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PubMed2002

Role of high-voltage activated potassium currents in high-frequency neuronal firing: evidence from a basal metazoan.

Certain neurons of vertebrates are specialized for high-frequency firing. Interestingly, high-frequency firing is also seen in central neurons in basal bilateral metazoans. Recently, the role of potassium currents with rightward-shifted activation curves in producing high-frequency firing has come under scrutiny. We apply intracellular recording, patch-clamp techniques, and compartmental modeling to examine the roles of rightward-shifted potassium currents in repetitive firing and shaping of action potentials in central neurons of the flatworm, Notoplana atomata (Phylum Platyhelminthes). The kinetic properties of potassium and sodium currents were determined from patch-clamp experiments on dissociated brain cells. To predict the effects of changing the steady-state and kinetic properties of these potassium currents, these data were incorporated into a computer model of a 30-microm spherical cell with the levels of current adjusted to approximate the values recorded in voltage-clamp experiments. The model was able to support regenerative spikes at high frequencies in response to injected current. Current-clamp recordings of cultured cells and of neurons in situ also showed evidence of very-high-frequency firing. Adjusting the ratio of inactivating to non-inactivating potassium currents had little effect upon the firing pattern of the cell or its ability to fire at high frequencies, whereas the presence of the non-inactivating current was necessary for repetitive firing. Computer simulations suggested that the rightward shift in voltage sensitivity confers a raised firing threshold, while rapid channel kinetics underlie high frequency firing, and the large activation range enhances the coding range of the cell.

Action Potentials↗

PubMed2006

Cooperative gating between single HCN pacemaker channels.

HCN pacemaker channels (I(f), I(q), or I(h)) play a fundamental role in the physiology of many excitable cell types, including cardiac myocytes and central neurons. While cloned HCN channels have been studied extensively in macroscopic patch clamp experiments, their extremely small conductance has precluded single channel analysis to date. Nevertheless, there remain fundamental questions about HCN gating that can be resolved only at the single channel level. Here we present the first detailed single channel study of cloned mammalian HCN2. Excised patch clamp recordings revealed discrete hyperpolarization-activated, cAMP-sensitive channel openings with amplitudes of 150-230 fA in the activation voltage range. The average conductance of these openings was approximately 1.5 pS at -120 mV in symmetrical 160 mM K(+). Some traces with multiple channels showed unusual gating behavior, characterized by a variable long delay after a voltage step followed by runs of openings. Noise analysis on macroscopic currents revealed fluctuations whose magnitudes were systematically larger than predicted from the actual single channel current size, consistent with cooperativity between single HCN channels.

Cell Line↗

PubMed1998

Ethanol reduces spontaneous firing and potentiates GABA-induced currents in acutely dissociated rat medial vestibular nucleus neurons.

Effects of ethanol on acutely dissociated medial vestibular nucleus (MVN) neurons were examined using whole-cell patch clamp technique to elucidate the mechanism underling the inhibitory effects of this drug on the neurons observed in in vivo studies. Dissociated MVN neurons obtained from male Wistar rats were superfused with extracellular solution continuously at a flow rate of 1-3 ml/min. Whole-cell patch clamp recording was performed according to standard procedures. GABA was applied by pressure from a pipette placed near the neuron recorded. Ethanol was applied via pipette by pressure or through bath perfusion. Acutely dissociated MVN neurons regularly showed spontaneous firing. Under current-clamp conditions, bath application of ethanol at 0.1% caused hyperpolarization and reduced spontaneous firing in MVN neurons, while 0.1% ethanol did not affect spontaneous firing. Pulse application of higher concentrations of ethanol (0.1-1%) caused similar hyperpolarization. Under voltage-clamp conditions at a holding potential of -30 mV, GABA induced outward currents in a concentration-dependent manner. GABA-induced currents were potentiated in the presence of 0.01% ethanol. These results indicate that high concentrations of ethanol (0.1-1%) directly induce inhibition of spontaneous firing and low concentrations (0.01%) enhance GABA-induced inhibition in the MVN neurons.

Action Potentials↗

PubMed2001

Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.

Base Sequence↗

PubMed2001

Ca(2+) influx through nonselective cation channels plays an essential role in endothelin-1-induced mitogenesis in C6 glioma cells.

Ca(2+) channels activated by endothelin-1 (ET-1) in C6 glioma cells (C6 cells) were characterized using whole-cell patch-clamps and by monitoring the intracellular free Ca(2+) concentration ([Ca(2+)](i)), when administering Ca(2+) channel blockers such as LOE 908 and SK&F 96365. Using this methodology, the Ca(2+) channels involved in ET-1-induced mitogenesis were identified. The patch-clamp study and [Ca(2+)](i) monitoring showed that 10 nM ET-1 activated two types of Ca(2+)-permeable nonselective cation channels (NSCC); one was sensitive to LOE 908 but resistant to SK&F 96365 (NSCC-1) and the other was sensitive to both LOE 908 and SK&F 96365 (NSCC-2). Conversely, 0.1 nM ET-1 activated only NSCC-1.ET-1-induced mitogenesis in a concentration-dependent manner, with the maximum effect arising at concentrations > or =10 nM. LOE 908 completely suppressed the 10 nM ET-1-induced mitogenesis, whereas SK&F 96365 only partially suppressed it. The IC(50) values of these blockers for the ET-1-induced mitogenesis were similar to those for the 10 nM ET-1-induced increase in [Ca(2+)](i). In contrast, LOE 908 completely suppressed 0.1 nM ET-1-induced mitogenesis, whereas SK&F 96365 did not affect it.Collectively, these results demonstrate that the sustained increase in [Ca(2+)](i), via NSCC-1 and NSCC-2, may be essential for ET-1-induced mitogenesis in C6 cells. Moreover, the sensitivity of NSCC-1 to ET-1 is higher than that of NSCC-2 to ET-1.

Acetamides↗

PubMed2006

Calcium spikes in basal dendrites of layer 5 pyramidal neurons during action potential bursts.

Patch-clamp recording from dendrites has lead to a significant increase in our understanding of the mechanisms underlying signal integration and propagation in neurons. The majority of synaptic input to neurons, however, is made onto small-diameter dendrites, currently beyond the scope of patch-clamp recording techniques. Here we use both calcium and voltage imaging to investigate propagation of action potentials (APs) in fine basal dendrites of cortical layer 5 pyramidal neurons. High-frequency (200 Hz) AP bursts caused supralinear increases in dendritic calcium at distal, but not proximal, basal locations. Supralinear increases in dendritic calcium were also observed at distal basal locations during AP trains above a critical frequency (approximately 100 Hz). Using voltage imaging, we show that single APs undergo significant attenuation as they propagate into basal dendrites, whereas AP bursts lead to generation of dendritic calcium spikes. Focal and bath application of 4-AP increased the amplitude of calcium transients evoked by APs at distal, but not proximal, locations, suggesting that A-type potassium channels regulate AP backpropagation into basal dendrites. Finally, we show that pairing EPSPs with AP bursts is an effective means of activating synaptic NMDA receptors in basal dendrites. The experimental observations on the role of A-type potassium channels in regulation of AP backpropagation in basal dendrites, as well as the generation of dendritic calcium spikes during AP bursts, were reproduced in a morphologically realistic neuronal model with uniform distributions of dendritic sodium, calcium, and potassium channels. Together, these findings have important implications for understanding dendritic integration and synaptic plasticity in cortical basal dendrites.

Action Potentials↗

PubMed2005

Long-term intracellular recordings of respiratory neuronal activities in situ during eupnea, gasping and blockade of synaptic transmission.

For a definitive evaluation of the hypothesis that different neurophysiological mechanisms underlie the neurogenesis of eupnea and gasping, long-term continuous intracellular recordings of respiratory neuronal activities during both respiratory patterns are required. Such recordings in vivo are technically difficult, especially in small mammals, due to mechanical instability of the brainstem and cardiovascular depression that accompany hypoxia-induced gasping. Respiratory-related rhythmic activities of in vitro preparations are confounded by the lack of a clear correspondence with both eupnea and gasping. Here, we describe new methodologies and report on whole cell patch clamp recordings from the ventrolateral medulla and the hypoglossal motor nucleus in situ during multiple bouts of hypoxia-induced gasping. The longevity of recordings (range 20--35 min) also allowed subsequent analysis of neuronal behaviour after blockade of inhibitory and excitatory synaptic activities. We conclude that whole cell patch clamp recordings in the in situ preparation will allow an analysis of both synaptic and ionic conductances of respiratory neurons during defined eupnea and gasping, providing an additional approach to in vitro preparations.

Animals↗

PubMed1999

Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine.

1. We have investigated the inhibition of inwardly rectifying potassium channels by the alpha-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flexor digitorum brevis muscle and whole-cell patch-clamp of cell lines transfected with Kir2.1 (IRK1). 2. In skeletal muscle and at a membrane potential of -50 mV, chloroethylclonidine (CEC), an agonist at alpha2-adrenergic receptors and an antagonist at alpha1x-receptors, was found to inhibit the inward rectifier current with a Ki of 30 microM. 3. The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of alpha1-(prazosin) or alpha2-(rauwolscine) antagonists. 4. The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at -50 mV c.f. approximately 10% block at -190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be approximately 8 s at 0 mV, and the rate of unblock was described by the relationship tau=exp((Vm+149)/22) s. 5. This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine. 6. The data suggest that the clonidine-like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of alpha-receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.

Animals↗

PubMed1997

Functional coupling in bovine ciliary epithelial cells is modulated by carbachol.

The functional coupling of the ciliary epithelium was studied in isolated pairs (couplets) of pigmented ciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE) cells using the whole cell patch clamp and the fluorescent dye lucifer yellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2-5 M omega electrode, containing 1-2 mM lucifer yellow, in the whole cell configuration of the patch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells were coupled. The electrical coupling of the cells was also studied by calculating the capacitance (using the time-domain technique), assuming a "supercell" model for coupled cells. The mean capacitance of coupled pairs was 79.8 +/- 4.3 (SE) pF (n = 47) compared with single cell capacitances of 36.8 +/- 3.4 pF (n = 10) for PCE cells and 38.1 +/- 3.1 pF (n = 15) for NPCE cells. Octanol, carbachol (CCh), and raised extracellular Ca2+ concentration ([Ca2+]o) all caused uncoupling in pairs (couplets) of coupled NPCE and PCE cells. At room temperature (22-24 degrees C), the capacitance of the couplets decreased from 70.5 +/- 8.0 to 48.0 +/- 5.2 pF (n = 5) when exposed to octanol (1 mM), from 73.8 +/- 9.2 to 43.2 +/- 9.5 pF (n = 4) when exposed to CCh (100 microM), and from 80.5 +/- 6.7 to 49.9 +/- 7.8 pF (n = 4) when exposed to 10 mM [Ca2+]o. The response to CCh was dose dependent; at higher temperatures of 34-37 degrees C, 10 microM CCh caused a 38% reduction in capacitance, from 53.7 +/- 9.7 to 33.5 +/- 3.3 pF (n = 7) with a half-time of 249 s, and 100 microM CCh caused a 49% reduction in capacitance, from 51.3 +/- 5.6 to 26.0 +/- 2.4 pF (n = 7) with a half-time of 124 s. After pairs uncoupled and the uncoupling agent was washed out, the cell pairs often exhibited an increase in capacitance that we interpreted as "recoupling" or a reopening of the gap junctional communication pathway; the half-time for this process was 729 s after uncoupling with 100 microM CCh and 211 s after uncoupling with 10 microM CCh. This interpretation was confirmed optically by the spread of lucifer yellow into both cells of an uncoupled pair with a time course corresponding to the increase in electrical coupling. The controllable coupling of ciliary epithelial cells extends the idea of a functional syncytium involved in active transport. PCE cells take up solute and water from the blood, which then cross to NPCE cells via gap junctions and from there are secreted into the posterior chamber of the eye. Modulation of the coupling between NPCE and PCE cells may provide a mechanism to control secretion.

Animals↗

PubMed2003

Role of L-type calcium channels in pacing-induced short-term and long-term cardiac memory in canine heart.

BACKGROUND: We tested the hypothesis that ICa,L is important to the development of cardiac memory. METHODS AND RESULTS: The effects of L-type Ca2+ channel blockade and beta-blockade were tested on acutely anesthetized and on chronically instrumented, conscious dogs. Short-term memory (STM) was induced by 2 hours of ventricular pacing and long-term memory (LTM) by ventricular pacing for 21 days. STM dogs received placebo, nifedipine, or propranolol, and LTM dogs received placebo, atenolol, or amlodipine. AT1 receptor blockade (candesartan) and ACE inhibition (trandolapril) were also tested in LTM. Microelectrodes were used to record transmembrane potentials from isolated epicardial and endocardial slabs using a protocol simulating STM in intact animals. Left ventricular epicardial myocytes from LTM or sham control dogs were dissociated, and ICa,L was recorded (whole-cell patch-clamp technique). Evolution of STM and LTM was attenuated by ICa,L blockers but not beta-blockers. Neither AT1 receptor blockade nor ACE inhibition suppressed LTM. In microelectrode experiments, pacing induced an epicardial-endocardial gradient change mimicking STM that was suppressed by nifedipine. In patch-clamp experiments, peak ICa,L density in LTM and control were equivalent, but activation was more positive and time constants of inactivation longer in LTM (P<0.05). CONCLUSIONS: ICa,L blockade but not beta-adrenergic blockade suppresses cardiac memory. LTM evolution is unaffected by angiotensin II blockade and is associated with altered ICa,L kinetics.

Action Potentials↗

PubMed1997

Modulation of intrinsic circuits by serotonin 5-HT3 receptors in developing ferret visual cortex.

Serotonergic projections are widespread in the developing neocortex, but their functions are obscure. The effects of 5-HT3 receptor agonists on cortical circuit response properties were studied in slices of ferret primary visual cortex using high-speed optical imaging of voltage-sensitive dye signals and whole-cell patch-clamp recording. Activation of the 5-HT3 receptor decreased the amplitude and lateral extent of excitation throughout postnatal development. This effect peaks after eye opening, which indicates a function for serotonergic modulation of circuit responses during the period of refinement of cortical connections. Whole-cell patch-clamp recordings from single neurons revealed that synaptic responses evoked by white matter stimulation were reduced by 5-HT3 receptor agonists, whereas the frequency of spontaneous GABAergic synaptic currents was enhanced dramatically. This indicates that the modulation of spontaneous synaptic activity by fast-acting serotonin receptors is reflected in an inhibition of the circuit response, in line with the notion of background synaptic activity altering the spatiotemporal integration properties of cortical cells by changing their membrane potential and their electrotonic structure. These mechanisms may regulate the response properties of intrinsic circuits in both the adult and developing neocortex.

2-Amino-5-phosphonovalerate↗

PubMed2004

Nitric oxide activates glibenclamide-sensitive K+ channels in urinary bladder myocytes through a c-GMP-dependent mechanism.

In the present investigation, we used standard patch clamp techniques to test whether nitric oxide (NO) generation has any role to play with either activation or inhibition of ATP-sensitive (KATP) channels in guinea-pig urinary bladder. We found that NO generation leads to activation of KATP channels through a cyclic guanosine monophosphate (c-GMP)-dependent protein kinase. 3-Morpholinosydnonimine (SIN, 100 microM) potentiated activation of an inward current in whole cell patch clamp experiments. Glibenclamide (10 microM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) inhibited the SIN-activated current. Both in cell-attached and in inside out patches, SIN (200 microM) potentiated KATP channel activity, and the increased channel activity in inside out patches was suppressed by glibenclamide (50 microM), ATP (1 mM) and (9s,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12,-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6] benzodiazocine-10-carboxylic acid, methyl ester (KT-5823, 10 nM). 8-Br-cGMP (100 microM) increased the KATP channel activity in cell-attached patches, and this was suppressed by glibenclamide (50 microM). These results suggest that the NO-c-GMP-PKG pathway contributes to activation of K(ATP) channels in guinea-pig urinary bladder myocytes.

Adenosine Triphosphate↗

PubMed1997

Expression and functional analysis of voltage-activated Na+ channels in human prostate cancer cell lines and their contribution to invasion in vitro.

Ion channels are important for many cellular functions and disease states including cystic fibrosis and multidrug resistance. Previous work in the Dunning rat model of prostate cancer has suggested a relationship between voltage-activated Na+ channels (VASCs) and the invasive phenotype in vitro. The objectives of this study were to 1) evaluate the expression of VASCs in the LNCaP and PC-3 human prostate cancer cell lines by Western blotting, flow cytometry, and whole-cell patch clamping, 2) determine their role in invasion in vitro using modified Boyden chambers with and without a specific blocker of VASCs (tetrodotoxin). A 260-kd protein representing VASCs was found only in the PC-3 cell line, and these were shown to be membrane expressed on flow cytometry. Patch clamping studies indicated that functional VASCs were present in 10% of PC-3 cells and blocking these by tetrodotoxin (600 nmol/L) reduced their invasiveness by 31% (P = 0.02) without affecting the invasiveness of the LNCaP cells. These results indicate that the reduction of invasion is a direct result of VASC blockade and not a nonspecific action of the drug. This is the first report of VASCs in a human prostatic cell line. VASCs are present in PC-3 but not LNCaP cells as determined by both protein and functional studies. Tetrodotoxin reduced the invasiveness of PC-3 but not LNCaP cells, and these data suggest that ion channels may play an important functional role in tumor invasion.

Animals↗

PubMed1999

Identification of different putative neuronal subtypes in cultures of the superior region of the hippocampus using electrophysiological parameters.

Cultured neurons offer many advantages over a slice preparation for whole-cell patch-clamp studies, such as better control over the environment and space clamp control. However, heterogeneous cultures of neurons present problems in distinguishing the cell type from which recordings are made. The present study uses correlations with data obtained in the hippocampal slice preparation to determine the feasibility of "identifying" different neuronal subtypes in cultures obtained from the superior region of postnatal two- to 13-day-old rat hippocampus. Whole-cell patch-clamp recording in the current-clamp mode after 24-96 h in culture was used to determine if the action potential duration would be a useful criterion in distinguishing cell types. Single action potentials were elicited by a 0.1-0.2 ms, 2-4 nA depolarizing pulse. The average membrane potential and input resistance were -46.8+/-1.2 mV (n = 58) and 576+/-56 Mohms (n = 57), respectively. A frequency distribution of the action potential duration measured at half-maximal amplitude showed four distinct groups of neurons (group 1, 1.36+/-0.03 ms, n = 17; group 2, 2.19+/-0.05 ms, n = 20; group 3, 3.17+/-0.10 ms, n = 16; group 4, 4.36+/-0.13, n = 5). Based on correlations with previous studies using intracellular recording in identified cells in slices, the data suggest that group 1 represents basket cells, group 2 represents vertical cells, group 3 represents a combination of stellate cells and pyramidal cells, and group 4 represents another unidentified class of cells. Further analysis of the fast afterhyperpolarization allows distinction between pyramidal cells and stellate cells in group 3. In contrast to the interneurons in a slice preparation, these cells offer good voltage control and environmental control. Future studies will record from these cells in current-clamp mode to quickly characterize the action potential before switching to voltage-clamp recording to characterize the currents present in the different types of interneurons.

Action Potentials↗

PubMed1998

Regulation of the epithelial Na+ channel by membrane tension.

The sensitivity of alphabetagamma rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2-5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at -100 mV) from -3.42 +/- 0.34 to -2.02 +/- 0.23 microA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that alpha beta gamma rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

Actins↗

PubMed1994

Metal interactions with voltage- and receptor-activated ion channels.

Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions.

Animals↗

PubMed1992

Stretch-activated anion currents of rabbit cardiac myocytes.

1. Stretch-activated anion currents were studied in sino-atrial and atrial cells using the whole-cell patch clamp technique. With continuous application of positive pressure (5-15 cmH2O) through the patch clamp electrode, the cell was inflated and the membrane conductance was increased. 2. Voltage clamp steps revealed that the stretch-activated currents had time-independent characteristics. The increase in membrane conductance was reversible on subsequent application of negative pressure to the electrode. 3. The reversal potential of the stretch-activated currents was shifted by 60 mV for a 10-fold change in intracellular Cl- concentration, while it was unaffected by replacement of Na+ in the extracellular solution by N-methyl-D-glucamine. Cell superfusion with Cl(-)-deficient solution (10 mM Cl-) reduced the amplitude of outward current. These findings indicate that the stretch-activated conductance is Cl- selective. 4. The sequence of anion permeability through the stretch-activated conductance was determined to be I-(1.7) > NO3-(1.5) > Br-(1.2) > Cl-(1.0) > and F-(0.6). SCN- appeared to be more permeant than I-. 5. The stretch-activated conductance was reduced by the Cl- channel blockers, 4,4'-dinitrostilbene-2,2'-disulphonic acid disodium salt, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid or anthracene-9-carboxylate (9-AC). Administration of furosemide or bumetanide had no effect. 6. The stretch-activated Cl- current was recorded even though intracellular Ca2+ ions were chelated by including 10 mM EGTA in the pipette solution. Neither the specific peptide inhibitor of cyclic AMP-dependent protein kinase (50 microM), nor the non-selective blocker of protein kinases, H-7 (20 microM), was effective in reducing the stretch-activated Cl- current, suggesting that the stretch-activated Cl- current is a novel type of cardiac Cl- current, which shows a different modulatory mechanism from that of other cardiac Cl- currents.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed2001

A novel function of sphingosine-1-phosphate to activate a non-selective cation channel in human endothelial cells.

1. The Ca2+ entry pathway activated by sphingosine-1-phosphate (S1P) was examined in primary cultured vascular endothelial cells dispersed from human umbilical vein (HUVECs) by measuring intracellular Ca2+ concentration ([Ca2+]i), whole-cell membrane currents and single channel activity. 2. Application of S1P to HUVECs induced a slowly developing, sustained increase in [Ca2+]i. When Ca2+ was absent from the bathing solution, no S1P-induced changes in [Ca2+]i were observed. Tert-butylhydroquinone (BHQ), an inhibitor of Ca2+ pumps in endoplasmic reticulum, and histamine induced a transient elevation of [Ca2+]i in HUVECs. 3. Pretreatment of HUVECs with 100 ng x ml(-1) pertussis toxin (PTX) for 15 h almost abolished the S1P effect on [Ca2+]i and reduced the histamine effect to 40% of the control. The BHQ-induced elevation of [Ca2+]i was insensitive to PTX. 4. When whole-cell membrane currents were recorded using the amphotericin B-perforated-patch clamp technique while monitoring [Ca2+]i, application of S1P induced a tiny inward current (I(S1P)) which was followed by the elevation of [Ca2+]i. I(S1P) reversed at +20.0 +/- 2.7 mV under these experimental conditions. 5. When S1P was included in the pipette solution in the excised inside-out patch clamp configuration, single channel activity with a conductance of 17 pS was activated. This channel activity depended on the presence of intracellular GTP. 6. In summary, these results show that S1P has a novel effect in mammalian cardiovascular endothelium to activate a non-selective cation (NSC) channel in a GTP-dependent manner via a PTX-sensitive G-protein. This S1P-sensitive NSC channel acts as a Ca2+ entry pathway in endothelium.

Calcium↗