scholarly journals Modification of Na channel gating by an alpha scorpion toxin from Tityus serrulatus.

1989 ◽  
Vol 93 (1) ◽  
pp. 67-83 ◽  
Author(s):  
G E Kirsch ◽  
A Skattebøl ◽  
L D Possani ◽  
A M Brown
Keyword(s):  
Cortical Neurons ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Neuroblastoma Cells ◽  
Na Channel ◽  
External Application ◽  
Whole Cell ◽  
Peak Current ◽  
Open Time ◽  
Tityus Serrulatus

The effects of TsIV-5, a toxin isolated from the Brazilian scorpion Tityus serrulatus, on whole-cell and single-channel Na currents were determined in N18 neuroblastoma cells. In whole-cell records at a test potential of -10 mV, external application of 500 nM TsIV-5 slowed inactivation 20-fold and increased peak current by about one-third without changing time-to-peak. Both the steady-state activation and inactivation curves were shifted to more negative potentials. Other alpha scorpion toxins produce similar effects but the single-channel mechanism is not known. TsIV-5 caused a voltage-dependent prolongation of mean single-channel open time such that at a test potential of -60 mV no change was observed, whereas at -20 mV mean open time increased about threefold and prolonged bursting was observed. Macroscopic current reconstructed from summed single-channel records showed a characteristic toxin-induced potentiation of peak current and a 20-fold slowing of the decay phase. TsIV-5 does not discriminate between tissue-specific Na channel subtypes. Prolonged open times and bursting were also observed in toxin-treated Na channels from rat ventricular myocytes, rat cortical neurons, and mouse skeletal muscle. The toxin effects are shown to be consistent with a kinetic model in which TsIV-5 selectively interferes with the ability of the channel to reach the inactivated state.

scholarly journals Kinetic properties of single sodium channels in rat heart and rat brain.

1989 ◽  
Vol 93 (1) ◽  
pp. 85-99 ◽  
Author(s):  
G E Kirsch ◽  
A M Brown
Keyword(s):  
Cortical Neurons ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Na Channel ◽  
Kinetic Properties ◽  
Open Time ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Near Threshold

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.

Unitary conductance of Na+ channel isoforms in cardiac and NB2a neuroblastoma cells

1995 ◽  
Vol 269 (6) ◽  
pp. C1356-C1363 ◽  
Author(s):  
C. M. Baumgarten ◽  
S. C. Dudley ◽  
R. B. Rogart ◽  
H. A. Fozzard
Keyword(s):  
Rate Constants ◽  
Ventricular Myocytes ◽  
Neuroblastoma Cells ◽  
Cardiac Cells ◽  
Na Channel ◽  
Pipette Solution ◽  
Open Time ◽  
Channel Opening ◽  
Rabbit Ventricular Myocytes ◽  
Reversal Potentials

Unitary conductances of native Na+ channel isoforms (gamma Na) have been determined under a variety of conditions, making comparisons of gamma Na difficult. To allow direct comparison, we measured gamma Na in cell-attached patches on NB2a neuroblastoma cells and rabbit ventricular myocytes under identical conditions [pipette solution (in mM): 280 Na+ and 2 Ca2+, pH 7.4; 10 degrees C]. gamma Na of NB2a channels, 22.9 +/- 0.9 pS, was 21% greater than that of cardiac channels, 18.9 +/- 0.9 pS. In contrast, respective extrapolated reversal potentials, +62.4 +/- 4.6 and +57.9 +/- 5.1 mV, were not significantly different. Several kinetic differences between the channel types were also noted. Negative to -20 mV, mean open time (MOT) of the NB2a isoform was significantly less than that of cardiac channels, and, near threshold, latency to channel opening decayed more rapidly in NB2a. On the basis of analysis of MOT between -60 and 0 mV, the rate constants at 0 mV for the open-to-closed (O-->C) and open-to-inactivated (O-->I) transitions were 0.42 +/- 0.11 and 0.47 +/- 0.11 ms-1 in NB2a and 0.10 +/- 0.06 and 1.19 +/- 0.07 ms-1 in myocytes. The slope factors were -38.9 +/- 8.7 and +10.7 +/- 6.1 mV in NB2a and -27.3 +/- 7.1 and +23.7 +/- 4.9 mV in myocytes. Transition rate constants were significantly different in NB2a and cardiac cells, but voltage dependence was not.

scholarly journals Veratridine modifies open sodium channels.

1988 ◽  
Vol 91 (3) ◽  
pp. 421-443 ◽  
Author(s):  
S Barnes ◽  
B Hille
Keyword(s):  
Single Channel ◽  
Neuroblastoma Cells ◽  
Cell Voltage ◽  
State Dependence ◽  
Na Channel ◽  
Na Channels ◽  
Pipette Solution ◽  
Whole Cell ◽  
Current Activation ◽  
Normal Current

The state dependence of Na channel modification by the alkaloid neurotoxin veratridine was investigated with single-channel and whole-cell voltage-clamp recording in neuroblastoma cells. Several tests of whole-cell Na current behavior in the presence of veratridine supported the hypothesis that Na channels must be open in order to undergo modification by the neurotoxin. Modification was use dependent and required depolarizing pulses, the voltage dependence of production of modified channels was similar to that of normal current activation, and prepulses that caused inactivation of normal current had a parallel effect on the generation of modified current. This hypothesis was then examined directly at the single-channel level. Modified channel openings were easily distinguished from normal openings by their smaller current amplitude and longer burst times. The modification event was often seen as a sudden, dramatic reduction of current through an open Na channel and produced a somewhat flickery channel event having a mean lifetime of 1.6 s at an estimated absolute membrane potential of -45 mV (23 degrees C). The modified channel had a slope conductance of 4 pS, which was 20-25% the size of the slope conductance of normal channels with the 300 mM NaCl pipette solution used. Most modified channel openings were initiated by depolarizing pulses, began within the first 10 ms of the depolarizing step, and were closely associated with the prior opening of single normal Na channels, which supports the hypothesis that modification occurs from the normal open state.

scholarly journals Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels.

1985 ◽  
Vol 86 (5) ◽  
pp. 691-719 ◽  
Author(s):  
D L Kunze ◽  
A E Lacerda ◽  
D L Wilson ◽  
A M Brown
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Waiting Times ◽  
Potential Range ◽  
Slow Inactivation ◽  
Na Channels ◽  
Whole Cell ◽  
Whole Cells ◽  
Open Time ◽  
Na Currents

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.

scholarly journals Regulation of the Epithelial Na+ Channel by Membrane Tension

1998 ◽  
Vol 112 (2) ◽  
pp. 97-111 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Muthangi Subramanyam
Keyword(s):  
Cytochalasin B ◽  
Single Channel ◽  
Direct Injection ◽  
Expression System ◽  
Cell Swelling ◽  
Na Channel ◽  
Bathing Solution ◽  
Membrane Tension ◽  
Whole Cell ◽  
Epithelial Na Channel

The sensitivity of αβγ 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 μA (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 αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

scholarly journals Stretch-activated whole cell currents in adult rat cardiac myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. H548-H557 ◽  
Author(s):  
Tao Zeng ◽  
Glenna C. L. Bett ◽  
Frederick Sachs
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Cellular Level ◽  
Whole Cell ◽  
Adult Rat ◽  
Longitudinal Stretch ◽  
Single Channel Currents ◽  
Channel Currents

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 μM Gd3+ but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately −6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 − 5.0SL (μm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.

scholarly journals Sodium channel subconductance levels measured with a new variance-mean analysis.

1988 ◽  
Vol 92 (4) ◽  
pp. 413-430 ◽  
Author(s):  
J B Patlak
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Na Channel ◽  
Na Channels ◽  
Open Probability ◽  
Open Time ◽  
Flexor Digitorum Brevis ◽  
Dissociated Cells ◽  
A New Technique ◽  
Flexor Digitorum

The currents through single Na+ channels were recorded from dissociated cells of the flexor digitorum brevis muscle of the mouse. At 15 degrees C the prolonged bursts of Na+ channel openings produced by application of the drug DPI 201-106 had brief sojourns to subconductance levels. The subconductance events were relatively rare and brief, but could be identified using a new technique that sorts amplitude estimates based on their variance. The resulting "levels histogram" had a resolution of the conductance levels during channel activity that was superior to that of standard amplitude histograms. Cooling the preparation to 0 degrees C prolonged the subconductance events, and permitted further quantitative analysis of their amplitudes, as well as clear observations of single-channel subconductance events from untreated Na+ channels. In all cases the results were similar: a subconductance level, with an amplitude of roughly 35% of the fully open conductance and similar reversal potential, was present in both drug-treated and normal Na+ channels. Drug-treated channels spent approximately 3-6% of their total open time in the subconductance state over a range of potentials that caused the open probability to vary between 0.1 and 0.9. The summed levels histograms from many channels had a distinctive form, with broader, asymmetrical open and substate distributions compared with those of the closed state. Individual subconductance events to levels other than the most common 35% were also observed. I conclude that subconductance events are a normal subset of the open state of Na+ channels, whether or not they are drug treated. The subconductance events may represent a conformational alteration of the channel that occurs when it conducts ions.

scholarly journals Modulation of Calcium Channels in Human Erythroblasts by Erythropoietin

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 92-100 ◽  
Author(s):  
Joseph Y. Cheung ◽  
Xue-Qian Zhang ◽  
Krister Bokvist ◽  
Douglas L. Tillotson ◽  
Barbara A. Miller
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Erythropoietin Receptor ◽  
Single Channels ◽  
Open Probability ◽  
Whole Cell ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Open Time ◽  
High Doses

Abstract Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca2+ ([Ca2+]i ) in human erythroblasts, which is dependent on extracellular Ca2+ and blocked by high doses of nifedipine or Ni2+. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca2+]i increase, indicating the specificity of the Ca2+ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca2+ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca2+ current. Using Ba2+ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba2+ concentrations. Channel open probability did not appreciably change over the voltage range (−50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 ± 0.75 to 11.15 ± 1.80 ms) and open probability (from 0.26 ± 0.06 to 2.56 ± 0.59%) of this Ba2+-permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca2+]i increase in human erythroblasts is mediated via Ca2+ entry through a voltage-independent Ca2+ channel.

scholarly journals Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells

1998 ◽  
Vol 111 (6) ◽  
pp. 825-846 ◽  
Author(s):  
Toru Ishikawa ◽  
Yoshinori Marunaka ◽  
Daniela Rotin
Keyword(s):  
Single Channel ◽  
Mdck Cells ◽  
Na Channel ◽  
Channel Activity ◽  
Channel Conductance ◽  
Na Current ◽  
Single Channel Conductance ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
A Charge

The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na+ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-d-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be ∼5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (Po), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nPo) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca2+ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.

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