Symmetry and asymmetry of permeation through toxin-modified Na+ channels.

Single Na+ channels from rat skeletal muscle were inserted into planar lipid bilayers in the presence of either 200 nM batrachotoxin (BTX) or 50 microM veratridine (VT). These toxins, in addition to their ability to shift inactivation of voltage-gated Na+ channels, may be used as probes of ion conduction in these channels. Channels modified by either of the toxins have qualitatively similar selectivity for the alkali cations (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). Biionic reversal potentials, for example, were concentration independent for all ions studied. Na+/K+ and Na+/Rb+ reversal potentials, however, were dependent on the orientation of the ionic species with respect to the intra- or extracellular face of the channel, whereas Na+/Li+ biionic reversal potentials were not orientation dependent. A simple, four-barrier, three-well, single-ion occupancy model was used to generate current-voltage relationships similar to those observed in symmetrical solutions of Na, K, or Li ions. The barrier profiles for Na and Li ions were symmetric, whereas that for K ions was asymmetric. This suggests the barrier to ion permeation for K ions may be different than that for Na and Li ions. With this model, these hypothetical energy barrier profiles could predict the orientation-dependent reversal potentials observed for Na+/K+ and Na+/Rb+. The energy barrier profiles, however, were not capable of describing biionic Na/Li ion permeation. Together these results support the hypothesis that Na ions have a different rate determining step for ion permeation than that of K and Rb ions.     

Na+ block and permeation in a K+ channel of known structure

The effects of intracellular Na(+) were studied on K(+) and Rb(+) currents through single KcsA channels. At low voltage, Na(+) produces voltage-dependent block, which becomes relieved at high voltage by a "punchthrough" mechanism representing Na(+) escaping from its blocking site through the selectivity filter. The Na(+) blocking site is located in the wide, hydrated vestibule, and it displays unexpected selectivity for K(+) and Rb(+) against Na(+). The voltage dependence of Na(+) block reflects coordinated movements of the blocker with permeant ions in the selectivity filter.     

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

1. Macroscopic and single-channel currents through several types of cloned rat brain Na⁺ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na⁺ channels in chromaffin cells, intracellular Mg²⁺ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na⁺ channel (Pusch et al. 1989). 3. A sodium-channel mutant (cZ-2) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg²⁺. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg²⁺ and a voltage-dependence of e-fold per 39 mV (in 125 mM [Na] _i ); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg²⁺ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg] _i . This indicates that Mg²⁺ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 10⁸ M^–1 s^–1 and 3.6 · 10⁵ s^–1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg²⁺ strongly depends on [Na] _i . This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg²⁺ block at physiological levels of [Mg] _i .abbreviations [Na]_i intracellular Na⁺ concentration - [K] _i intracellular K⁺ concentration - [Mg] _i intracellular Mg²⁺ concentration - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis-[\-amino-ethyl ether] N,N-tetra acetic acid - TEA tetraethylammonium     

The N-linker region of hERG1a upregulates hERG1b potassium channels

A major physiological role of hERG1 (human Ether-á-go-go-Related Gene 1) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the potassium current I_Kr in cardiomyocytes. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, long QT syndrome, and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood. Here, we report that the hERG1a N-terminal region expressed in trans with hERG1b markedly increased hERG1b currents and increased biotin-labeled hERG1b protein at the membrane surface. hERG1b channels with a deletion of the N-terminal 1b domain did not have a measurable increase in current or biotinylated protein when coexpressed with hERG1a N-terminal regions, indicating that the 1b domain was required for the increase in hERG1b. Using a biochemical pull-down interaction assay and a FRET hybridization experiment, we detected a direct interaction between the hERG1a N-terminal region and the hERG1b N-terminal region. Using engineered deletions and alanine mutagenesis, we identified a short span of amino acids at positions 216 to 220 within the hERG1a “N-linker” region that were necessary for the upregulation of hERG1b. We propose that direct structural interactions between the hERG1a N-linker region and the hERG1b 1b domain increase hERG1b at the plasma membrane. Mechanisms regulating hERG1a and hERG1b are likely critical for cardiac function, may be disrupted by long QT syndrome mutants, and serve as potential targets for therapeutics.     

Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.     

State-dependent block of wild-type and inactivation-deficient Na+ channels by flecainide

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/DeltaKPQ 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 beta1 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 micro M when the cell was not stimulated for 1,000 s. At 100 micro M flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an approximately 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 micro M, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 micro M-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 approximately 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.     

Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to - 80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.     

Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon

Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.     

Proton block of the pore underlies the inhibition of hERG cardiac K+ channels during acidosis

Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.     

Enhanced inhibitory effect of acidosis on hERG potassium channels that incorporate the hERG1b isoform

Extracellular acidosis occurs in the heart during myocardial ischemia and can lead to dangerous arrhythmias. Potassium channels encoded by hERG (human ether-à-go-go-related gene) mediate the cardiac rapid delayed rectifier K⁺ current (I_Kr), and impaired hERG function can exacerbate arrhythmia risk. Nearly all electrophysiological investigations of hERG have centred on the hERG1a isoform, although native I_Kr channels may be comprised of hERG1a and hERG1b, which has a unique shorter N-terminus. This study has characterised for the first time the effects of extracellular acidosis (an extracellular pH decrease from 7.4 to 6.3) on hERG channels incorporating the hERG1b isoform. Acidosis inhibited hERG1b current amplitude to a significantly greater extent than that of hERG1a, with intermediate effects on coexpressed hERG1a/1b. I_hERG tail deactivation was accelerated by acidosis for both isoforms. hERG1a/1b activation was positively voltage-shifted by acidosis, and the fully-activated current–voltage relation was reduced in amplitude and right-shifted (by ∼10 mV). Peak I_hERG1a/1b during both ventricular and atrial action potentials was both suppressed and positively voltage-shifted by acidosis. Differential expression of hERG isoforms may contribute to regional differences in I_Kr in the heart. Therefore inhibitory effects of acidosis on I_Kr could also differ regionally, depending on the relative expression levels of hERG1a and 1b, thereby increasing dispersion of repolarization and arrhythmia risk.     

Relief of Na+ block of Ca2+-activated K+ channels by external cations

The flickery block of single Ca2+-activated K+ channels that is produced by internally applied Na+ can be relieved by millimolar concentrations of external K+. This effect of K+ on the kinetics of Na+ block was studied by the method of amplitude distribution analysis described in the companion paper (Yellen, G., 1984b, J. Gen. Physiol., 84:157-186). It appears that K+ relieves block by increasing the exit rate of the blocking ion from the channel, not by competitively slowing its entrance rate. This suggests that a K ion that enters the channel from the outside can expel the blocking Na ion, which entered the channel from the inside. Cs+, which cannot carry current through the channel, and Rb+, which carries a reduced current through the channel, are just as effective as K+ in relieving the block by internal Na+. The kinetics of block by internal nonyltriethylammonium (C9) are unaffected by the presence of these ions in the external bathing solution.     

Ion permeation and block of M-type and delayed rectifier potassium channels. Whole-cell recordings from bullfrog sympathetic neurons

Ion permeation and conduction were studied using whole-cell recordings of the M-current (I(M)) and delayed rectifier (IDR), two K+ currents that differ greatly in kinetics and modulation. Currents were recorded from isolated bullfrog sympathetic neurons with 88 mM [K+]i and various external cations. Selectivity for extracellular monovalent cations was assessed from permeability ratios calculated from reversal potentials and from chord conductances for inward current. PRb/PK was near 1.0 for both channels, and GRb/GK was 0.87 +/- 0.01 for IDR but only 0.35 +/- 0.01 for I(M) (15 mM [Rb+]o or [K+]o). The permeability sequences were generally similar for I(M) and IDR: K+ approximately Rb+ > NH4+ > Cs+, with no measurable permeability to Li+ or CH3NH3+. However, Na+ carried detectable inward current for IDR but not I(M). Nao+ also blocked inward K+ current for IDR (but not IM), at an apparent electrical distance (delta) approximately 0.4, with extrapolated dissociation constant (KD) approximately 1 M at 0 mV. Much of the instantaneous rectification of IDR in physiologic ionic conditions resulted from block by Nao+. Extracellular Cs+ carried detectable inward current for both channel types, and blocked I(M) with higher affinity (KD = 97 mM at 0 mV for I(M), KD) approximately 0.2 M at 0 mV for IDR), with delta approximately 0.9 for both. IDR showed several characteristics reflecting a multi-ion pore, including a small anomalous mole fraction effect for PRb/PK, concentration-dependent GRb/GK, and concentration- dependent apparent KD's and delta's for block by Nao+ and Cso+. I(M) showed no clear evidence of multi-ion pore behavior. For I(M), a two- barrier one-site model could describe permeation of K+ and Rb+ and block by Cso+, whereas for IDR even a three-barrier, two-site model was not fully adequate.     

Drug block of the hERG potassium channel: insight from modeling

Many commonly used, structurally diverse, drugs block the human ether-a-go-go-related gene (hERG) K(+) channel to cause acquired long QT syndrome, which can lead to sudden death via lethal cardiac arrhythmias. This undesirable side effect is a major hurdle in the development of safe drugs. To gain insight about the structure of hERG and the nature of drug block we have produced structural models of the channel pore domain, into each of which we have docked a set of 20 hERG blockers. In the absence of an experimentally determined three-dimensional structure of hERG, each of the models was validated against site-directed mutagenesis data. First, hERG models were produced of the open and closed channel states, based on homology with the prokaryotic K(+) channel crystal structures. The modeled complexes were in partial agreement with the mutagenesis data. To improve agreement with mutagenesis data, a KcsA-based model was refined by rotating the four copies of the S6 transmembrane helix half a residue position toward the C-terminus, so as to place all residues known to be involved in drug binding in positions lining the central cavity. This model produces complexes that are consistent with mutagenesis data for smaller, but not larger, ligands. Larger ligands could be accommodated following refinement of this model by enlarging the cavity using the inherent flexibility about the glycine hinge (Gly648) in S6, to produce results consistent with the experimental data for the majority of ligands tested.     

Actin modifies Ca2+ block of epithelial Na+ channels in planar lipid bilayers

The mechanism by which the cytoskeletal protein actin affects the conductance of amiloride-sensitive epithelial sodium channels (ENaC) was studied in planar lipid bilayers. In the presence of monomeric actin, we found a decrease in the single-channel conductance of alpha-ENaC that did not occur when the internal [Ca2+]free was buffered to <10 nM. An analysis of single-channel kinetics demonstrated that Ca2+ induced the appearance of long-lived closed intervals separating bursts of channel activity, both in the presence and in the absence of actin. In the absence of actin, the duration of these bursts and the time spent by the channel in its open, but not in its short-lived closed state, were inversely proportional to [Ca2+]. This, together with a lengthening of the interburst intervals, translated into a dose-dependent decrease in the single-channel open probability. In contrast, a [Ca2+]-dependent decrease in alpha-ENaC conductance in the presence of actin was accompanied by lengthening of the burst intervals with no significant changes in the open or closed (both short- and long-lived) times. We conclude that Ca2+ acts as a "fast-to-intermediate" blocker when monomeric actin is present, producing a subsequent attenuation of the apparent unitary conductance of the channel.     

Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Extracellular polyvalent cation block of slow Na+ channels in Xenopus laevis oocytes

Sustained depolarization of the Xenopus oocyte membrane elicits a slowly activating Na+ current, thought to be due to the opening of sodium selective channels. These channels are induced to become voltage gated by the depolarization. They show unconventional gating properties and are insensitive to tetrodotoxin (TTX). The present study was undertaken to evaluate the effect of extracellular multivalent cations (Ca2+, Co2+, Cd2+, La3+, Mg2+, Mn2+, Ni2+, Sr2+ and Zn2+) on these TTX-resistant channels, either on membrane potential responses or on current responses. Our data show that all the polyvalent cations used blocked Na+ channels in a concentration-dependent manner. The order of potency of the most efficient cations was Co2+ < Ni2+ < Cd2+ < Zn2+, the respective concentration required to cause a 50% decrease of Na+ current was 0.9+/-0.29; 0.47+/-0.15; 0.36+/-0.09 and 0.06+/-0.02 mmol/l. The comparison of the activation curves from controls and after treatment with the cations indicated a shift towards more positive voltages. These results can be interpreted as due to the screening effect of divalent cations together with an alteration of the Na+ channel gating properties. We also show that divalent cations blocked Na+ channels in an open state without interfering with the induction mechanism of the channels. The possibility that cation block was due to a possible interaction between cations and SH-groups was investigated, but a sulphydryl alkylating reagent failed to abolish Zn2+ block.     

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

A voltage-gated, small, persistent Na⁺ current (I_Na) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent I_Na that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on I_Na in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the -subunit (hH1) of human cardiac Na⁺ channels (hNav1.5) plus ₁-subunits. Extracellular application of 5 µM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited I_Na. The late portion of I_Na (I_{Na late}, measured near the end of each pulse) was almost completely suppressed. I_Na returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on I_Na was concentration dependent, with IC₅₀ values of 4.0 ± 0.4 µM for I_Na peak (I_{Na peak}) and 0.9 ± 0.1 µM for I_{Na late}. EPA shifted the steady-state inactivation of I_{Na peak} by –19 mV in the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na⁺ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, all at 5 µM concentration, also significantly inhibited I_Na. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 µM concentration had no effect on I_Na. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 induce inactivation-deficient I_Na and that n-3 PUFAs inhibit mutant I_Na. human cardiac sodium channel