scholarly journals Evidence for a population of sleepy sodium channels in squid axon at low temperature.

1982 ◽  
Vol 79 (5) ◽  
pp. 739-758 ◽  
Author(s):  
D R Matteson ◽  
C M Armstrong
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Reversal Potential ◽  
Current Amplitude ◽  
Na Channel ◽  
Na Current ◽  
Current Increase ◽  
Temperature Changes ◽  
Na Currents ◽  
Steady State Current

We have studied the effects of temperature changes on Na currents in squid giant axons. Decreases in temperature in the 15-1 degrees C range decrease peak Na current with a Q10 of 2.2. Steady state currents, which are tetrodotoxin sensitive and have the same reversal potential as peak currents, are almost unaffected by temperature changes. After removal of inactivation by pronase treatment, steady state current amplitude has a Q10 of 2.3. Na currents generated at large positive voltages sometimes exhibit a biphasic activation pattern. The first phase activates rapidly and partially inactivates and is followed by a secondary slow current increase that lasts several milliseconds. Peak Na current amplitude can be increased by delivering large positive prepulses, an effect that is more pronounced at low temperatures. The slow activation phase is eliminated after a positive prepulse. The results are consistent with the hypothesis that there are two forms of the Na channel: (a) rapidly activating channels that completely inactivate, and (b) slowly activating "sleepy" channels that inactivate slowly if at all. Some fast channels are assumed to be converted to sleepy channels by cooling, possibly because of a phase transition in the membrane. The existence of sleepy channels complicates the determination of the Q10 of gating parameters and single-channel conductance.

scholarly journals Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve.

1985 ◽  
Vol 86 (5) ◽  
pp. 739-762 ◽  
Author(s):  
G K Wang ◽  
G Strichartz
Keyword(s):  
Steady State ◽  
Ionic Currents ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Current ◽  
Toxin Concentration ◽  
Toxin Molecule ◽  
Channel Inactivation ◽  
Na Currents ◽  
Voltage Dependent

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.

scholarly journals Na Self Inhibition of Human Epithelial Na Channel

2002 ◽  
Vol 120 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Ahmed Chraïbi ◽  
Jean-Daniel Horisberger
Keyword(s):  
Steady State ◽  
Reversal Potential ◽  
Outward Current ◽  
Inactivation Rate ◽  
Na Channel ◽  
Temperature Dependent ◽  
Open Probability ◽  
Inward Current ◽  
Steady State Current ◽  
Epithelial Na Channel

The regulation of the open probability of the epithelial Na+ channel (ENaC) by the extracellular concentration of Na+, a phenomenon called “Na+ self inhibition,” has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na+ self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na+ concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q10act = 1.5) activation rate and a strongly temperature-dependant (Q10inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na+ self inhibition depended only on the extracellular Na+ concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na+ as well as a reduction of Na+ outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na+ self inhibition is an intrinsic property of sodium channels resulting from the expression of the α, β, and γ subunits of human ENaC in Xenopus oocyte. The extracellular Na+-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.

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.

Relationship between Na+ current expression and cell-cell coupling in astrocytes cultured from rat hippocampus

1991 ◽  
Vol 65 (4) ◽  
pp. 989-1002 ◽  
Author(s):  
H. Sontheimer ◽  
S. G. Waxman ◽  
B. R. Ransom
Keyword(s):  
Lucifer Yellow ◽  
Na Channel ◽  
Dye Coupling ◽  
Cell Coupling ◽  
Na Current ◽  
Na Currents ◽  
Coupled Cells ◽  
Hippocampal Astrocytes ◽  
Cell Cell

1. Cell-cell coupling between hippocampal astrocytes in culture was studied by following the intracellular spread of the low molecular weight fluorescent dye Lucifer yellow (LY). Dye coupling appeared as early as 24 h after plating, at which time approximately 20% of all astrocytes that physically contacted neighboring cells showed dye coupling. 2. The percentage of coupled cells increased with time in culture and peaked after 10 days in vitro (DIV) when approximately 50% of astrocytes showed coupling. Further time in culture, up to 20 DIV, did not increase the percentage of coupled cells. Thus, coupled and noncoupled astrocytes coexist in hippocampal cultures in approximately equal numbers. 3. Na+ currents were expressed in a subpopulation of hippocampal astrocytes and changed characteristics during in vitro development. A "neuronal type" of Na+ current, so called because of an h alpha curve that had a midpoint near -60 mV, was observed within the first 5 days post-plating. A "glial type" of Na+ current, characterized by a -25 mV shift in its h alpha curve, was only expressed after 6 days in culture. 4. Na+ current expression was restricted to hippocampal astrocytes that did not exhibit dye coupling; astrocytes that exhibited dye coupling (n = 39) did not show measurable Na+ currents. 5. The failure to see Na+ currents in coupled astrocytes cannot be explained by insufficient space-clamp since astrocytes acutely uncoupled with octanol (10 microM) did not reveal Na+ current expression. Control experiments showed that low concentrations of octanol (i.e., 10-100 microM) did not block Na+ currents; blockage of Na+ currents by octanol was only observed at high concentrations (e.g., 50-fold the concentration used for uncoupling). These observations support the idea that Na(+)-channel expression was restricted to noncoupled astrocytes. 6. The time courses for the development of cell coupling and Na+ current expression appeared to be inversely correlated and suggested a gradual increase in cell coupling in concert with a loss in Na+ current expression with time in culture.

Cholinergic modulation of apical Na+ channels in turtle colon: analysis of CDPC-induced fluctuations

1990 ◽  
Vol 259 (4) ◽  
pp. C668-C674 ◽  
Author(s):  
D. J. Wilkinson ◽  
D. C. Dawson
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Rate Coefficients ◽  
Corner Frequency ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Na Channels ◽  
Na Current

Current fluctuation analysis was used to investigate the properties of apical Na+ channels during muscarinic inhibition of active Na+ absorption. A reversible Na+ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 microM. The on (k'on) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'on = 11.1 +/- 0.8 rad.s-1.microM-1 and k(off) = 744 +/- 53 rad/s, and the microscopic inhibition constant was 67 microM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of Isc by 5 microM serosal carbachol. Single-channel Na+ current (iNa) and the density of open and blocked Na+ channels (N(ob)) were estimated from the fluctuations induced by 40 microM CDPC. Under control conditions, iNa was 0.43 +/- 0.05 pA and N(ob) was 251 +/- 42 X 10(6)/cm2 (n = 10). After exposure to serosal carbachol (2-10 microM) for 60 min, Na+ current and N(ob) were reduced by approximately 50%, but iNa was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na+ absorption was associated with a reduction in the number of open Na+ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.

Quantification of K+ secretion through apical low-conductance K channels in the CCD

2005 ◽  
Vol 289 (1) ◽  
pp. F117-F126 ◽  
Author(s):  
Daniel A. Gray ◽  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Collecting Duct ◽  
Reversal Potential ◽  
Inward Rectification ◽  
Na Channel ◽  
Sk Channels ◽  
Maximal Conductance ◽  
Channel Density ◽  
Sk Channel ◽  
High K

Outward and inward currents through single small-conductance K+ (SK) channels were measured in cell-attached patches of the apical membrane of principal cells of the rat cortical collecting duct (CCD). Currents showed mild inward rectification with high [K+] in the pipette (Kp+), which decreased as Kp+ was lowered. Inward conductances had a hyperbolic dependence on Kp+ with half-maximal conductance at ∼20 mM. Outward conductances, measured near the reversal potential, also increased with Kp+ from 15 pS (Kp+ = 0) to 50 pS (Kp+ = 134 mM). SK channel density was measured as the number of conducting channels per patch in cell-attached patches. As reported previously, channel density increased when animals were on a high-K diet for 7 days. Addition of 8-cpt-cAMP to the bath at least 5 min before making a seal increased SK channel density to an even greater extent, although this increase was not additive with the effect of a high-K diet. In contrast, increases in Na channel activity, assessed as the whole cell amiloride-sensitive current, due to K loading and 8-cpt-cAMP treatment were additive. Single-channel conductances and channel densities were used as inputs to a simple mathematical model of the CCD to predict rates of transepithelial Na+ and K+ transport as a function of apical Na+ permeability and K+ conductance, basolateral pump rates and K+ conductance, and the paracellular conductance. With measured values for these parameters, the model predicted transport rates that were in good agreement with values measured in isolated, perfused tubules. The number and properties of SK channels account for K+ transport by the CCD under all physiological conditions tested.

scholarly journals Na channel distribution in vertebrate skeletal muscle.

1986 ◽  
Vol 87 (6) ◽  
pp. 907-932 ◽  
Author(s):  
J H Caldwell ◽  
D T Campbell ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Current Density ◽  
Sharp Decrease ◽  
Na Channel ◽  
Na Current ◽  
Na Currents ◽  
Current Densities ◽  
Kinetics Of ◽  
A Current ◽  
Vertebrate Skeletal Muscle

The loose patch voltage clamp has been used to map Na current density along the length of snake and rat skeletal muscle fibers. Na currents have been recorded from (a) endplate membrane exposed by removal of the nerve terminal, (b) membrane near the endplate, (c) extrajunctional membrane far from both the endplate and the tendon, and (d) membrane near the tendon. Na current densities recorded directly on the endplate were extremely high, exceeding 400 mA/cm2 in some patches. The membrane adjacent to the endplate has a current density about fivefold lower than that of the endplate, but about fivefold higher than the membrane 100-200 micron from the endplate. Small local variations in Na current density are recorded in extrajunctional membrane. A sharp decrease in Na current density occurs over the last few hundred micrometers from the tendon. We tested the ability of tetrodotoxin to block Na current in regions close to and far from the endplate and found no evidence for toxin-resistant channels in either region. There was also no obvious difference in the kinetics of Na current in the two regions. On the basis of the Na current densities measured with the loose patch clamp, we conclude that Na channels are abundant in the endplate and near-endplate membrane and are sparse close to the tendon. The current density at the endplate is two to three orders of magnitude higher than at the tendon.

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 Patch recordings from the electrocytes of Electrophorus electricus. Na currents and PNa/PK variability.

1991 ◽  
Vol 97 (5) ◽  
pp. 1013-1041 ◽  
Author(s):  
S Shenkel ◽  
F J Sigworth
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Reversal Potential ◽  
Fluctuation Analysis ◽  
Open Probability ◽  
Na Currents ◽  
Before And After ◽  
Steady State Inactivation ◽  
K Current ◽  
Reversal Potentials

Sodium currents were recorded in cell-attached and inside-out patches from the innervated membrane of Electrophorus electrocytes. Electrocytes from Sachs and main electric organs were prepared as described by Pasquale et al. (1986. J. Membr. Biol. 93:195.). Maximal currents in the Sachs organ, measured with 1-2 microns diameter patch pipettes and at room temperature, were in the range of 20 to 300 pA (27 patches) and were obtained near +10 mV. This range of current corresponds to approximately 70 to 1,300 channels in a patch. Maximal current in main organ cells also occurred near +10 mV and were in the range of 100 to 400 pA. Delayed K current was observed in a few patches. The inactivation phase of the currents during maintained depolarizations appears to be a single-exponential relaxation. The time constant decreases from 1 ms near -55 mV to a minimum of 0.3 ms near 0 mV, and then gradually increases with stronger depolarization. The mean currents are half inactivated near -90 mV with an apparent voltage dependence of e-fold per 6 mV. No apparent differences were observed in the decay time course or steady-state inactivation of the currents in the same patch before and after excision. From ensemble fluctuation analysis the peak open probability was found to be approximately 0.5 at +25 mV and increased only gradually with larger depolarizations. The single channel conductances were approximately 20 pS with 200 mM Na outside and 200 mM K inside, and 40 pS in 400 mM solutions. Reversal potentials in the 200 Na parallel 200 K solutions ranged from +51 to +94 mV in multichannel patches, corresponding to selectivity ratios PNa/PK from 8 to 43. Large differences in reversal potentials were seen even among patches from the same cell. Several controls rule out obvious sources of error in the reversal potential measurements. It is concluded that there is heterogeneity in the selectivity properties of the Na channels.

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