scholarly journals BTX modification of Na channels in squid axons. I. State dependence of BTX action.

1991 ◽  
Vol 97 (3) ◽  
pp. 499-519 ◽  
Author(s):  
J Tanguy ◽  
J Z Yeh
Keyword(s):  
Time Constant ◽  
Slow Rate ◽  
Open Channel ◽  
Voltage Dependence ◽  
State Dependence ◽  
Na Channel ◽  
Na Channels ◽  
Fast Inactivation ◽  
Similar Time ◽  
Chloramine T

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.

scholarly journals State-dependent Block of Wild-type and Inactivation-deficient Na+ Channels by Flecainide

2003 ◽  
Vol 122 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Ging Kuo Wang ◽  
Corinna Russell ◽  
Sho-Ya Wang
Keyword(s):  
Time Constant ◽  
Open Channel ◽  
Inhibitory Concentration ◽  
Antiarrhythmic Agent ◽  
Na Channels ◽  
Fast Inactivation ◽  
Wild Type ◽  
Open Channel Block ◽  
State Dependent ◽  
Na Currents

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/ΔKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with β1 subunits in Hek293t cells. The flecainide-resting block at −140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 μM when the cell was not stimulated for 1,000 s. At 100 μM flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an ∼70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at −140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at −50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 μM, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 μM−1s−1 and 12.2 s−1, respectively. Upon repolarization to −140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was ∼20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.

scholarly journals Single Na+ channels activated by veratridine and batrachotoxin.

1987 ◽  
Vol 89 (3) ◽  
pp. 459-480 ◽  
Author(s):  
S S Garber ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
Open Channel ◽  
Voltage Dependence ◽  
Single Channel ◽  
Reversal Potential ◽  
Na Channel ◽  
Single Channels ◽  
Na Channels ◽  
Ionic Selectivity ◽  
Symmetrical Solution

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.

Isoform-selective Effects of Isoflurane on Voltage-gated Na+ Channels

2007 ◽  
Vol 107 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Wei OuYang ◽  
Hugh C. Hemmings
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
Fast Inactivation ◽  
Membrane Excitability ◽  
Voltage Gated ◽  
Hyperpolarizing Shift ◽  
Selective Effects

Abstract Background: Voltage-gated Na+ channels modulate membrane excitability in excitable tissues. Inhibition of Na+ channels has been implicated in the effects of volatile anesthetics on both nervous and peripheral excitable tissues. The authors investigated isoform-selective effects of isoflurane on the major Na+ channel isoforms expressed in excitable tissues. Methods: Rat Nav1.2, Nav1.4, or Nav1.5 α subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage clamp recording. The effects of isoflurane on Na+ current activation, inactivation, and recovery from inactivation were analyzed. Results: The cardiac isoform Nav1.5 activated at more negative potentials (peak INa at −30 mV) than the neuronal Nav1.2 (0 mV) or skeletal muscle Nav1.4 (−10 mV) isoforms. Isoflurane reversibly inhibited all three isoforms in a concentration- and voltage-dependent manner at clinical concentrations (IC50 = 0.70, 0.61, and 0.45 mm, respectively, for Nav1.2, Nav1.4, and Nav1.5 from a physiologic holding potential of −70 mV). Inhibition was greater from a holding potential of −70 mV than from −100 mV, especially for Nav1.4 and Nav1.5. Isoflurane enhanced inactivation of all three isoforms due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation. Inhibition of Nav1.4 and Nav1.5 by isoflurane was attributed primarily to enhanced inactivation, whereas inhibition of Nav1.2, which had a more positive V1/2 of inactivation, was due primarily to tonic block. Conclusions: Two principal mechanisms contribute to Na+ channel inhibition by isoflurane: enhanced inactivation due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation (Nav1.5 ≈ Nav1.4 > Nav1.2) and tonic block (Nav1.2 > Nav1.4 ≈ Nav1.5). These novel mechanistic differences observed between isoforms suggest a potential pharmacologic basis for discrimination between Na+ channel isoforms to enhance anesthetic specificity.

scholarly journals Kinetics of veratridine action on Na channels of skeletal muscle.

1986 ◽  
Vol 87 (1) ◽  
pp. 1-24 ◽  
Author(s):  
J B Sutro
Keyword(s):  
Time Constant ◽  
Time Course ◽  
Voltage Dependence ◽  
Na Channels ◽  
H Infinity ◽  
Tail Current ◽  
Time Integral ◽  
Activation Gating ◽  
Chloramine T ◽  
Kinetics Of

Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations.

scholarly journals Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

2013 ◽  
Vol 142 (3) ◽  
pp. 191-206 ◽  
Author(s):  
Amanda H. Lewis ◽  
Indira M. Raman
Keyword(s):  
Open Channel ◽  
Voltage Sensor ◽  
Channel Block ◽  
Na Channels ◽  
Fast Inactivation ◽  
Na Current ◽  
Open Channel Block ◽  
Channel Blocking ◽  
Open States ◽  
Resurgent Currents

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.

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.

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.

Two conformational states involved in the use-dependent TTX blockade of human cardiac Na+ channel

1996 ◽  
Vol 270 (6) ◽  
pp. H2029-H2037 ◽  
Author(s):  
R. Dumaine ◽  
H. A. Hartmann
Keyword(s):  
Direct Interaction ◽  
Na Channel ◽  
Fast Inactivation ◽  
Slow Recovery ◽  
Similar Time ◽  
Activated State ◽  
High Affinity Site ◽  
Action Potential Plateau ◽  
Time Courses ◽  
High Concentrations

We used a fast inactivation-deficient mutant (QQQ) of the human heart Na+ channel alpha-subunit (hH1a) to assess the influence of the inactivation gate on tetrodotoxin (TTX) use-dependent block (UDB) and postrepolarization block (PRB). PRB had similar time courses in both channels, suggesting no direct interaction of the inactivation gate with the TTX binding site. The UDB saturated with high concentrations of TTX in hH1a but not in QQQ, revealing the modulatory action of fast inactivation on UDB. TTX did not stabilize the inactivated states of QQQ, and the extra block developing during long depolarizations suggests a higher-affinity site involved in the gating of the channel. These results cannot be solely explained by a slow recovery from the block in the inactivated states. They suggest a common use-dependent block mechanism for hH1a and QQQ involving a high-affinity site. We propose that an activated state is primarily responsible for UDB during short depolarization in the range of the action potential plateau and that fast inactivation modulates the accessibility of the toxin to this site.

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.

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