scholarly journals Relation between veratridine reaction dynamics and macroscopic Na current in single cardiac cells.

1992 ◽  
Vol 99 (5) ◽  
pp. 683-697 ◽  
Author(s):  
X G Zong ◽  
M Dugas ◽  
P Honerjäger
Keyword(s):  
Time Constant ◽  
Maximum Amplitude ◽  
Cardiac Cells ◽  
Maximum Effect ◽  
Na Channels ◽  
Half Maximum ◽  
Na Current ◽  
Current Time ◽  
Tail Current ◽  
In Cells

Veratridine modification of Na current was examined in single dissociated ventricular myocytes from late-fetal rats. Extracellularly applied veratridine reduced peak Na current and induced a noninactivating current during the depolarizing pulse and an inward tail current that decayed exponentially (tau = 226 ms) after repolarization. The effect was quantitated as tail current amplitude, Itail (measured 10 ms after repolarization), relative to the maximum amplitude induced by a combination of 100 microM veratridine and 1 microM BDF 9145 (which removes inactivation) in the same cell. Saturation curves for Itail were predicted on the assumption of reversible veratridine binding to open Na channels during the pulse with reaction rate constants determined previously in the same type of cell at single Na channels comodified with BDF 9145. Experimental relationships between veratridine concentration and Itail confirmed those predicted by showing (a) half-maximum effect near 60 microM veratridine and no saturation up to 300 microM in cells with normally inactivating Na channels, and (b) half-maximum effect near 3.5 microM and saturation at 30 microM in cells treated with BDF 9145. Due to its known suppressive effect on single channel conductance, veratridine induced a progressive, but partial reduction of noninactivating Na current during the 50-ms depolarizations in the presence of BDF 9145, the kinetics of which were consistent with veratridine association kinetics in showing a decrease in time constant from 57 to 22 and 11 ms, when veratridine concentration was raised from 3 to 10 and 30 microM, respectively. As predicted for a dissociation process, the tail current time constant was insensitive to veratridine concentration in the range from 1 to 300 microM. In conclusion, we have shown that macroscopic Na current of a veratridine-treated cardiomyocyte can be quantitatively predicted on the assumption of a direct relationship between veratridine binding dynamics and Na current and as such can be successfully used to analyze molecular properties of the veratridine receptor site at the cardiac Na channel.

scholarly journals Mutually exclusive action of cationic veratridine and cevadine at an intracellular site of the cardiac sodium channel.

1992 ◽  
Vol 99 (5) ◽  
pp. 699-720 ◽  
Author(s):  
P Honerjäger ◽  
M Dugas ◽  
X G Zong
Keyword(s):  
Sodium Channel ◽  
Intracellular Ph ◽  
Time Constant ◽  
Ventricular Myocytes ◽  
Maximum Amplitude ◽  
Fold Increase ◽  
Na Current ◽  
Cardiac Sodium Channel ◽  
Fetal Rats ◽  
Protonated Molecules

Veratridine modification of Na current was examined in single dissociated ventricular myocytes from late-fetal rats by applying pulses to -30 mV for 50 ms every 2 or 5 s from a holding potential of -100 mV (20 degrees C) and measuring amplitude, Itail, and time constant, tau tail, of the post-repolarization inward tail current induced by the alkaloid. Increasing the pH of a 30 microM veratridine superfusate from 7.3 to 8.3 (which increases the fraction of uncharged veratridine molecules from 0.5 to 5% while decreasing that of protonated molecules from 99.5 to 95%) increased Itail by a factor of 2.5 +/- 0.5 (mean +/- SEM; n = 3). Switching from 100 microM veratridine superfusate at pH 7.3 to 10 microM at pH 8.3 did not affect the size of Itail (n = 4). Intracellular (pipette) application of 100 microM veratridine at pH 7.3 or 8.3 produced small Itail's suggesting transmembrane loss of alkaloid. If this was compensated for by simultaneous extracellular application of 100 microM veratridine at a pH identical to intracellular pH, Itail (measured relative to the maximum amplitude induced by a combination of 100 microM veratridine and 1 microM BDF 9145 in the same cell) at pHi 7.3 did not significantly differ from that at pHi 8.3 (84 +/- 4 vs. 70 +/- 6%; n = 3 each). Results from six control cells and five cells subjected to extra- and/or intracellularly increased viscosity by the addition of 0.5 or 1 molal sucrose showed that increasing intracellular viscosity 1.6- and 2.5-fold increased tau tail 1.5- and 2.3-fold, respectively, while a selective 2.5-fold increase of extracellular viscosity did not significantly affect tau tail.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage dependence and activation kinetics of pharmacologically defined components of the high-threshold calcium current in rat neocortical neurons

1993 ◽  
Vol 70 (4) ◽  
pp. 1530-1543 ◽  
Author(s):  
A. M. Brown ◽  
P. C. Schwindt ◽  
W. E. Crill
Keyword(s):  
Time Constant ◽  
Calcium Current ◽  
Voltage Dependence ◽  
Current Amplitude ◽  
Activation Kinetics ◽  
Maximum Slope ◽  
Current Time ◽  
Time Constants ◽  
Tail Current ◽  
Boltzmann Function

1. As a first step toward identification of the functional significance of the spatial distribution of calcium channels we examined the high voltage-activated calcium current (HVA current) in acutely isolated pyramidal neurons from rat sensorimotor cortex using whole-cell voltage clamp. The goals of this study were (1) to determine whether the pharmacologically separable components of the HVA current differed in voltage dependence or activation kinetics and (2) to develop an empirical model that could predict the HVA current evoked by action potentials or other physiological responses. 2. Cells with short dendrites were chosen for study. Input resistance averaged 3.5 +/- 0.4 (SE) G omega. Specific membrane resistance averaged 51.9 +/- 6.8 K omega-cm2 on the basis of estimated membrane surface area. 3. Using 2 mM calcium in the extracellular solution, we evoked the HVA current by depolarizations positive to -45 mV from a holding potential of -60 mV, a potential where the low-threshold calcium current is fully inactivated. Maximum HVA current amplitude (484.9 +/- 42.3 pA) occurred near -15 mV. The evoked current was completely and reversibly blocked by 200 microM cadmium. 4. Tail current amplitude at a fixed potential increased as a sigmoidal function of prepulse potential. A plot of normalized tail current amplitude, taken as the fraction of HVA channels open at each prepulse potential, was best described by a Boltzmann function (maximum slope: e-fold per 11.3 mV; half activation: -24.6 mV) raised to the power of 2. This relation was not altered by extracellular application of 5 microM nifedipine or 10 microM omega-conotoxin, each of which reduced a separate component of the HVA current uniformly at all potentials. We conclude that the pharmacologically separable components of the HVA current do not differ significantly in voltage dependence. 5. The time course of current onset during a step depolarization was best described by second-order activation kinetics. Activation time constants ranged from a maximum of 1.2 ms at -40 mV to 0.3 ms at +25 mV. Neither activation nor tail current time constants were altered by extracellular application of 5 microM nifedipine or 10 microM omega-conotoxin. After application of 1 microM Bay K 8644 tail current decay was best described by a fast time constant similar to control values and a slow time constant. We conclude that the pharmacologically separable components of the HVA current in these neurons do not differ significantly in kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)

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 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 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.

Participation of Na+ channels in the response of carotid body chemoreceptor cells to hypoxia

1994 ◽  
Vol 267 (3) ◽  
pp. C738-C744 ◽  
Author(s):  
A. Rocher ◽  
A. Obeso ◽  
M. T. Cachero ◽  
B. Herreros ◽  
C. Gonzalez
Keyword(s):  
Carotid Body ◽  
Neurotransmitter Release ◽  
Marked Reduction ◽  
Adult Rabbit ◽  
Na Channels ◽  
Na Current ◽  
Chemoreceptor Cells ◽  
Cell Depolarization ◽  
First Time ◽  
Mild Hypoxia

The role played by Na+ channels of carotid body (CB) chemoreceptor cells was investigated by studying the effects of tetrodotoxin (TTX) on the release of 3H-labeled catecholamines ([3H]CA) by adult rabbit CBs previously incubated with the precursor [3H]tyrosine. TTX inhibited partially the release of [3H]CA elicited by mild hypoxia (10 or 7% O2) or by depolarizing incubation medium containing 20 or 30 mM KCl, but the response to more intense hypoxia (5 or 2% O2) or to higher KCl concentration (40 or 50 mM) was not significantly affected. The release of [3H]CA elicited by acidic stimuli, either 20% CO2 (pH 6.6) or the protonophore dinitrophenol (100 microM), although comparable in magnitude to that elicited by mild hypoxia, was not modified by TTX. These results provide evidence for the first time that Na+ channels of chemoreceptor cells participate in the transduction of hypoxic stimuli into the neurotransmitter release response of these cells and suggest that Na+ current operates as an amplifying device that enhances the initial cell depolarization mediated by the closure of the O2-sensitive K+ channels. Sympathetic denervation of CBs was followed by a marked reduction in the release of [3H]CA elicited by veratridine or by 20 mM KCl, suggesting that the number of Na+ channels in chemoreceptor cells decreases after denervation.

Long-lasting reduction of excitability by a sodium-dependent potassium current in cat neocortical neurons

1989 ◽  
Vol 61 (2) ◽  
pp. 233-244 ◽  
Author(s):  
P. C. Schwindt ◽  
W. J. Spain ◽  
W. E. Crill
Keyword(s):  
Voltage Clamp ◽  
Time Course ◽  
Outward Current ◽  
Repetitive Firing ◽  
K Channel ◽  
Channel Blockers ◽  
Dantrolene Sodium ◽  
Current Time ◽  
Tail Current ◽  
Neocortical Neurons

1. The function and ionic mechanism of a slow outward current were studied in large layer V neurons of cat sensorimotor cortex using an in vitro slice preparation and single microelectrode voltage clamp. 2. With Ca2+ influx blocked, a slow relaxation ("tail") of outward current followed either (1) repetitive firing evoked for 1 s or (2) a small 1-s depolarizing voltage clamp step that activated the persistent Na+ current of neocortical neurons, INaP. When a depolarization that activated INaP was maintained, an outward current gradually developed and increased in amplitude over a period of tens of seconds to several minutes. An outward tail current of similar duration followed repolarization. The slow outward current was abolished by TTX, indicating it depended on Na+ influx. 3. With Ca2+ influx blocked, the onset of the slow Na+-dependent outward current caused spike frequency adaptation during current-evoked repetitive firing. Following the firing, the decay of the Na+-dependent current caused a slow afterhyperpolarization (sAHP) and a long-lasting reduction of excitability. It also was responsible for habituation of the response to repeated identical current pulses. 4. The Na+-dependent tail current had properties expected of a K+ current. Membrane chord conductance increased during the tail, and tail amplitude was reduced or reversed by membrane potential hyperpolarization and raised extracellular K+ concentration [( K+]0). 5. The current tail was reduced reversibly by the K+ channel blockers TEA (5-10 mM), muscarine (5-20 microM), and norepinephrine (100 microM). These agents also resulted in a larger, more sustained inward current during the preceding step depolarization. Comparison of current time course before and after the application of blocking agents suggested that, in spite of its capability for slow buildup and decay, the onset of the Na+-dependent outward current occurs within 100 ms of an adequate step depolarization. 6. With Ca2+ influx blocked, extracellular application of dantrolene sodium (30 microM) had no clear effect on the current tail or the corresponding sAHP.(ABSTRACT TRUNCATED AT 400 WORDS)

Do neurons from rat neostriatum express both a TTX-sensitive and a TTX-insensitive slow Na+ current?

1995 ◽  
Vol 74 (3) ◽  
pp. 934-941 ◽  
Author(s):  
T. I. Chao ◽  
C. Alzheimer
Keyword(s):  
Slow Inward Current ◽  
Medium Spiny Neurons ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Internal Solution ◽  
Voltage Range ◽  
Spiny Neurons ◽  
Current Voltage ◽  
Relationship Of ◽  
In Cells

1. The properties of a tetrodotoxin (TTX)-sensitive, persistent Na+ current and a purported TTX-insensitive slow Na+ current were studied in acutely isolated neurons from rat neostriatum with the use of the whole cell configuration of the patch-clamp technique. 2. A TTX-sensitive, persistent Na+ current (INaP) was activated positive to -60 mV and reached a peak amplitude of -40 to -120 pA at about -40 mV. As indicated by slow depolarizing voltage ramps, activation of INaP did not require preceding activation of the fast, rapidly inactivating Na+ current. 3. The current-voltage (I-V) relationship of INaP displayed an unexpected inflection after passing through its peak value near -40 mV. Between -40 and -10 mV, INaP declined more rapidly with depolarization than it did at more depolarized potentials. The corresponding conductance (GNaP) peaked at -40 mV and declined to a smaller limiting value at potentials positive to about -10 mV. 4. This behavior is not consistent with the notion that INaP arises solely from a bell-shaped window conductance that results from the overlapping steady-state activation and inactivation curves of the fast Na+ current in a narrow voltage range, nor with the notion that INaP is generated by a single uniform conductance independent of the fast Na+ current. 5. In addition to INaP, a second slow inward current (IS) was evoked when small monovalent cations were omitted from the internal solution. INaP and IS were present both in cells resembling medium spiny neurons and in cells resembling aspiny interneurons. 6. IS was insensitive to TTX (1.2 microM) and the Ca2+ channel blocker, cadmium.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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