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 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 Slow Inactivation Does Not Affect Movement of the Fast Inactivation Gate in Voltage-gated Na+ Channels

1998 ◽  
Vol 111 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Vasanth Vedantham ◽  
Stephen C. Cannon
Keyword(s):  
Na Channel ◽  
Slow Inactivation ◽  
Na Channels ◽  
Fast Inactivation ◽  
Voltage Gated ◽  
Cytoplasmic Face ◽  
Voltage Dependent ◽  
Tightly Coupled ◽  
Exclusive Processes ◽  
A Site

Voltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III–IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (μl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (h∞•) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2–10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

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 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.

Different voltage dependence of transient and persistent Na+ currents is compatible with modal-gating hypothesis for sodium channels

1994 ◽  
Vol 71 (6) ◽  
pp. 2562-2565 ◽  
Author(s):  
A. M. Brown ◽  
P. C. Schwindt ◽  
W. E. Crill
Keyword(s):  
Pyramidal Neurons ◽  
Voltage Dependence ◽  
Sufficient Evidence ◽  
Na Channel ◽  
Na Channels ◽  
Activation Curve ◽  
Neocortical Neurons ◽  
Na Currents ◽  
Flow Through ◽  
The Difference

1. These experiments tested the hypothesis that the differing voltage dependence of the transient (INa) and persistent (INaP) Na+ currents in neocortical neurons results from the state of inactivation of one type of Na+ channel rather than from the existence of different types of Na+ channels. This question was examined in acutely isolated pyramidal neurons from the sensorimotor cortex of rats by using papain to remove inactivation from INa and comparing the resulting activation curve with that of INaP. 2. In control cells, INaP activated at more negative potentials than INa. Inclusion of papain in the recording pipette removed inactivation from INa and caused the INa activation curve to be shifted leftward to the position of the curve for INaP measured in control cells. Papain greatly increased both INa amplitude and the time to reach peak INa during smaller depolarizations, whereas the difference between control and test currents was reduced during large depolarizations. 3. We conclude that differences in the voltage dependence of INa and INaP activation does not provide sufficient evidence that these currents flow through separate sets of Na+ channels. Instead, our results are consistent with the idea that INaP largely arises from a fraction of the transient Na+ channels that intermittently lose their inactivation.

Structure and function of voltage-dependent sodium channels: comparison of brain II and cardiac isoforms

1996 ◽  
Vol 76 (3) ◽  
pp. 887-926 ◽  
Author(s):  
H. A. Fozzard ◽  
D. A. Hanck
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Point Mutations ◽  
Amino Acid Sequences ◽  
Na Channel ◽  
Na Channels ◽  
Voltage Dependent ◽  
And Function ◽  
Relationship Of ◽  
The Relationship

Cardiac and nerve Na channels have broadly similar functional properties and amino acid sequences, but they demonstrate specific differences in gating, permeation, ionic block, modulation, and pharmacology. Resolution of three-dimensional structures of Na channels is unlikely in the near future, but a number of amino acid sequences from a variety of species and isoforms are known so that channel differences can be exploited to gain insight into the relationship of structure to function. The combination of molecular biology to create chimeras and channels with point mutations and high-resolution electrophysiological techniques to study function encourage the idea that predictions of structure from function are possible. With the goal of understanding the special properties of the cardiac Na channel, this review examines the structural (sequence) similarities between the cardiac and nerve channels and considers what is known about the relationship of structure to function for voltage-dependent Na channels in general and for the cardiac Na channels in particular.

Effects of Propofol on Sodium Channel-dependent Sodium Influx and Glutamate Release in Rat Cerebrocortical Synaptosomes

1997 ◽  
Vol 86 (2) ◽  
pp. 428-439 ◽  
Author(s):  
L. Ratnakumari ◽  
H. C. Hemmings
Keyword(s):  
Glutamate Release ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
General Anesthetic ◽  
Adult Rats ◽  
Sodium Influx ◽  
Voltage Dependent ◽  
Channel Dependent ◽  
Presynaptic Nerve Terminals

Background Previous electrophysiologic studies have implicated voltage-dependent Na+ channels as a molecular site of action for propofol. This study considered the effects of propofol on Na+ channel-mediated Na+ influx and neurotransmitter release in rat brain synaptosomes (isolated presynaptic nerve terminals). Methods Purified cerebrocortical synaptosomes from adult rats were used to determine the effects of propofol on Na+ influx through voltage-dependent Na+ channels (measured using 22Na+) and intracellular [Na+] (measured by ion-specific spectrofluorimetry). For comparison, the effects of propofol on synaptosomal glutamate release evoked by 4-aminopyridine (Na+ channel dependent), veratridine (Na+ channel dependent), KCi (Na+ channel independent) were studied using enzyme-coupled fluorimetry. Results Propofol inhibited veratridine-evoked 22Na+ influx (inhibitory concentration of 50% [IC50] = 46 microM; 8.9 microM free) and changes in intracellular [Na+] (IC50 = 13 microM; 6.3 microM free) in synaptosomes in a dose-dependent manner. Propofol also inhibited 4-aminopyridine-evoked (IC50 = 39 microM; 19 microM free) and veratridine (20 microM)-evoked (IC50 = 30 microM; 14 microM free), but not KCi-evoked (up to 100 microM) glutamate release from synaptosomes. Conclusions Inhibition of Na+ channel-mediated Na+ influx, increased in intracellular [Na+], and glutamate release occurred in synaptosomes at concentrations of propofol achieved clinically. These results support a role for neuronal voltage-dependent Na+ channels as a molecular target for presynaptic general anesthetic effects.

Resurgent Na Currents in Four Classes of Neurons of the Cerebellum

2004 ◽  
Vol 92 (5) ◽  
pp. 2831-2843 ◽  
Author(s):  
Fatemeh S. Afshari ◽  
Krzysztof Ptak ◽  
Zayd M. Khaliq ◽  
Tina M. Grieco ◽  
N. Traverse Slater ◽  
...  
Keyword(s):  
Open Channel ◽  
Granule Cells ◽  
Na Channel ◽  
Na Channels ◽  
Brush Cells ◽  
Cerebellar Neurons ◽  
Na Currents ◽  
Unipolar Brush Cells ◽  
Resurgent Current ◽  
Resurgent Currents

Action potential firing rates are generally limited by the refractory period, which depends on the recovery from inactivation of voltage-gated Na channels. In cerebellar Purkinje neurons, the kinetics of Na channels appear specialized for rapid firing. Upon depolarization, an endogenous open-channel blocker rapidly terminates current flow but prevents binding of the “fast” inactivation gate. Upon repolarization, unbinding of the blocker produces “resurgent” Na current while allowing channels to recover rapidly. Because other cerebellar neurons, including granule cells, unipolar brush cells, and neurons of the cerebellar nuclei, also fire rapidly, we tested whether these cells might also express Na channels with resurgent kinetics. Neurons were acutely isolated from mice and rats, and TTX-sensitive Na currents were recorded under voltage clamp. Unlike Purkinje cells, the other cerebellar neurons produced only tiny resurgent currents in solutions optimized for voltage-clamping Na currents (50 mM Na+; Co2+ substitution for Ca2+). Under more physiological ionic conditions (155 mM Na+; 2 mM Ca2+ with 0.03 mM Cd2+), however, granule cells, unipolar brush cells, and cerebellar nuclear cells all produced robust resurgent currents. The increase in resurgent current, which was greater than predicted by the Goldman-Hodgkin-Katz equation, appeared to result from a combination of knock-off of open-channel blockers by permeating ions as well as relief of divalent block at negative potentials. These results indicate that resurgent current is typical of many cerebellar neurons and suggest that rapid open-channel block and unblock may be a widespread mechanism for restoration of Na channel availability in rapidly firing neurons.

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.

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