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.

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

scholarly journals Inactivation viewed through single sodium channels.

1984 ◽  
Vol 84 (4) ◽  
pp. 535-564 ◽  
Author(s):  
C A Vandenberg ◽  
R Horn
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Gh3 Cells ◽  
Activation Process ◽  
Whole Cell ◽  
Open Time ◽  
Voltage Dependent

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.

Single-channel behavior of a purified epithelial Na+ channel subunit that binds amiloride

1992 ◽  
Vol 263 (5) ◽  
pp. C1111-C1117 ◽  
Author(s):  
S. Sariban-Sohraby ◽  
M. Abramow ◽  
R. S. Fisher
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Transport ◽  
Patch Clamp Technique ◽  
Transport Inhibitor ◽  
Open Time ◽  
The Mean ◽  
Epithelial Na Channel ◽  
Binding Subunit

The apical membrane of high electrical resistance epithelia, which is selectively permeable to Na+, plays an essential role in the maintenance of salt balance. Na+ entry from the apical fluid into the cells is mediated by amiloride-blockable Na(+)-specific channels. The channel protein, purified from both amphibian and mammalian sources, is composed of several subunits, only one of which the 150-kDa polypeptide, specifically binds the Na+ transport inhibitor amiloride. The goal of the present study was to investigate whether the isolated amiloride-binding subunit of the channel could conduct Na+. The patch-clamp technique was used to study the 150-kDa polypeptide incorporated into a lipid bilayer formed on the tip of a glass pipette. Unitary conductance jumps averaged 4.8 pS at 100 mM Na2HPO4. Open times ranged from 24 ms to several seconds. The channel spent most of the time in the closed state. Channel conductance and gating were independent of voltage between -60 and +100 mV. Amiloride (0.1 microM) decreased the mean open time of the channel by 98%. We conclude that the 150-kDa subunit of the amiloride-blockable Na+ channel conducts current and may be sufficient for the Na+ transport function of the whole channel.

Properties of veratridine-modified single Na+ channels in guinea pig ventricular myocytes

1993 ◽  
Vol 264 (2) ◽  
pp. H454-H463 ◽  
Author(s):  
A. Sunami ◽  
T. Sasano ◽  
A. Matsunaga ◽  
Z. Fan ◽  
T. Swanobori ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Simultaneous Occurrence ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
High Event ◽  
Event Times

Modification of single Na+ channels by the alkaloid neurotoxin veratridine was investigated in guinea pig ventricular myocytes using the cell-attached configuration of the patch-clamp technique. Pipette application of veratridine (50 microM) induced long-lasting openings with two different single-channel conductances of 7.6 and 3.0 pS, in addition to normal type of short openings with a single-channel conductance of 16 pS. The veratridine-modified high- and low-conductance channels appeared commonly, and they could coexist with the normal one in the same patch. The open-time distributions for the high- and low-conductance channels could be fitted by a single exponential. The mean open time for the high- and low-conductance events ranged between 19.1 ms at -120 mV and 86.0 ms at -10 mV and between 4.5 ms at -120 mV and 16.2 ms at -10 mV, respectively. The closed-time distributions for the two conductance channels consisted of at least two components, and their values and voltage dependence were similar. External Ca2+ block resulted in an apparent reduction of unitary current amplitudes with a similar voltage dependence and affinity for Ca2+ in the high- and low-conductance channels. However, the low-conductance channel was more resistant to tetrodotoxin than the high one. The probability of simultaneous occurrence of the high and low events was equal to the product of the probabilities of occurrence of the high event times that of the low event. Furthermore, we observed modified channel openings after a normal opening for the two conductance channels and a modified one turning into a normal one for the high-conductance channel. It is concluded that veratridine induces the two different types of modified Na+ channels in cardiac myocytes and these are correlated with normal openings.

Fast and Slow Activation Kinetics of Voltage-Gated Sodium Channels in Molluscan Neurons

1997 ◽  
Vol 77 (5) ◽  
pp. 2373-2384 ◽  
Author(s):  
William F. Gilly ◽  
Rhanor Gillette ◽  
Matthew McFarlane
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Na Channel ◽  
Molluscan Neurons ◽  
Na Channels ◽  
Activation Kinetics ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Voltage Dependent ◽  
Kinetics Of

Gilly, William F., Rhanor Gillette, and Matthew McFarlane. Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons. J. Neurophysiol. 77: 2373–2384, 1997. Whole cell patch-clamp recordings of Na current ( I Na) were made under identical experimental conditions from isolated neurons from cephalopod ( Loligo, Octopus) and gastropod ( Aplysia, Pleurobranchaea, Doriopsilla) species to compare properties of activation gating. Voltage dependence of peak Na conductance ( g Na) is very similar in all cases, but activation kinetics in the gastropod neurons studied are markedly slower. Kinetic differences are very pronounced only over the voltage range spanned by the g Na-voltage relation. At positive and negative extremes of voltage, activation and deactivation kinetics of I Na are practically indistinguishable in all species studied. Voltage-dependent rate constants underlying activation of the slow type of Na channel found in gastropods thus appear to be much more voltage dependent than are the equivalent rates in the universally fast type of channel that predominates in cephalopods. Voltage dependence of inactivation kinetics shows a similar pattern and is representative of activation kinetics for the two types of Na channels. Neurons with fast Na channels can thus make much more rapid adjustments in the number of open Na channels at physiologically relevant voltages than would be possible with only slow Na channels. This capability appears to be an adaptation that is highly evolved in cephalopods, which are well known for their high-speed swimming behaviors. Similarities in slow and fast Na channel subtypes in molluscan and mammalian neurons are discussed.

scholarly journals Dynamics of 9-aminoacridine block of sodium channels in squid axons.

1979 ◽  
Vol 73 (1) ◽  
pp. 1-21 ◽  
Author(s):  
J Z Yeh
Keyword(s):  
Ionic Channels ◽  
Na Channel ◽  
Na Channels ◽  
Drug Molecule ◽  
Na Currents ◽  
Voltage Dependent ◽  
A Site ◽  
Kinetics Of ◽  
Block Of Na Channels ◽  
Single Exponential Function

The interactions of 9-aminoacridine with ionic channels were studied in internally perfused squid axons. The kinetics of block of Na channels with 9-aminoacridine varies depending on the voltage-clamp pulses and the state of gating machinery of Na channels. In an axon with intact h gate, the block exhibits frequency- and voltage-dependent characteristics. However, in the pronase-perfused axon, the frequency-dependent block disappears, whereas the voltage-dependent block remains unchanged. A time-dependent decrease in Na currents indicative of direct block of Na channel by drug molecule follows a single exponential function with a time constant of 2.0 +/- 0.18 and 1.0 +/- 0.19 ms (at 10 degrees C and 80 m V) for 30 and 100 microM 9-aminoacridine, respectively. A steady-state block can be achieved during a single 8-ms depolarizing pulse when the h gate has been removed. The block in the h-gate intact axon can be achieved only with multiple conditioning pulses. The voltage-dependent block suggests that 9-aminoacridine binds to a site located halfway across the membrane with a dissociation constant of 62 microM at 0 m V. 9-Aminoacridine also blocks K channels, and the block is time- and voltage-dependent.

scholarly journals Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins.

1984 ◽  
Vol 84 (5) ◽  
pp. 687-704 ◽  
Author(s):  
E Moczydlowski ◽  
S Hall ◽  
S S Garber ◽  
G S Strichartz ◽  
C Miller
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Hydroxyl Groups ◽  
Binding Affinities ◽  
Na Channels ◽  
Plasma Membrane Vesicles ◽  
Toxin Binding ◽  
Voltage Dependent ◽  
Toxin Receptor

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field.

scholarly journals Gating of Acid-sensitive Ion Channel-1: Release of Ca2+ Block vs. Allosteric Mechanism

2006 ◽  
Vol 127 (2) ◽  
pp. 109-117 ◽  
Author(s):  
Ping Zhang ◽  
Fred J. Sigworth ◽  
Cecilia M. Canessa
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Single Channel ◽  
Allosteric Mechanism ◽  
Open Time ◽  
Channel Opening ◽  
Voltage Dependent ◽  
High Affinity Binding Site ◽  
The Mean ◽  
High Concentrations

The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at −60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.

Properties of single Na+ channels in cut-open Myxicola giant axons

1987 ◽  
Vol 65 (4) ◽  
pp. 568-573 ◽  
Author(s):  
C. L. Schauf
Keyword(s):  
Patch Clamp ◽  
Time Dependence ◽  
Voltage Dependence ◽  
Single Channel ◽  
Single Channels ◽  
Channel Conductance ◽  
Dependent Behavior ◽  
Open Time ◽  
Voltage Dependent ◽  
Steady State Inactivation

Time- and voltage-dependent behavior of the Na+ conductance in dialyzed intact Myxicola axons was compared with cut-open axons subjected to loose-patch clamp of the interior and to axons where Gigaseals were formed after brief enzyme digestion. Voltage and time dependence of activation, inactivation, and reactivation were identical in whole-axons and loose-patch preparations. Single channels observed in patch-clamp axons had a conductance of 18.3 ± 2.3 pS and a mean open time of 0.84 ± 0.12 ms. The time-dependence of Na+ currents found by averaging patch-clamp records was similar to intact axons, as was the voltage dependence of activation. Steady-state inactivation in patch-clamped axons was shifted by an average of 15 mV from that seen in loose-patch or intact axons. Substitution of D2O for H2O decreased single channel conductance by 24 ± 6% in patch-clamped axons compared with 28 ± 4% in intact axons, slowed inactivation by 58 ± 8% compared with 49 ± 6%, and increased mean open time by 52 ± 7%. The results confirm observations on macroscopic channel behavior in Myxicola and resemble that seen in other excitable tissues.

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.

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