scholarly journals Properties of single cardiac Na channels at 35 degrees C.

1994 ◽  
Vol 104 (5) ◽  
pp. 801-820 ◽  
Author(s):  
K Benndorf
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Band Width ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Total Noise ◽  
Na Currents ◽  
Transition Points

Single Na channel currents were recorded in cell-attached patches of mouse ventricular myocytes with an improved patch clamp technique. Using patch pipettes with a pore diameter in the range of 200 nm, seals with a resistance of up to 4 T omega were obtained. Under those conditions, total noise could be reduced to levels as low as 0.590 pA rms at 20 kHz band width. At this band width, properties of single-channel Na currents were studied at 35 degrees C. Six out of a total of 23 patches with teraohm seals contained channel activity and five of these patches contained one and only one active channel. Amplitude histograms excluding transition points showed heterogenous distributions of levels. In one patch, part of the openings was approximately Gaussian distributed at different potentials yielding a slope conductance of 27 pS. The respective peak open probability at -10 mV was 0.26. The mean open time was determined at voltages between -60 and -10 mV by evaluation of the distribution of the event-related gaps in the center of the baseline noise to be approximately 40 microseconds at -60 mV and 50-74 microseconds between -50 and -10 mV. It is concluded that single cardiac Na channels open at 35 degrees C frequently with multiple levels and with open times in the range of several tens of microseconds.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry

1993 ◽  
Vol 264 (3) ◽  
pp. F565-F574 ◽  
Author(s):  
G. Frindt ◽  
R. B. Silver ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Homeostasis ◽  
Feedback Regulation ◽  
Feedback Mechanism ◽  
Acute Administration ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Single Channel Currents

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and single-channel currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of amiloride (10 microM) or benzamil (0.5 microM) to the superfusate resulted in a twofold increase in the mean number of open channels (NPo) after 2 min. The effect closely paralleled an increase in i, presumably reflecting membrane hyperpolarization. The effects on both i and NPo reversed within 3 min after removal of amiloride. The increase in NPo was accounted for, at least in part, by an increase in Po. Several cellular events may contribute to this phenomenon. Channels could be activated directly by membrane hyperpolarization and by cell shrinkage, both of which are known to occur during acute administration of amiloride. In addition, benzamil elicited a 30% decrease in intracellular Ca compared with control levels as measured by fura-2 fluorescence. A comparable decrease observed after reducing extracellular Ca did not increase NPo. No changes in cell pH, measured with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, were observed. The modulation of channel Po by the rate of Na entry into the cell will act as a feedback mechanism to maintain cellular ion homeostasis, and this may also serve to distribute Na reabsorption more evenly along the nephron.

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 Block of Neuronal Na+Channels by Antidepressant Duloxetine in a State-dependent Manner

2010 ◽  
Vol 113 (3) ◽  
pp. 655-665 ◽  
Author(s):  
Sho-Ya Wang ◽  
Joanna Calderon ◽  
Ging Kuo Wang
Keyword(s):  
Open Channel ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Dependent Manner ◽  
Channel Block ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Channel Block ◽  
Human Embryonic Kidney Cells ◽  
Na Currents

Background Duloxetine is a mixed serotonin-norepinephrine reuptake inhibitor used for major depressive disorder. Duloxetine is also beneficial for patients with diabetic peripheral neuropathy and with fibromyalgia, but how it works remains unclear. Methods We used the whole cell, patch clamp technique to test whether duloxetine interacts with the neuronal Nav1.7 Na+ channel as a potential target. Resting and inactivated Nav1.7 Na+ channel block by duloxetine were measured by conventional pulse protocols in transfected human embryonic kidney cells. The open-channel block was determined directly using inactivation-deficient mutant Nav1.7 Na+ channels. Results The 50% inhibitory concentration (IC50) of duloxetine for the resting and inactivated wild-type hNav1.7 Na+ channel were 22.1+/-0.4 and 1.79+/-0.10 microM, respectively (mean+/-SE, n=5). The IC50 for the open Na+ channel was 0.25+/-0.02 microM (n=5), as determined by the block of persistent late Nav1.7 Na+ currents. Similar open-channel block by duloxetine was found in the muscle Nav1.4 isoform (IC50=0.51+/-0.05 microM; n=5). Block by duloxetine appeared via the conserved local anesthetic receptor as determined by site-directed mutagenesis. Finally, duloxetine elicited strong use-dependent block of neuronal transient Nav1.7 Na+ currents during repetitive stimulations. Conclusions Duloxetine blocks persistent late Nav1.7 Na+ currents preferentially, which may in part account for its analgesic action.

scholarly journals Kinetics of 9-aminoacridine block of single Na channels.

1984 ◽  
Vol 84 (3) ◽  
pp. 361-377 ◽  
Author(s):  
D Yamamoto ◽  
J Z Yeh
Keyword(s):  
Rate Constant ◽  
Voltage Dependence ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

Apical Na+ conductance in maturing rabbit principal cell

1996 ◽  
Vol 270 (3) ◽  
pp. F391-F397 ◽  
Author(s):  
L. M. Satlin ◽  
L. G. Palmer
Keyword(s):  
Principal Cell ◽  
Na Channel ◽  
Postnatal Life ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Pipette Solution ◽  
Principal Cells ◽  
Inward Currents ◽  
The Mean

Net Na+ absorption in microperfused rabbit cortical collecting ducts (CCDs) is low during the 1st wk of postnatal life, increasing substantially thereafter [L. M. Satlin. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F57-F65, 1994]. To establish whether the low rate of Na+ absorption observed immediately after birth is due to a low apical Na+ permeability of the neonatal principal cell, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to estimate conductance, number (N), and open probability (Po) of apical Na+ channels in principal cells. With LiCl in the pipette and a NaCl or potassium gluconate solution, warmed to 37 degrees C, in the bath, inward currents with a conductance of approximately 11 pS (n = 23) were observed in 17% of cell-attached patches at 1 wk, 41% of patches at 2 wk, and 43% of patches at 5 wk. The mean N per patch in the 1st wk (0.22 +/- 0.09; n = 36) was significantly less than that observed in the 2nd (1.38 +/- 0.39; n = 34) and 5th (1.24 +/- 0.37; n = 21) wk of life. Po, studied at positive pipette voltages, was significantly lower in the 1st wk (0.085 +/- 0.035; n = 5) than in the 2nd wk (0.345 +/- 0.063; n = 9) and 5th wk (0.291 +/- 0.058; n = 4). To confirm that the 11-pS channel represented the amiloride-sensitive apical Na+ channel, cell-attached patches in CCDs isolated from 2-wk-old rabbits were studied with 0.5 microM amiloride added to the LiCl pipette solution. Amiloride led to > 90% reductions in mean open and closed times of the 11-pS conductance, consistent with blockade of the channel. These data indicate that N and Po of apical amiloride-sensitive Na+ channels in principal cells increase significantly after birth.

Development of inwardly rectifying K+ channel family in rat ventricular myocytes

1997 ◽  
Vol 272 (4) ◽  
pp. H1741-H1750 ◽  
Author(s):  
L. H. Xie ◽  
M. Takano ◽  
A. Noma
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
K Current ◽  
Inwardly Rectifying

The ATP-sensitive K+ current (I(K,ATP)), the inward rectifier K+ current (I(K1)), and the acetylcholine-activated K+ current (I(K,ACh)) were recorded in fetal, neonatal, and adult rat ventricular myocytes using the patch-clamp technique. The density (pA/pF) of I(K1) increased from gestation day 10 through neonatal day 1 and then decreased after neonatal day 30. The density of I(K,ATP) activated maximally by metabolic inhibition changed in parallel with the I(K1) density, and the density of I(K,ATP) channel distribution was 1.3 times higher than that of I(K1) throughout the development. We failed to observe developmental changes in the single-channel conductance and the mean open time of I(K1) and I(K,ATP) channels. However, the open probability of the I(K,ATP) channel was lower in fetuses, and the sensitivity to ATP was highest in 1-day neonates. I(K,ACh) were present in the ventricle at all stages of development but at a much lower density than in atrium. The relationship between the resting membrane potential and the development of the inwardly rectifying K-channel family is discussed.

Characterization of the isoform-specific differences in the gating of neuronal and muscle sodium channels

1998 ◽  
Vol 76 (10-11) ◽  
pp. 1041-1050 ◽  
Author(s):  
Michael E O'Leary
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Cultured Cells ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Rate Of Recovery

Human heart (hH1), human skeletal muscle (hSkM1), and rat brain (rIIA) Na channels were expressed in cultured cells and the activation and inactivation of the whole-cell Na currents measured using the patch clamp technique. hH1 Na channels were found to activate and inactivate at more hyperpolarized voltages than hSkM1 and rIIA. The conductance versus voltage and steady state inactivation relationships have midpoints of -48 and -92 mV (hH1), -28 and -72 mV (hSkM1), and -22 and -61 mV (rIIA). At depolarized voltages, where Na channels predominately inactivate from the open state, the inactivation of hH1 is 2-fold slower than that of hSkM1 and rIIA. The recovery from fast inactivation of all three isoforms is well described by a single rapid component with time constants at -100 mV of 44 ms (hH1), 4.7 ms (hSkM1), and 7.6 ms (rIIA). After accounting for differences in voltage dependence, the kinetics of activation, inactivation, and recovery of hH1 were found to be generally slower than those of hSkM1 and rIIA. Modeling of Na channel gating at hyperpolarized voltages where the channel does not open suggests that the slow rate of recovery from inactivation of hH1 accounts for most of the differences in the steady-state inactivation of these Na channels.Key words: cardiac, neuronal, skeletal muscle, sodium channel.

scholarly journals Transfer of twelve charges is needed to open skeletal muscle Na+ channels.

1995 ◽  
Vol 106 (6) ◽  
pp. 1053-1068 ◽  
Author(s):  
B Hirschberg ◽  
A Rovner ◽  
M Lieberman ◽  
J Patlak
Keyword(s):  
Skeletal Muscle ◽  
Membrane Potential ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Fast Inactivation ◽  
Open Probability ◽  
Single Channel Analysis ◽  
Voltage Dependent ◽  
Function Models

Voltage-dependent Na+ channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Measurement of open probability (Po) as a function of membrane potential gives a quantitative indication of the number of such charges that move through the field in opening the channel. We have used single-channel recording to measure skeletal muscle Na+ channel open probability at its most negative extreme, where channels may open as seldom as once per minute. To prevent fast inactivation from masking the voltage dependence of Po, we have generated a clone of the rat skeletal muscle Na+ channel that is lacking in fast inactivation (IFM1303QQQ). Using this mutant channel expressed in Xenopus oocytes, and the extra resolution afforded by single-channel analysis, we have extended the resolution of the hyperpolarized tail of the Po curve by four orders of magnitude. We show that previous measurements, which indicated a minimum of six effective gating charges, may have been made in a range of Po values that had not yet arrived at its limiting slope. In our preparation, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models based on six charges, and they have major implications for the interpretation of S4 mutagenesis studies and structure/function models of the Na+ channel.

scholarly journals Two modes of gating during late Na+ channel currents in frog sartorius muscle.

1986 ◽  
Vol 87 (2) ◽  
pp. 305-326 ◽  
Author(s):  
J B Patlak ◽  
M Ortiz
Keyword(s):  
Reversal Potential ◽  
Current Amplitude ◽  
Na Channel ◽  
Sartorius Muscle ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Na Currents ◽  
Inward Currents ◽  
Channel Currents

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.

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