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 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 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.

The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Nav1.6 voltage-dependent sodium channel

2007 ◽  
Vol 293 (2) ◽  
pp. C783-C789 ◽  
Author(s):  
Christian Rosker ◽  
Birgit Lohberger ◽  
Doris Hofer ◽  
Bibiane Steinecker ◽  
Stefan Quasthoff ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channels ◽  
Therapeutic Intervention ◽  
Time Course ◽  
Specific Interaction ◽  
Xenopus Laevis Oocytes ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Invaluable Tool

The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Nav1.8). Generally, TTX was 40–231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Nav1.6, where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC50 values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 ± 1.3/1,260 ± 121 (Nav1.2), 2.8 ± 2.3/341 ± 36 (Nav1.3), 4.5 ± 1.0/988 ± 62 (Nav1.4), 1,970 ± 565/78,500 ± 11,600 (Nav1.5), 3.8 ± 1.5/7.8 ± 2.3 (Nav1.6), 5.5 ± 1.4/1,270 ± 251 (Nav1.7), and 1,330 ± 459/>30,000 (Nav1.8). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Nav1.6 subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Nav1.5 subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Nav1.1 to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Nav1.6-mediated function, but also for therapeutic intervention.

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.

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 The Human Heart and Rat Brain IIA Na+ Channels Interact with Different Molecular Regions of the β1 Subunit

2002 ◽  
Vol 120 (6) ◽  
pp. 887-895 ◽  
Author(s):  
Thomas Zimmer ◽  
Klaus Benndorf
Keyword(s):  
Rat Brain ◽  
Human Heart ◽  
Xenopus Oocytes ◽  
Extracellular Domain ◽  
Na Channel ◽  
Na Channels ◽  
Membrane Anchor ◽  
Α Subunit ◽  
Extracellular Region ◽  
Recovery From Inactivation

The α subunit of voltage-gated Na+ channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory β1, but not the β2 subunit. In the present study, we used β1/β2 chimeras to identify molecular regions within the β1 subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na+ channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the β1/β2 chimeras with rat brain IIA channels. In agreement with previous studies, the β1 extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the β1 subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the β1 subunit was required. An exchange of the β1 membrane anchor by the corresponding β2 subunit region almost completely abolished the effects of the β1 subunit on hH1, suggesting that the β1 membrane anchor plays a crucial role for the modulation of the cardiac Na+ channel isoform. It is concluded that the β1 subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.

scholarly journals Voltage-dependent gating of veratridine-modified Na channels.

1986 ◽  
Vol 87 (1) ◽  
pp. 25-46 ◽  
Author(s):  
M D Leibowitz ◽  
J B Sutro ◽  
B Hille
Keyword(s):  
Time Course ◽  
Drug Binding ◽  
Na Channels ◽  
Current Voltage ◽  
Instantaneous Current ◽  
Activation Gating ◽  
Voltage Dependent ◽  
Ca Ions ◽  
Rate Of Recovery ◽  
Gating Process

Na channels of frog muscle fibers treated with 100 microM veratridine became transiently modified after a train of repetitive depolarizations. They open and close reversibly with a gating process whose midpoint lies 93 mV more negative than the midpoint of normal activation gating and whose time course shows no appreciable delay in the opening or closing kinetics but still requires more than two kinetic states. Like normal activation, the voltage dependence of the modified gating can be shifted by changing the bathing Ca2+ concentration. The instantaneous current-voltage relation of veratridine-modified channels is curved at potentials negative to -90 mV, as if external Ca ions produced a voltage-dependent block but also permeated. Modified channels probably carry less current than normal ones. When the concentration of veratridine is varied between 5 and 100 microM, the initial rate of modification during a pulse train is directly proportional to the concentration, while the rate of recovery from modification after the train is unaffected. These are the properties expected if drug binding and modification of channels can be equated. Hyperpolarizations that close modified channels slow unbinding. Allethrin and DDT also modify channels. They bind and unbind far faster than veratridine does, and their binding requires open channels.

Slow inward Ca current in frog heart: theoretical evidence against a voltage-dependent inactivation

1982 ◽  
Vol 60 (9) ◽  
pp. 1185-1192 ◽  
Author(s):  
Rodolphe Fischmeister ◽  
Magda Horackova
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Frog Heart ◽  
Type Model ◽  
Actual Behavior ◽  
Ca Current ◽  
Recovery From Inactivation ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Theoretical Evidence

The validity of a Hodgkin–Huxley type voltage-dependent inactivation of slow inward Ca current (Isi) was tested in frog heart using a computer simulation. The time course of Isi, was calculated during the development of a frog atrial action potential (AP). With a time constant of inactivation (τf) of 55 ms at a membrane potential (Em) of –15 mV, the variation of Isi was biphasic; after a transient increase followed by a decrease to zero, Isi partially "reactivated" (at the beginning of the AP repolarization phase) and then fully deactivated. The "reactivation" phase of Isi developed whether τf was an increasing, decreasing, U-shaped, or bell-shaped function of Em. The addition of an independent and slower process responsible for the recovery from inactivation only partly suppressed the "reactivation" phase. However, until now there was no experimental evidence supporting such a biphasic variation of Isi during AP repolarization. Thus our results indicate that the Hodgkin–Huxley type model of the voltage-dependence of Isi-inactivation process may not correctly represent the actual behavior of frog cardiac muscle.

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart

1995 ◽  
Vol 269 (1) ◽  
pp. H203-H214 ◽  
Author(s):  
A. I. Undrovinas ◽  
G. S. Shander ◽  
J. C. Makielski
Keyword(s):  
Membrane Proteins ◽  
Actin Polymerization ◽  
Integral Membrane Proteins ◽  
Na Channel ◽  
Na Channels ◽  
Open Probability ◽  
Whole Cell ◽  
Voltage Dependent ◽  
D 20 ◽  
Inside Out

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

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