in vitro patch-clamp studies have examined the cellular mechanisms underlying the sensory abilities of these specialized CNS neurons. The primary limitation of these studies, however, has been the inability to identify homogeneous groups of SFO neurons for such investigation. We report here the development of techniques to permit patch clamp recording from dissociated SFO neurons identified according to their in vivo projection site. SFO neurons were labeled by injection of fluorescently labeled, retrogradely transported microspheres into the hypothalamic paraventricular nucleus (PVN) 3 days prior to cell dissociation. Patch-clamp recordings from these SFO-PVN neurons revealed both sodium currents, potassium currents, action potentials, input resistance and membrane potential which were all similar to SFO cells prepared from animals with no prior tracer injection. Labeled SFO-->PVN cells were also found to be osmosensitive and responsive to angiotensin II, suggesting specific functional roles for this anatomically defined group of SFO neurons. Intriguingly, our post hoc analysis also demonstrated that all labeled neurons demonstrated a unique electrophysiological profile dominated by a large transient potassium conductance such that the transient/sustained potassium current ratio, or degree of inactivation was, on average, greater than 4.0. Utilization of these tracing techniques to permit the in vitro recording from cells with known in vivo connections will permit study of intrinsic mechanisms that underlie physiological responses of anatomically defined populations of neurons.

Action Potentials↗

PubMed2005

Atrial natriuretic peptide and cGMP activate sodium transport through PKA-dependent pathway in the urinary bladder of the Japanese tree frog.

The effects of atrial natriuretic peptide (ANP) and cGMP on transepithelial ion transport were examined in the urinary bladder of the Japanese tree frog, Hyla japonica, using Ussing chamber voltage-clamp and whole-cell patch-clamp techniques. When the bladders were exposed to 4.4 x 10(-11) to 10(-6) M ANP or 10(-7) to 3 x 10(-4) M 8-Br-cGMP, both the transepithelial potential difference (PD) and the short-circuit current (Isc) were significantly increased in a concentration-response manner. The cGMP-dependent responses were inhibited in a Na+-free bath solution and in the presence of amiloride. The cGMP-dependent increases in Isc were significantly inhibited by specific PKA inhibitors (5 x 10(-7) M KT-5720 and >10(-5) M H-89), but not by a specific PKG inhibitor (5 x 10(-7) M KT-5823). ANP-dependent increases in Isc were also significantly inhibited by KT-5720. In the patch-clamp study, ANP and cGMP significantly increased in inward currents involving Na+ uptake. These results suggest that a cross-talk mechanism exists between cAMP and cGMP signaling pathways, which leads to Na+ transport in the frog urinary bladder. In addition, the cGMP-dependent increases in Isc were partially inhibited by 10(-4) M l-cis-diltiazem, a specific inhibitor of cyclic nucleotide-gated (CNG) channels. These results also suggest a relation between CNG channels and the cGMP-dependent increases in Na+ absorption of the frog urinary bladder.

Animals↗

PubMed2001

Cell-to-cell communication via nitric oxide modulation of oscillatory Cl(-) currents in rat intact cerebral arterioles.

1. Diffusion-mediated changes in ion channel function within blood vessels have not been demonstrated directly in a patch-clamp study. Here, we examined the hypothesis that endothelium-derived diffusible bioactive substances would modify endothelin-1 (ET-1)-evoked membrane currents in smooth muscle cells situated within intact arterioles. 2. In pieces of arterioles dissected from the rat cerebral pial membrane, patch electrodes were placed on single smooth muscle cells identified under the microscope. Under perforated patch-clamp conditions, ET-1 evoked an oscillatory inward current at negative potentials in such cells in the presence of the gap junction disrupter 18alpha-glycyrrhetinic acid. ET-1 also elicited an oscillation superimposed on a membrane depolarization in current-clamp mode. 3. The oscillatory current exhibited an outwardly rectifying current-voltage relationship, a sensitivity to niflumic acid, a requirement for inositol 1,4,5-trisphosphate (IP(3))- and caffeine-sensitive Ca(2+) stores and for external Ca(2+) and a rank order of anion permeabilities characteristic of Ca(2+)-activated Cl(-) currents (I(Ca(Cl))). 4. This oscillatory response was inhibited by bradykinin (an effect distinct from the electrical propagation of hyperpolarization) and this effect was attenuated by the NO-synthase inhibitor N(omega)-nitro-L-arginine and by the NO scavenger oxyhaemoglobin but not by the cyclo-oxygenease inhibitor indomethacin. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and nitroprusside closely mimicked the effect of bradykinin. 5. The present patch-clamp study has revealed diffusion-mediated cell-to-cell interaction in an intact blood vessel: bradykinin appears to cause NO to move from endothelium to smooth muscle, there to inhibit an ET-1-evoked oscillatory I(Ca(Cl)) via the NO-cGMP pathway.

Animals↗

PubMed1997

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Animals↗

PubMedDate not supplied

Current concepts in nuclear pore electrophysiology.

Over 4 decades ago, microelectrode studies of in situ nuclei showed that, under certain conditions, the nuclear envelope (NE) behaves as a barrier opposing the nucleocytoplasmic flow of physiological ions. As the nuclear pore complexes (NPCs) of the NE are the only pathways for direct nucleocytoplasmic flow, those experiments implied that the NPCs are capable of restricting ion flow. These early studies validated electrophysiology as a useful approach to quantify some of the mechanisms by which NPCs mediate gene activity and expression. Since electron microscopy (EM) and other non-electrophysiological investigations, showed that the NPC lumen is a nanochannel, the opinion prevailed that the NPC could not oppose the flow of ions and, therefore, that electrophysiological observations resulted from technical artifacts. Consequently, the initial enthusiasm with nuclear electrophysiology faded out in less than a decade. In 1990, nuclear electrophysiology was revisited with patch-clamp, the most powerful electrophysiological technique to date. Patch-clamp has consistently demonstrated that the NE has intrinsic ion channel activity. Direct demonstrations of the NPC on-off ion channel gating behavior were published for artificial conditions in 1995 and for intact living nuclei in 2002. This on-off switching/gating behavior can be interpreted in terms of a metastable energy barrier. In the hope of advancing nuclear electrophysiology, and to complement the other papers contained in this special issue of the journal, here I review some of the main technical, experimental, and theoretical issues of the field, with special focus on NPCs.

Animals↗

PubMed2006

PTH-induced actin depolymerization increases mechanosensitive channel activity to enhance mechanically stimulated Ca2+ signaling in osteoblasts.

UNLABELLED: Disruption of the actin cytoskeleton with cytochalasin D enhanced the mechanically induced increase in intracellular Ca(2+) ([Ca(2+)](i)) in osteoblasts in a manner similar to that of PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Patch-clamp analyses show that the MSCC is directly influenced by alterations in actin integrity. INTRODUCTION: PTH significantly enhances the fluid shear-induced increase in [Ca(2+)](i) in osteoblasts, in part, through increased activation of both the mechanosensitive, cation-selective channel (MSCC) and L-type voltage-sensitive Ca(2+) channel (L-VSCC). Both stimuli have been shown to produce dynamic changes in the organization of the actin cytoskeleton. In this study, we examined the effects of alterations in actin polymerization on [Ca(2+)](i) and MSCC activity in MC3T3-E1 and UMR-106.01 osteoblasts in response to shear +/- PTH pretreatment. MATERIALS AND METHODS: MC3T3-E1 or UMR-106.01 cells were plated onto type I collagen-coated quartz slides, allowed to proliferate to 60% confluency, and mounted on a modified parallel plate chamber and subjected to 12 dynes/cm(2). For patch-clamp studies, cells were plated on collagen-coated glass coverslips, mounted on the patch chamber, and subjected to pipette suction. Modulators of actin cytoskeleton polymerization were added 30 minutes before the experiments, whereas channel inhibitors were added 10 minutes before mechanical stimulation. All drugs were maintained in the flow medium for the duration of the experiment. RESULTS AND CONCLUSIONS: Depolymerization of actin with 1-5 microM cytochalasin D (cyto D) augmented the peak [Ca(2+)](i) response and increased the number of cells responding to shear, similar to the increased responses induced by pretreatment with 50 nM PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Inhibition of the MSCC with Gd(3+) significantly blocked both the peak Ca(2+) response and the number of cells responding to shear in cells pretreated with either PTH or cyto D. Inhibition of the L-VSCC reduced the peak [Ca(2+)](i) response to shear in cells pretreated with PTH, but not with cyto D. Patch-clamp analyses found that addition of PTH or cyto D significantly increased the MSCC open probability in response to mechanical stimulation, whereas phalloidin significantly attenuated the PTH-enhanced MSCC activation. These data indicate that actin reorganization increases MSCC activity in a manner similar to PTH and may be one mechanism through which PTH may reduce the mechanical threshold of osteoblasts.

Actins↗