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

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.

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

The Journal of General Physiology ◽

10.1085/jgp.201912390 ◽

2019 ◽

Vol 151 (11) ◽

pp. 1300-1318 ◽

Cited By ~ 7

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.

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

The Journal of General Physiology ◽

10.1085/jgp.200308857 ◽

2003 ◽

Vol 122 (3) ◽

pp. 365-374 ◽

Cited By ~ 34

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.

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Cross-Species Conservation of Open-Channel Block by Na Channel 4 Peptides Reveals Structural Features Required for Resurgent Na Current

Journal of Neuroscience ◽

10.1523/jneurosci.1428-11.2011 ◽

2011 ◽

Vol 31 (32) ◽

pp. 11527-11536 ◽

Cited By ~ 20

Author(s):

A. H. Lewis ◽

I. M. Raman

Keyword(s):

Open Channel ◽

Species Conservation ◽

Structural Features ◽

Na Channel ◽

Channel Block ◽

Na Current ◽

Open Channel Block

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Block of Neuronal Na+Channels by Antidepressant Duloxetine in a State-dependent Manner

Anesthesiology ◽

10.1097/aln.0b013e3181e89a93 ◽

2010 ◽

Vol 113 (3) ◽

pp. 655-665 ◽

Cited By ~ 17

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.

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Bimodal Action of Protons on ATP Currents of Rat PC12 Cells

The Journal of General Physiology ◽

10.1085/jgp.200308825 ◽

2003 ◽

Vol 122 (1) ◽

pp. 33-44 ◽

Cited By ~ 9

Author(s):

Andrei Skorinkin ◽

Andrea Nistri ◽

Rashid Giniatullin

Keyword(s):

Pc12 Cells ◽

Open Channel ◽

Secretory Vesicles ◽

Channel Block ◽

Receptor Model ◽

High Concentration ◽

Open Channel Block ◽

Central Neurons ◽

Dual Action ◽

Open States

The mode of action of extracellular protons on ATP-gated P2X2 receptors remains controversial as either enhancement or depression of ATP-mediated currents has been reported. By investigating, at different pH, the electrophysiological effect of ATP on P2X2 receptors and complementing it with receptor modelling, the present study suggests a unified mechanism for both potentiation and inactivation of ATP receptors by protons. Our experiments on patch-clamped PC12 cells showed that, on the same cell, mild acidification potentiated currents induced by low ATP concentrations (<0.1 mM) and attenuated responses to high ATP concentrations (>1 mM) with emergence of current fading and rebound. To clarify the nature of the ATP/H+ interaction, we used the Ding and Sachs's “loop” receptor model which best describes the behavior of such receptors with two open states linked via one inactivated state. No effects by protons could be ascribed to H+-mediated open channel block. However, by assuming that protons facilitated binding of ATP to resting as well as open receptors, the model could closely replicate H+-induced potentiation of currents evoked by low ATP doses plus fading and rebound induced by high ATP doses. The latter phenomenon was due to receptor transition to the inactive state. The present data suggest that the high concentration of protons released with ATP (and catecholamines) from secretory vesicles may allow a dual action of H+ on P2X2 receptors. This condition might also occur on P2X2 receptors of central neurons exposed to low pH during ischemia.

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Open-channel block of Na+ channels by intracellular Mg2+

European Biophysics Journal ◽

10.1007/bf00196922 ◽

1990 ◽

Vol 18 (6) ◽

Cited By ~ 9

Author(s):

M. Pusch

Keyword(s):

Open Channel ◽

Channel Block ◽

Na Channels ◽

Open Channel Block ◽

Block Of Na Channels

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Flecainide sensitivity of a Na channel long QT mutation shows an open-channel blocking mechanism for use-dependent block

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01317.2005 ◽

2006 ◽

Vol 291 (1) ◽

pp. H29-H37 ◽

Cited By ~ 10

Author(s):

Yujie Zhu ◽

John W. Kyle ◽

Peter J. Lee

Keyword(s):

Steady State ◽

Open Channel ◽

Na Channel ◽

Channel Block ◽

Long Qt ◽

Open Channel Block ◽

Cardiac Sodium Channel ◽

Channel Blocking ◽

A Minor ◽

Blocking Mechanism

A long QT mutation in the cardiac sodium channel, D1790G (DG), shows enhanced flecainide use-dependent block (UDB). The relative importance of open and inactivated states of the channel in flecainide UDB has been controversial. We used a modifiable, inactivation-deficient mutant channel that contains the F1486C mutation in the IFM motif to investigate the UDB difference between the wild-type (WT-ICM) and DG (DG-ICM) channels. UDB at 5 Hz was greater in DG-ICM than WT-ICM, and IC50 values for steady-state UDB were 7.19 and 18.06 μM, respectively. When [2-(trimethyammonium) ethyl]methanethiosulfonate bromide (MTSET) was included in the pipette and fast inactivation was disabled, IC50 was 5.04 μM for DG-ICM and 12.63 μM for WT-ICM. We measured open-channel block by flecainide directly in MTSET-treated, noninactivating ICM channels. Steady-state block was higher for DG-ICM than WT-ICM (IC50 was 2.34 μM for DG-ICM and 5.87 μM for WT-ICM), suggesting that open-channel block is an important determinant of flecainide UDB. We obtained association ( kon) and dissociation ( koff) rates for open-channel block by the Langmuir-isotherm model. They were koff = 31.37 s−1, kon = 5.83 s−1·μM−1, and calculated Kd = 5.38 μM for WT-ICM (where Kd = koff/ kon); and koff = 24.88 s−1, kon = 9.54 s−1·μM−1, and calculated Kd = 2.61 μM for DG-ICM. These Kd values were similar to IC50 measured from steady-state open-channel block. Furthermore, we modeled UDB mathematically by using these kinetic rates and found that the model predicted experimental UDB accurately. The recovery from UDB had a minor contribution to UDB. Flecainide UDB is predominantly determined by an open-channel blocking mechanism, and DG-ICM channels appeared to have an altered open-channel state with higher flecainide affinity than WT-ICM.

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Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells

The Journal of General Physiology ◽

10.1085/jgp.200409128 ◽

2004 ◽

Vol 124 (6) ◽

pp. 691-701 ◽

Cited By ~ 36

Author(s):

Sho-Ya Wang ◽

Jane Mitchell ◽

Edward Moczydlowski ◽

Ging Kuo Wang

Keyword(s):

Local Anesthetics ◽

Open Channel ◽

Time Dependent ◽

Hek293 Cells ◽

Alternative View ◽

Channel Block ◽

Na Channels ◽

Open Channel Block ◽

Channel Activation ◽

High Affinity

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+ channels with higher affinities. However, an alternative view suggests that activation of Na+ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50 values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na+ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na+ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.

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