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 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 Effects of FGF14 and NaVβ4 deletion on transient and resurgent Na current in cerebellar Purkinje neurons

2019 ◽  
Vol 151 (11) ◽  
pp. 1300-1318 ◽  
Author(s):  
Hayley V. White ◽  
Spencer T. Brown ◽  
Thomas C. Bozza ◽  
Indira M. Raman
Keyword(s):  
Purkinje Cell ◽  
Purkinje Cells ◽  
Open Channel ◽  
Na Channel ◽  
Channel Block ◽  
Na Channels ◽  
Na Current ◽  
Open Channel Block ◽  
Resurgent Current ◽  
Α Subunits

Voltage-gated Na channels of Purkinje cells are specialized to maintain high availability during high-frequency repetitive firing. They enter fast-inactivated states relatively slowly and undergo a voltage-dependent open-channel block by an intracellular protein (or proteins) that prevents stable fast inactivation and generates resurgent Na current. These properties depend on the pore-forming α subunits, as well as modulatory subunits within the Na channel complex. The identity of the factors responsible for open-channel block remains a question. Here we investigate the effects of genetic mutation of two Na channel auxiliary subunits highly expressed in Purkinje cells, NaVβ4 and FGF14, on modulating Na channel blocked as well as inactivated states. We find that although both NaVβ4 and the FGF14 splice variant FGF14-1a contain sequences that can generate resurgent-like currents when applied to Na channels in peptide form, deletion of either protein, or both proteins simultaneously, does not eliminate resurgent current in acutely dissociated Purkinje cell bodies. Loss of FGF14 expression does, however, reduce resurgent current amplitude and leads to an acceleration and stabilization of inactivation that is not reversed by application of the site-3 toxin, anemone toxin II (ATX). Tetrodotoxin (TTX) sensitivity is higher for resurgent than transient components of Na current, and loss of FGF14 preferentially affects a highly TTX-sensitive subset of Purkinje α subunits. The data suggest that NaV1.6 channels, which are known to generate the majority of Purkinje cell resurgent current, bind TTX with high affinity and are modulated by FGF14 to facilitate open-channel block.

scholarly journals Time-Dependent Block and Resurgent Tail Currents Induced by Mouse β4154–167 Peptide in Cardiac Na+ Channels

2006 ◽  
Vol 127 (3) ◽  
pp. 277-289 ◽  
Author(s):  
Ging Kuo Wang ◽  
Thomas Edrich ◽  
Sho-Ya Wang
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Time Dependent ◽  
Purkinje Neurons ◽  
Na Channels ◽  
Wild Type ◽  
Na Currents ◽  
Resurgent Currents ◽  
Lysine Substitution

Resurgent tail Na+ currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse β4 subunit, β4154–167, acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons (Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005. Neuron. 45:233–244). To explore these phenotypes in vitro, we characterized β4154–167 actions in inactivation-deficient cardiac hNav1.5 Na+ channels expressed in human embryonic kidney 293t cells. Intracellular β4154–167 from 25–250 μM elicited a conspicuous time-dependent block of inactivation-deficient Na+ currents at 50 mV in a concentration-dependent manner. On and off rates for β4154–167 binding were estimated at 10.1 μM−1s−1 and 49.1 s−1, respectively. Upon repolarization, large tail currents emerged with a slight delay at −140 mV, probably as a result of the rapid unblocking of β4154–167. Near the activation threshold (approximately −70 mV), resurgent tail currents were robust and long lasting. Likewise, β4154–167 induces resurgent currents in wild-type hNav1.5 Na+ channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na+ channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na+ channels opened persistently but became resistant to β4154–167 and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na+ channels resistant to β4154–167. Together, our in vitro studies identify a putative S6-binding site for β4154–167 within the inner cavity of hNav1.5 Na+ channels. Such an S6 receptor readily explains (1) why β4154–167 gains access to its receptor as an open-channel blocker, (2), why bound β4154–167 briefly prevents the activation gate from closing by a “foot-in-the-door” mechanism during deactivation, (3) why BTX inhibits β4154–167 binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates β4154–167 binding.

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.

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.

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 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 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.

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