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

2006 ◽  
Vol 291 (1) ◽  
pp. H29-H37 ◽  
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.

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.

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 Identification of the Ca2+ Blocking Site of Acid-sensing Ion Channel (ASIC) 1

2004 ◽  
Vol 124 (4) ◽  
pp. 383-394 ◽  
Author(s):  
Martin Paukert ◽  
Elena Babini ◽  
Michael Pusch ◽  
Stefan Gründer
Keyword(s):  
Ion Channels ◽  
Open Channel ◽  
Transmembrane Domain ◽  
Na Channel ◽  
Channel Block ◽  
Open Channel Block ◽  
Apparent Affinity ◽  
Acid Sensing Ion Channels ◽  
Ion Pore ◽  
Epithelial Na Channel

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 ∼ 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.

Open-channel-block induced by the neurotransmitter glycine in homomeric alpha1-GlyR and heteromeric alpha1/beta1- GlyR

2008 ◽  
Vol 35 (S 01) ◽  
Author(s):  
Y.P Song ◽  
F Schlesinger ◽  
S Petri ◽  
R Dengler ◽  
K Krampfl
Keyword(s):  
Open Channel ◽  
Channel Block ◽  
Open Channel Block

GABAA receptor activation and open-channel block by volatile anaesthetics: a new principle of receptor modulation?

2002 ◽  
Vol 451 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Rainer Haseneder ◽  
Gerhard Rammes ◽  
Walter Zieglgänsberger ◽  
Eberhard Kochs ◽  
Gerhard Hapfelmeier
Keyword(s):  
Gabaa Receptor ◽  
Open Channel ◽  
Receptor Activation ◽  
Channel Block ◽  
Volatile Anaesthetics ◽  
Open Channel Block ◽  
Receptor Modulation

scholarly journals Membrane Lipid Modulations Remove Divalent Open Channel Block from TRP-Like and NMDA Channels

2009 ◽  
Vol 29 (8) ◽  
pp. 2371-2383 ◽  
Author(s):  
M. Parnas ◽  
B. Katz ◽  
S. Lev ◽  
V. Tzarfaty ◽  
D. Dadon ◽  
...  
Keyword(s):  
Open Channel ◽  
Membrane Lipid ◽  
Channel Block ◽  
Open Channel Block ◽  
Nmda Channels
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