scholarly journals Simulation of Na channel inactivation by thiazine dyes.

1982 ◽  
Vol 80 (5) ◽  
pp. 641-662 ◽  
Author(s):  
C M Armstrong ◽  
R S Croop
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Na Channel ◽  
Equilibration Time ◽  
Na Channels ◽  
Dye Molecules ◽  
Gating Current ◽  
Thiazine Dyes ◽  
Opening And Closing ◽  
Dye Molecule

Some dyes of the methylene blue family serve as artificial inactivators of the sodium channels when present inside squid axons at a concentration of approximately 0.1 mM. The dyes restore a semblance of inactivation after normal inactivation has been destroyed by pronase. In fibers that inactivate normally, the dyes hasten the decay of sodium current. Many dye-blocked channels conduct transiently on exit of the dye molecule after repolarization to the holding potential. In contrast, normally inactivated channels do not conduct during recovery from inactivation. Kinetic evidence shows that inactivation of a dye-blocked channel is unlikely or impossible, which suggests that dye molecules compete with inactivation "particles" for the same site. In the absence of tetrodotoxin, the dyes do not affect the ON gating current unless the interpulse interval is very short. If sufficient equilibration time is allowed during a pulse, the initial amplitude of the OFF gating current is reduced to near zero. This suggests that a dye molecule is a Na channel completely blocks that channel's gating current, even the fraction that is resistant to normal inactivation. Dyes block INa and Ig with the same time course. This provides the strongest evidence to date that virtually all of recorded "gating current" is associated with Na channels. Tetrodotoxin greatly slows dissociation of dye molecules from Na channels and reduced gating current during both opening and closing of the channels.

scholarly journals Charge Movement Associated with the Opening and Closing of the Activation Gates of the Na Channels

1974 ◽  
Vol 63 (5) ◽  
pp. 533-552 ◽  
Author(s):  
Clay M. Armstrong ◽  
Francisco Bezanilla
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Close Association ◽  
Outward Current ◽  
Transient Outward Current ◽  
Activation Process ◽  
Charge Movement ◽  
Gating Current ◽  
Dipolar Molecules ◽  
Opening And Closing

The sodium current (INa) that develops after step depolarization of a voltage clamped squid axon is preceded by a transient outward current that is closely associated with the opening of the activation gates of the Na pores. This "gating current" is best seen when permeant ions (Na and K) are replaced by relatively impermeant ones, and when the linear portion of capacitative current is eliminated by adding current from positive steps to that from exactly equal negative ones. During opening of the Na pores gating current is outward, and as the pores close there is an inward tail of current that decays with approximately the same time-course as INa recorded in Na-containing medium. Both outward and inward gating current are unaffected by tetrodotoxin (TTX). Gating current is capacitative in origin, the result of relatively slow reorientation of charged or dipolar molecules in a suddenly altered membrane field. Close association with the Na activation process is clear from the time-course of gating current, and from the fact that three procedures that reversibly block INa also block gating current: internal perfusion with Zn2+, prolonged depolarization of the membrane, and inactivation of INa with a short positive prepulse.

Is there a second external lidocaine binding site on mammalian cardiac cells?

1989 ◽  
Vol 257 (1) ◽  
pp. H79-H84 ◽  
Author(s):  
L. A. Alpert ◽  
H. A. Fozzard ◽  
D. A. Hanck ◽  
J. C. Makielski
Keyword(s):  
Binding Site ◽  
Cardiac Arrhythmias ◽  
Local Anesthetics ◽  
Sodium Current ◽  
Cardiac Cells ◽  
Na Channel ◽  
Functional Difference ◽  
Na Channels ◽  
Internal Perfusion ◽  
Cardiac Purkinje Cells

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

scholarly journals Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification.

1992 ◽  
Vol 99 (1) ◽  
pp. 1-20 ◽  
Author(s):  
G K Wang ◽  
S Y Wang
Keyword(s):  
Time Course ◽  
Cell Voltage ◽  
Gh3 Cells ◽  
Na Channel ◽  
Na Channels ◽  
H Infinity ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Pharmacological Modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.

scholarly journals Augmentation of Recovery from Inactivation by Site-3 Na Channel Toxins

1999 ◽  
Vol 113 (2) ◽  
pp. 333-346 ◽  
Author(s):  
G. Richard Benzinger ◽  
Gayle S. Tonkovich ◽  
Dorothy A. Hanck
Keyword(s):  
Time Course ◽  
Genetic Diseases ◽  
Low Frequency ◽  
Periodic Paralysis ◽  
Na Channel ◽  
Na Channels ◽  
Cell Recovery ◽  
Reverse Transcription Pcr ◽  
Recovery From Inactivation ◽  
Voltage Dependent

Site-3 toxins isolated from several species of scorpion and sea anemone bind to voltage-gated Na channels and prolong the time course of INa by interfering with inactivation with little or no effect on activation, effects that have similarities to those produced by genetic diseases in skeletal muscle (myotonias and periodic paralysis) and heart (long QT syndrome). Some published reports have also reported the presence of a noninactivating persistent current in site-3 toxin-treated cells. We have used the high affinity site-3 toxin Anthopleurin B to study the kinetics of this current and to evaluate kinetic differences between cardiac (in RT4-B8 cells) and neuronal (in N1E-115 cells) Na channels. By reverse transcription–PCR from N1E-115 cell RNA multiple Na channel transcripts were detected; most often isolated were sequences homologous to rBrII, although at low frequency sequences homologous to rPN1 and rBrIII were also detected. Toxin treatment induced a voltage-dependent plateau current in both isoforms for which the relative amplitude (plateau current/peak current) approached a constant value with depolarization, although the magnitude was much greater for neuronal (17%) than cardiac (5%) INa. Cell-attached patch recordings revealed distinct quantitative differences in open times and burst durations between isoforms, but for both isoforms the plateau current comprised discrete bursts separated by quiescent periods, consistent with toxin induction of an increase in the rate of recovery from inactivation rather than a modal failure of inactivation. In accord with this hypothesis, toxin increased the rate of whole-cell recovery at all tested voltages. Moreover, experimental data support a model whereby recovery at negative voltages is augmented through closed states rather than through the open state. We conclude that site-3 toxins produce qualitatively similar effects in cardiac and neuronal channels and discuss implications for channel kinetics.

scholarly journals Slowing of sodium channel opening kinetics in squid axon by extracellular zinc.

1982 ◽  
Vol 79 (6) ◽  
pp. 935-964 ◽  
Author(s):  
W F Gilly ◽  
C M Armstrong
Keyword(s):  
Rate Constants ◽  
Membrane Surface ◽  
Outward Current ◽  
Forward Rate ◽  
Na Channel ◽  
Na Channels ◽  
Gating Current ◽  
Channel Opening ◽  
The Mean ◽  
Kinetics Of

The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e., the amount of additional depolarization required to overcome the Zn2+ effect. In these terms the mean shifts caused by 30 mM Zn2+ were +29.5 mV for Na channel opening (on) kinetics (t1/2 on), +2 mV for closing (off) kinetics (tau off), and +8.4 mV for the gNa-V curve. Zn2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of or approximately 0.67. Outward current is unaffected. Effects of Zn2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNa. On gating current kinetics are shifted by +27.5 mV, off kinetics by +6 mV, and the Q-V distribution by +6.5 mV. Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants. More than one of the several steps in activation must be affected. The results are not compatible with the usual simple theory of uniform fixed surface charge. They suggest instead that Zn2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation.

scholarly journals Inactivation of cloned Na channels expressed in Xenopus oocytes.

1990 ◽  
Vol 96 (4) ◽  
pp. 689-706 ◽  
Author(s):  
D S Krafte ◽  
A L Goldin ◽  
V J Auld ◽  
R J Dunn ◽  
N Davidson ◽  
...  
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Voltage Dependence ◽  
Single Channel ◽  
Single Amino Acid ◽  
Na Channel ◽  
Low Molecular Weight ◽  
Cdna Clones ◽  
Na Channels ◽  
Single Amino Acid Residue

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.

scholarly journals Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels.

1995 ◽  
Vol 106 (6) ◽  
pp. 1193-1209 ◽  
Author(s):  
H B Nuss ◽  
G F Tomaselli ◽  
E Marbán
Keyword(s):  
Skeletal Muscle ◽  
Local Anesthetics ◽  
Time Course ◽  
Large Fraction ◽  
Expression System ◽  
Na Channel ◽  
Na Channels ◽  
Na Current ◽  
Unexpected Findings ◽  
Cardiac Sodium Channels

When lidocaine is given systemically, cardiac Na channels are blocked preferentially over those in skeletal muscle and nerve. This apparent increased affinity is commonly assumed to arise solely from the fact that cardiac Na channels spend a large fraction of their time in the inactivated state, which exhibits a high affinity for local anesthetics. The oocyte expression system was used to compare systematically the sensitivities of skeletal (mu 1-beta 1) and cardiac (hH1-beta 1) Na channels to block by lidocaine, under conditions in which the only difference was the choice of alpha subunit. To check for differences in tonic block, Na currents were elicited after 3 min of exposure to various lidocaine concentrations at -100 mV, a potential at which both hH1-beta 1 and mu 1-beta 1 channels were fully reprimed. Surprisingly, hH1-beta 1 Na channels were threefold more sensitive to rested-state block by lidocaine (402 +/- 36 microM, n = 4-22) than were mu 1-beta 1 Na channels (1,168 +/- 34 microM, n = 7-19). In contrast, the inactivated state binding affinities determined at partially depolarized holding potentials (h infinity approximately 0.2) were similar (Kd = 16 +/- 1 microM, n = 3-9 for hH1-beta 1 and 12 +/- 2 microM, n = 4-11 for mu 1-beta 1). Lidocaine produced more use-dependent block of peak hH1-beta 1 Na current elicited by trains of short-(10 ms) or long- (1 s) duration step depolarizations (0.5 Hz, -20 mV) than of mu 1-beta 1 Na current. During exposure to lidocaine, hH1-beta 1 channels recover from inactivation at -100 mV after a prolonged delay (20 ms), while mu 1-beta 1 channels begin repriming immediately. The overall time course of recovery from inactivation in the presence of lidocaine is much slower in hH1-beta 1 than in mu 1-beta 1 channels. These unexpected findings suggest that structural differences in the alpha subunits impart intrinsically different lidocaine sensitivities to the two isoforms. The differences in steady state affinities and in repriming kinetics are both in the correct direction to help explain the increased potency of cardiac Na channel block by local anesthetics.

scholarly journals A perspective on Na and K channel inactivation

2017 ◽  
Vol 150 (1) ◽  
pp. 7-18 ◽  
Author(s):  
Clay M. Armstrong ◽  
Stephen Hollingworth
Keyword(s):  
Action Potential ◽  
Time Course ◽  
Salt Water ◽  
Normal Function ◽  
K Channel ◽  
Na Channel ◽  
Fast Time ◽  
Na Channels ◽  
K Channels ◽  
Channel Inactivation

We are wired with conducting cables called axons that rapidly transmit electrical signals (e.g., “Ouch!”) from, for example, the toe to the spinal cord. Because of the high internal resistance of axons (salt water rather than copper), a signal must be reinforced after traveling a short distance. Reinforcement is accomplished by ion channels, Na channels for detecting the signal and reinforcing it by driving it further positive (to near 50 mV) and K channels for then restoring it to the resting level (near −70 mV). The signal is called an action potential and has a duration of roughly a millisecond. The return of membrane voltage (Vm) to the resting level after an action potential is facilitated by “inactivation” of the Na channels: i.e., an internal particle diffuses into the mouth of any open Na channel and temporarily blocks it. Some types of K channels also show inactivation after being open for a time. N-type inactivation of K channels has a relatively fast time course and involves diffusion of the N-terminal of one of the channel’s four identical subunits into the channel’s inner mouth, if it is open. This mechanism is similar to Na channel inactivation. Both Na and K channels also display slower inactivation processes. C inactivation in K channels involves changes in the channel’s outer mouth, the “selectivity filter,” whose normal function is to prevent Na+ ions from entering the K channel. C inactivation deforms the filter so that neither K+ nor Na+ can pass.

scholarly journals Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin.

1995 ◽  
Vol 106 (4) ◽  
pp. 617-640 ◽  
Author(s):  
M F Sheets ◽  
D A Hanck
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Na Channel ◽  
Total Charge ◽  
Na Channels ◽  
Gating Currents ◽  
Gating Charge ◽  
Cardiac Purkinje Cells ◽  
Voltage Dependent ◽  
Cardiac Sodium Channels

The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage-dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O-->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.

Abstract 17570: Rescue of Dilated Cardiomyopathy in Nav1.5 Mutant Mice Defective in Calmodulin Binding by Inhibition of Late Na+ Current

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Hana Cho ◽  
Takeshi Aiba ◽  
Deborah DiSilvestre ◽  
Victoria Halperin ◽  
Eiki Takimoto ◽  
...  
Keyword(s):  
Heart Failure ◽  
Dilated Cardiomyopathy ◽  
Sodium Current ◽  
Ionic Currents ◽  
Na Channel ◽  
Binding Motif ◽  
Na Channels ◽  
Na Current ◽  
Down Regulation ◽  
K Currents

Introduction: Nav1.5, the main voltage-gated Na+ channel in the heart, has been shown to be involved in many cardiac diseases such as long QT syndrome, Brugada syndrome, and heart failure. Na+ channels are importantly regulated by Ca2+ calmodulin (CaM) mediated signaling: however, a fundamental understanding and physiological significance of CaM regulation of Na+ channel is incomplete. Here, we have created a transgenic mouse that harbors a mutated CaM binding motif (NaV1.5-IQ/AA), which is critical for Na+ channel regulation by Ca2+-CaM. Methods: Ventricle mass and function were analyzed with electrocardiogram, echocardiogram, and detailed invasive pressure-volume analysis. Additionally, single ventricular myocytes were obtained. Whole cell patch clamp was used to record membrane ionic currents, including sodium current, Ca2+ current, K+ currents and NCX current. Results: Homozygous mice are embryonic lethal and IQ/AA+/- mice exhibit a dramatic phenotype consisting of dilated cardiomyopathy (DCM) at 4-6 months of age with prolongation of QT. The Na+ current in IQ mice exhibits an enhanced slowly inactivating late component (INa,L) with concomitant up regulation of Na+/Ca2+ exchanger currents. Consistent with other models of DCM and heart failure, DCM in IQ/AA+/- mice was associated with a down regulation of transient outward K+ currents (Ito) and an increase in T-type Ca2+ currents. Chronic treatment with ranolazine designed to block INa,L prevented electrical remodeling of the hearts including an increase in INa,L and a down regulation of Ito. Consistent with the changes in INa,L and Ito, in ranolazine-fed IQ/AA+/- mice, the QT interval was decreased compared to vehicle (p<0.05). Further, the contractile dysfunction, cardiac hypertrophy, and myocardial fibrosis were attenuated in all ranolazine- fed animals, while ventricular dysfunction persisted in animals not fed drug (p<0.05). Conclusions: The data suggest that loss of CaM-mediated regulation of Na+ channel induces dilated cardiomyopathy by enhancing late Na+ current. Taken together, our data demonstrate a dynamic interplay for Ca2+ and Na+ signaling via the CaM binding motif of Na+ channels and highlight the critical importance of late Na+ currents to myopathy and arrhythmia.

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