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Phyllobates terribilis frog is listed as an endangered species, endemic to the Pacific coast of Colombia. Through diets, these golden poison frogs sequester lethal amounts of batrachotoxin in their skin for self-defense. Batrachotoxin activates voltage-gated Na+ channels and keeps them open persistently with deadly consequences. This study addresses how golden poison frogs may avoid poisoning themselves via a naturally occurring substitution at the batrachotoxin receptor within their muscle Na+ channel. An equivalent asparagine-to-threonine substitution not only preserved the functional integrity of rat muscle Na+ channels but also rendered them exceptionally resistant to batrachotoxin. Such a switch could evolve via a single nucleotide mutation.

Miniature persistent late Na(+) currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na(+) currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na(+) channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na(+) currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na(+) currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na(+) currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na(+) channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na(+) currents were blocked by tetrodotoxin with an IC(50) value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na(+) currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na(+) currents are suitable for studying as well as screening potent open-channel blockers.

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.

Articaine is widely used as a local anesthetic (LA) in dentistry, but little is known regarding its blocking actions on Na+ channels. We therefore examined the state-dependent block of articaine first in rat skeletal muscle rNav1.4 Na+ channels expressed in Hek293t cells. Articaine exhibited a weak block of resting rNav1.4 Na+ channels at -140 mV with a 50% inhibitory concentration (IC(50)) of 378 +/- 26 microM (n = 5). The affinity was higher for inactivated Na+ channels measured at -70 mV with an IC50 value of 40.6 +/- 2.7 microM (n = 5). The open-channel block by articaine was measured using inactivation-deficient rNav1.4 Na+ channels with an IC50 value of 15.8 +/- 1.5 microM (n = 5). Receptor mapping demonstrated that articaine interacted strongly with a D4S6 phenylalanine residue, which is known to form a part of the LA receptor. Thus the block of rNav1.4 Na+ channels by articaine is via the conserved LA receptor in a highly state-dependent manner, with a ranking order of open (23.9x) > inactivated (9.3x) > resting (1x) state. Finally, the open-channel block by articaine was likewise measured in inactivation-deficient hNav1.7 and rNav1.8 Na+ channels, with IC(50) values of 8.8 +/- 0.1 and 22.0 +/- 0.5 microM, respectively (n = 5), indicating that the high-affinity open-channel block by articaine is indeed preserved in neuronal Na+ channel isoforms.

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.

Sequence alignment of four S6 segments in the human cardiac Na+ channel suggests that serine-401 (hNav1.5-S401) at D1S6 along with asparagine-927 (N927) at D2S6, serine-1458 (S1458) at D3S6, and phenylalanine-1760 (F1760) at D4S6 may jointly form a pore-facing S(401)N(927)S(1458)F(1760) ring. Importantly, this pore-facing structure is adjacent to the putative gating-hinge (G(400)G(926)G(1457)S(1759)) and close to the selectivity filter. Within this SNSF ring, only S401 has not yet been identified as a batrachotoxin (BTX) sensing residue. We therefore created S401 mutants with 12 substitutions (S401C,W,P,A,K,F,R,E,L,N,D,G) and assayed their BTX sensitivity. All S401 mutants expressed Na+ currents but often with altered gating characteristics. Ten mutants were found sensitive to 5 muM BTX, which eliminated Na+ channel fast inactivation after repetitive pulses. However, S401K and S401R became BTX resistant. In addition, the block of open and inactivated hNav1.5-S401K Na+ channels by local anesthetic bupivacaine was reduced by approximately 8-10-fold, but not the block of resting Na+ channels. Qualitatively, these ligand-sensing phenotypes of hNav1.5-S401K channels resemble those of S1458K and F1760K channels reported earlier. Together, our results support that residue hNav1.5-S401 at D1S6 is facing the inner cavity and is in close proximity to the receptor sites for BTX and for local anesthetics.

Resurgent tail Na(+) currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse beta4 subunit, beta4(154-167), 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 beta4(154-167) actions in inactivation-deficient cardiac hNav1.5 Na(+) channels expressed in human embryonic kidney 293t cells. Intracellular beta4(154-167) from 25-250 microM elicited a conspicuous time-dependent block of inactivation-deficient Na(+) currents at 50 mV in a concentration-dependent manner. On and off rates for beta4(154-167) binding were estimated at 10.1 microM(-1)s(-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 beta4(154-167). Near the activation threshold (approximately -70 mV), resurgent tail currents were robust and long lasting. Likewise, beta4(154-167) 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 beta4(154-167) 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 beta4(154-167). Together, our in vitro studies identify a putative S6-binding site for beta4(154-167) within the inner cavity of hNav1.5 Na(+) channels. Such an S6 receptor readily explains (1) why beta4(154-167) gains access to its receptor as an open-channel blocker, (2), why bound beta4(154-167) briefly prevents the activation gate from closing by a "foot-in-the-door" mechanism during deactivation, (3) why BTX inhibits beta4(154-167) binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates beta4(154-167) binding.

A voltage-gated, small, persistent Na+ current ( INa) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent INa that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on INa in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the α-subunit (hH1α) of human cardiac Na+ channels (hNav1.5) plus β1-subunits. Extracellular application of 5 μM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited INa. The late portion of INa ( INa late, measured near the end of each pulse) was almost completely suppressed. INa returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on INa was concentration dependent, with IC50 values of 4.0 ± 0.4 μM for INa peak ( INa peak) and 0.9 ± 0.1 μM for INa late. EPA shifted the steady-state inactivation of INa 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 INa. 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 INa. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1α induce inactivation-deficient INa and that n-3 PUFAs inhibit mutant INa.

State-dependent blockade of human cardiac hNav1.5 sodium channels by propafenone was studied using whole-cell patch clamp techniques. Both a direct investigation using cells with inactivation-deficient sodium channels and an algorithmic approach used on cells with wild-type channels revealed a rapid binding of propafenone to the open state. This occurs approximately 4000 and 700 times faster than the binding to the resting and inactivated state, respectively. An established mathematical "gating" model was modified to represent the experimental data.

Voltage-gated Na(+) channels display rapid activation gating (opening) as well as fast and slow inactivation gating (closing) during depolarization. We substituted residue S1759 (serine), a putative D4S6 gating hinge of human cardiac hNav1.5 Na(+) channels with A (alanine), D (aspartate), K (lysine), L (leucine), P (proline), and W (tryptophan). Significant shifts in gating parameters for activation and steady-state fast inactivation were observed in A-, D-, K-, and W-substituted mutant Na(+) channels. No gating shifts occurred in the L-substituted mutant, whereas the P-substituted mutant did not yield sufficient Na(+) currents. Wild-type, A-, D-, and L-substituted mutant Na(+) channels showed little or no slow inactivation with a 10-s conditioning pulse ranging from -180 to 0 mV. Unexpectedly, W- and K-substituted mutant Na(+) channels displayed profound maximal slow inactivation around -100 mV ( approximately 85% and approximately 70%, respectively). However, slow inactivation was progressively reversed in magnitude from -70 to 0 mV. This regression was minimized in inactivation-deficient hNav1.5-S1759W/L409C/A410W Na(+) channels, indicating that the intracellular fast-inactivation gate caused such a reversal. Our data suggest that the hNav1.5-S1759 residue plays a critical role in slow inactivation. Possible mechanisms for S1759 involvement in slow inactivation and for antagonism between fast and slow inactivation are discussed.

Voltage-gated Na+ channels are dynamic transmembrane proteins responsible for the rising phase of the action potential in excitable membranes. Local anesthetics (LAs) and structurally related antiarrhythmic and anticonvulsant compounds target specific sites in voltage-gated Na+ channels to block Na+ currents, thus reducing excitability in neuronal, cardiac, or central nervous tissue. A high-affinity LA block is produced by binding to open and inactivated states of Na+ channels rather than to resting states and suggests a binding site that converts from a low- to a high-affinity conformation during gating. Recent findings using site-directed mutagenesis suggest that multiple S6 segments together form an LA binding site within the Na+ channel. While the selectivity filter may form the more extracellular-located part of this binding site, the role of the fast inactivation gate in LA binding has not yet been resolved. The receptor of the neurotoxin batrachotoxin (BTX) is adjacent to or even overlaps with the LA binding site. The close proximity of the LA and BTX binding sites to residues critical for inactivation, together with gating transitions through S6 segments, might explain the strong impact of LAs and BTX on inactivation of voltage-gated Na+ channels and might help elucidate the mechanisms underlying voltage- and frequency-dependent LA block.

Recent reports suggest that four S6 C-termini may jointly close the voltage-gated cation channel at the cytoplasmic side, probably as an inverted teepee structure. In this study we substituted individually a total of 18 residues at D1S6 and D4S6 C-terminal ends of the rNav1.4 Na(+) channel alpha-subunit with tryptophan (W) and examined their corresponding gating properties when expressed in Hek293t cells along with beta1 subunit. Several W-mutants displayed significant changes in activation, fast inactivation, and/or slow inactivation gating. In particular, five S6 W-mutants showed incomplete fast inactivation with noninactivating maintained currents present. Cysteine (C) substitutions of these five residues resulted in two mutants with slightly more maintained currents. Multiple substitutions at these five positions yielded two mutants (L437C/A438W, L435W/L437C/A438W) that exhibited phenotypes with minimal fast inactivation. Unexpectedly, such inactivation-deficient mutants expressed Na(+) currents as well as did the wild-type. Furthermore, all mutants with impaired fast inactivation exhibited an enhanced slow inactivation phenotype. Implications of these results will be discussed in terms of indirect allosteric modulations via amino acid substitutions and/or a direct involvement of S6 C-termini in Na(+) channel gating.

Local anesthetics (LAs) block voltage-gated sodium channels. Parts of the LA binding site are located in the pore-lining transmembrane segments 6 of domains 1, 3, and 4 (D1-S6, D3-S6, D4-S6). We suggested previously that residue N434 in D1-S6 interacts directly with bupivacaine enantiomers in inactivated channels because side-chain properties of different residues substituted at N434 correlated with changes in blocking potencies of bupivacaine enantiomers. Furthermore, mutation N434R exhibited significant stereoselectivity for block of inactivated channels that resulted from a selective decrease in block by S(–)-bupivacaine. In the present study, we analyzed the role of residue L1280 in D3-S6 of the rat skeletal muscle Nav1.4 channel in interactions with the enantiomers of bupivacaine. We substituted native leucine at L1280 with amino acids of different physicochemical properties. Wild-type and mutant channels were expressed transiently in human embryonic kidney 293t cells and were investigated under whole-cell voltage clamp. Block of resting mutant channels by bupivacaine enantiomers revealed little difference compared with wild-type channels. Block of inactivated channels was increased in a mutation containing an aromatic group (L1280W) and decreased in mutations containing a positive charge (L1280K, L1280R). Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels. More surprisingly, stereoselectivity resulted from a selective decrease in block by R(+)-bupivacaine, in contrast to mutation N434R in D1-S6. We propose that in inactivated channels, residues L1280 in D3-S6 and N434 in D1-S6 interact directly with LAs and thereby face each other in the ion-conducting pore.

The Vanilloid Receptor TRPV1 is a non-selective cation channel with a high relative Ca 2+ permeability. TRPV1 exhibits slow desensitization, a potential mechanism regulating adaptation of peripheral sensory neurons to noxious stimuli. The predicted folding pattern of TRPV1 resembles that of voltage-gated channels. Sequence alignment of segments 6 of TRPV1 and voltage-gated Na + channels reveals a conserved aromatic amino acid that in Na + channels is involved in fast inactivation and pharmacological block. We found that replacing this tyrosine Y671 by positively charged lysine (K) completely abrogated Ca 2+ -dependent desensitization. Y671K also exhibited significant reduction in Ca 2+ permeability that was not responsible for the lack in desensitization. Substitution of Y671 with negatively charged aspartate or uncharged alanine slightly altered desensitization but left Ca 2+ permeability unchanged. Substitution of Y671 with positively charged arginine produced a phenotype similar to Y671K. We propose that residue Y671 is critical for the high relative Ca 2+ permeability of TRPV1 and participates in the structural rearrangements of the channel protein leading to Ca 2+ -dependent desensitization.

Amitriptyline, nortriptyline, imipramine, doxepin, desipramine, protriptyline, trimipramine, and maprotiline are tricyclic antidepressants (TCAs) used orally in treating major depressive disorders. Recent studies showed that amitriptyline is more potent in blocking the sciatic nerve functions in vivo by local injection than bupivacaine, a long-acting local anesthetic. We therefore tested whether various TCAs could likewise act as local anesthetics in vivo after single injection via the rat sciatic notch. The duration of complete sciatic nerve blockade by TCAs and the time to reach full recovery were measured with neurobehavioral assays and compared with results from bupivacaine. Amitriptyline, doxepin, and imipramine at 5mM elicited a longer complete sciatic nerve blockade than did bupivacaine at 15.4mM (0.5%), whereas trimipramine and desipramine at 5mM produced a shorter blockade. In contrast, nortriptyline, protriptyline, and maprotiline failed to elicit complete sciatic nerve blockade. Thus, TCAs have very different efficacy as local anesthetics in vivo. The duration of rat sciatic nerve blockade in vivo by TCAs is not well correlated with the 50% inhibitory concentration (IC(50)) of TCAs in blocking human cardiac Nav1.5 Na(+) channels expressed in human embryonic kidney cells. With this in vitro expression system, TCAs appear more potent than bupivacaine as Na(+) channel blockers in Nav1.5 Na(+) channels. We suggest that the ability of TCAs to pass through various membrane barriers within peripheral nerve trunks is crucial to their local anesthetic efficacy in vivo. TCAs with a tertiary amine appear more effective in penetrating these membrane barriers than TCAs with a secondary amine.

We have shown previously that prenylamine, a calcium channel blocker, has potent local anesthetic activity in vivo and in vitro. We now characterize the tonic and use-dependent block of prenylamine on wild-type human cardiac voltage-gated sodium channels (hNav1.5) transiently expressed in human embryonic kidney 293t cells under whole-cell voltage-clamp condition. We also determine whether prenylamine and local anesthetics interact with a common binding site on the Nav1.5 channel by analyzing prenylamine block on mutant hNav1.5 channels that have substitution mutations in amino acids at the putative local anesthetic binding sites. Prenylamine exhibits tonic block at both hyperpolarizing and depolarizing potentials on hNav1.5 channels with 50% inhibitory concentrations of 9.67 +/- 0.25 microM and 0.72 +/- 0.02 microM, respectively. Substitutions of the amino acids at the putative local anesthetic binding site (i.e., F1760, N1765, Y1767, and N406) with lysine had much lesser effects on prenylamine block of the mutant hNav1.5 channels compared with local anesthetic block. The affinity of prenylamine was reduced at most by 5.8-fold, whereas that of bupivacaine, a known local anesthetic, was reduced by as much as 68-fold compared with wild-type by the mutations at the local anesthetic receptor site. Furthermore, equilibrium results between prenylamine-bupivacaine mixtures suggest two independent receptors. Thus, the data demonstrate that prenylamine has both tonic and use-dependent block of hNav1.5 channels similar to that of local anesthetics, but the location of the prenylamine binding site on hNav1.5 differs from that of the local anesthetic binding site.

Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na(+) channels for its therapeutic action. Cardiac Na(+) channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na(+) channels. Lacosamide showed little effect on hNav1.5 Na(+) currents at 300 µM when cells were held at -140 mV. With 30-second conditioning pulses from -90 to -50 mV; however, hNav1.5 Na(+) channels became sensitive to lacosamide with IC50 (50% inhibitory concentration) around 70-80 µM. Higher IC50 values were found at -110 and -30 mV. The development of lacosamide block at -70 mV was slow in wild-type Na(+) channels (τ; 8.04 ± 0.39 seconds, n = 8). This time constant was significantly accelerated in hNav1.5-CW inactivation-deficient counterparts. The recovery from lacosamide block at -70 mV for 10 seconds was relatively rapid in wild-type Na(+) channels (τ; 639 ± 90 milliseconds, n = 8). This recovery was accelerated further in hNav1.5-CW counterparts. Unexpectedly, lacosamide elicited a time-dependent block of persistent hNav1.5-CW Na(+) currents with an IC50 of 242 ± 19 µM (n = 5). Furthermore, both hNav1.5-CW/F1760K mutant and batrachotoxin-activated hNav1.5 Na(+) channels became completely lacosamide resistant, indicating that the lacosamide receptor overlaps receptors for local anesthetics and batrachotoxin. Our results together suggest that lacosamide targets the intermediate preopen and open states of hNav1.5 Na(+) channels. Lacosamide may thus track closely the conformational changes at the hNav1.5-F1760 region along the activation pathway.

Batrachotoxin (BTX) stabilizes the voltage-gated Na(+) channels in their open conformation, whereas local anesthetics (LAs) block Na(+) conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha-subunit Na(+) channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na(+) channel ((mu)1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na(+) currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na(+) currents during repetitive pulses. Two of these four mutants, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na(+) channels (K(I)) of approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.

Suppression of cardiac voltage-gated Na + currents is probably one of the important factors for the cardioprotective effects of the n-3 polyunsaturated fatty acids (PUFAs) against lethal arrhythmias. The α subunit of the human cardiac Na + channel (hH1 α ) and its mutants were expressed in human embryonic kidney (HEK293t) cells. The effects of single amino acid point mutations on fatty acid-induced inhibition of the hH1 α Na + current (I Na ) were assessed. Eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I Na in HEK293t cells expressing the wild type, Y1767K, and F1760K of hH1 α Na + channels. The inhibition was voltage and concentration-dependent with a significant hyperpolarizing shift of the steady state of I Na . In contrast, the mutant N406K was significantly less sensitive to the inhibitory effect of EPA. The values of the shift at 1, 5, and 10 μM EPA were significantly smaller for N406K than for the wild type. Coexpression of the β 1 subunit and N406K further decreased the inhibitory effects of EPA on I Na in HEK293t cells. In addition, EPA produced a smaller hyperpolarizing shift of the V 1/2 of the steady-state inactivation in HEK293t cells coexpressing the β 1 subunit and N406K. These results demonstrate that substitution of asparagine with lysine at the site of 406 in the domain-1-segment-6 region (D1-S6) significantly decreased the inhibitory effect of PUFAs on I Na , and coexpression with β 1 decreased this effect even more. Therefore, asparagine at the 406 site in hH1 α may be important for the inhibition by the PUFAs of cardiac voltage-gated Na + currents, which play a significant role in the antiarrhythmic actions of PUFAs.

Dietary polyunsaturated fatty acids (PUFAs) prevent ischemia-induced fatal cardiac arrhythmias in animals and probably in humans. This action results from inhibition of ion currents for Na+, Ca2+, and possibly other ions. To extend understanding of this protection we are seeking a possible binding site for the PUFAs on the alpha-subunit of the human cardiac Na+ channel, hH1alpha, transiently expressed in HEK293t cells. Three mutated single amino acid substitutions with lysine were made in the alpha-subunit at Domain 4-Segment 6 (D4-S6) for F1760, Y1767 and at D1-S6 for N406. These are in the putative sites of binding of local anesthetics and batrachotoxin, respectively. The mutants F1760K, Y1767K, and N406K, separately and to different extents, affected the current density, the steady-state inactivation potential, accelerated inactivation, delayed recovery from inactivation, and affected voltage-dependent block, but did not affect activation of the hH1alpha. It is essential to learn that single point mutations in D1-S6 and D4-S6 alone significantly modify the kinetics of human cardiac hH1alpha Na+ currents. The effects of PUFAs on these mutant channels will be the subject of subsequent reports.

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.

Voltage-gated cardiac Na+channels are composed of α- and β1-subunits. In this study β1-subunit was cotransfected with the α-subunit of the human cardiac Na+channel (hH1α) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na+currents were assessed. Current density was significantly greater in HEK293t cells coexpressing α- and β1-subunits ( INa,αβ) than in HEK293t cells expressing α-subunit alone ( INa,α). Compared with INa,α, the voltage-dependent inactivation and activation of INa,αβwere significantly shifted in the depolarizing direction. In addition, coexpression with β1-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n–3)] significantly reduced INa,αβin a concentration-dependent manner and at 5 μM shifted the midpoint voltage of the steady-state inactivation by −22 ± 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n–3)], α-linolenic acid [C18:3(n–3)], and conjugated linoleic acid [C18:2(n–6)] at 5 μM significantly inhibited both INa,αβand INa,α.In contrast, saturated and monounsaturated fatty acids had no effects on INa,αβ. This finding differs from the results for INa,α, which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of β1-subunit with hH1αmodifies the kinetics and fatty acid block of the Na+channel.

Voltage-gated Na(+) channels are the primary targets of local anesthetics (LAs). Amino acid residues in domain 4, transmembrane segment 6 (D4-S6) form part of the LA binding site. LAs inhibit binding of the neurotoxin batrachotoxin (BTX). Parts of the BTX binding site are located in D1-S6 and D4-S6. The affinity of BTX-resistant Na(+) channels mutated in D1-S6 (mu1-N434K, mu1-N437K) toward several LAs is significantly decreased. We have studied how residue mu1-N434 influences LA binding. By using site-directed mutagenesis, we created mutations at mu1-N434 that vary the hydrophobicity, aromaticity, polarity, and charge and investigated their influence on state-dependent binding and stereoselectivity of bupivacaine. Wild-type and mutant channels were transiently expressed in human embryonic kidney 293t cells and investigated under whole-cell voltage-clamp. For resting channels, bupivacaine enantiomers showed a higher potency in all mutant channels compared with wild-type channels. These changes were not well correlated with the physical properties of the substituted residues. Stereoselectivity was small and almost unchanged. In inactivated channels, the potency of bupivacaine was increased in mutations containing a quadrupole of an aromatic group (mu1-N434F, mu1-N434W, mu1-N434Y), a polar group (mu1-N434C), or a negative charge (mu1-N434D) and was decreased in a mutation containing a positive charge (mu1-N434K). In mutation mu1-N434R, containing the positively charged arginine, the potency of S(-)-bupivacaine was selectively decreased, resulting in a stereoselectivity (stereopotency ratio) of 3. Similar results were observed with cocaine but not with RAC 109 enantiomers. We propose that in inactivated channels, residue mu1-N434 interacts directly with the positively charged moiety of LAs and that D1-S6 and D4-S6 form a domain-interface site for binding of BTX and LAs in close proximity.

Local anesthetics (LAs) are noncompetitive antagonists of batrachotoxin (BTX) in voltage-gated Na+ channels. The putative LA receptor has been delineated within the transmembrane segment S6 in domain IV of voltage-gated Na+ channels, whereas the putative BTX receptor is within segment S6 in domain I. In this study, we created BTX-resistant muscle Na+ channels at segment I-S6 (micro1-N434K, micro1-L437K) to test whether these residues modulate LA binding. These mutant channels were expressed in transiently transfected human embryonic kidney 293T cells, and their sensitivity to lidocaine, QX-314, etidocaine, and benzocaine was assayed under whole-cell, voltage-clamp conditions. Our results show that LA binding in BTX-resistant micro1 Na+ channels was reduced significantly. At -100 mV holding potential, the reduction in LA affinity was maximal for QX-314 (by 17-fold) and much less for neutral benzocaine (by 2-fold). Furthermore, this reduction was residue specific; substitution of positively charged lysine with negatively charged aspartic acid (micro1-N434D) restored or even enhanced the LA affinity. We conclude that micro1-N434K and micro1-L437K residues located near the middle of the I-S6 segment of Na+ channels can reduce the LA binding affinity without BTX. Thus, this reduction of the LA affinity by point mutations at the BTX binding site is not caused by gating changes induced by BTX alone. We surmise that the BTX receptor and the LA receptor within segments I-S6 and IV-S6, respectively, may align near or within the Na+ permeation pathway.

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na + current (I Na,α ) in human embryonic kidney (HEK293t) cells transfected with the α-subunit of the human cardiac Na + channel (hH1 α ). Extracellular application of 0.01 to 30 μM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I Na,α with an IC 50 of 0.51 ± 0.06 μM. The EPA-induced suppression of I Na,α was concentration- and voltage-dependent. EPA at 5 μM significantly shifted the steady-state inactivation relationship by −27.8 ± 1.2 mV ( n = 6, P < 0.0001) at the V 1/2 point. In addition, EPA blocked I Na,α with a higher “binding affinity” to hH1 α channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 μM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 μM EPA, from 2.1 ± 0.8 ms for control to 34.8 ± 2.1 ms ( n = 5, P < 0.001). The effects of EPA on I Na,α were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), α-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 μM and all- trans -retinoic acid at 10 μM had similar effects on I Na,α as EPA. Even 5 μM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited I Na,α . In contrast, 5 μM EPA ethyl ester did not alter I Na,α (8 ± 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress I Na,α with high “binding affinity” to hH1 α channels in the inactivated state and prolong the duration of recovery from inactivation.

Batrachotoxin (BTX) is a steroidal alkaloid that causes Na + channels to open persistently. This toxin has been used widely as a tool for studying Na + channel gating processes and for estimating Na + channel density. In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine → lysine substitution of the rat muscle Na + channel α-subunit, μ1-N434K, renders the channel completely insensitive to 5 μM BTX when expressed in mammalian cells. This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, μ1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified μ1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional. This finding implies that the μ1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the μ1–434 position apparently diminishes the efficacy of BTX on eliminating Na + channel inactivation. Mutants of two adjacent residues, μ1-I433K and μ1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the μ1-I433, μ1-N434, and μ1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor.

According to Hille's modulated receptor hypothesis, benzocaine shares a common receptor with all other local anesthetics (LAs) in the voltage-gated Na+ channel. We tested this single receptor hypothesis using mutant muscle Na+ channels of mu1-I1575A, F1579A, and N1584A transiently expressed in Hek-293t cells. Both benzocaine and etidocaine are more effective at blocking mu1-N1584A current than the wild-type current, while they are less potent at blocking mu1-F1579A current. Such concurrent changes of both benzocaine and etidocaine potency towards F1579A and N1584A mutants suggest that they share a common LA receptor. Consistent with results found in studies of native Na+ channels, permanently charged QX-314 at 1 mM is not effective at blocking wild-type, F1579A, and N1584A current via external application. In contrast, QX-314 is relatively potent at blocking I1575A current when applied externally. This increased potency of external QX-314 against the mu1-I1575A mutant has been reported previously in a study of the brain counterpart. Mutant I1575A also appears to be highly sensitive to the external divalent cation Cd2+, probably because of the presence of cysteine residues near the mu1-I1575 position in the IV-S6 segment. To our surprise, neutral benzocaine becomes more effective at blocking mu1-I1575A current than the wild-type current, whereas the opposite is found for etidocaine. We hypothesize that an increase in accessibility of external QX-314 to the mu1-I1575A mutant is accompanied by a reduction of binding towards the charged amine component.

Bulleyaconitine A (BLA) is an analgesic and antiinflammatory drug isolated from Aconitum plants. BLA has several potential targets, including voltage-gated Na+ channels. We tested whether BLA elicited long-lasting cutaneous analgesia, when co-injected with lidocaine and epinephrine, as a model for prolonged infiltration anesthesia.The local anesthetic properties of BLA were assessed by the patch-clamp technique in HEK293t cells expressing Nav1.7 and Nav1.8 neuronal Na+ channels, both crucial for nociception. Drug solutions (0.6 mL) were injected subcutaneously via rat shaved dorsal skin. Inhibition of the cutaneous trunci muscle reflex was evaluated by pinpricks. Skin cross-sections were stained with hematoxylin and eosin or with antibodies against PGP9.5.BLA at 10 microM interacted minimally with resting or inactivated Nav1.7 and Nav1.8 Na+ channels when infrequently stimulated to +50 mV for 3 ms. However, when stimulated at 2 Hz for 1000 pulses, their peak Na+ currents were90% reduced by BLA. This use-dependent inhibition was not significantly reversed after 15-min washing. Complete nociceptive blockade after injection of lidocaine (0.5%)/epinephrine (1:200,000) lasted for approximately 1 h in rats; full recovery occurred after approximately 6 h. Co-injection of 0.125 mM BLA with lidocaine/epinephrine increased the duration of complete nociceptive blockade to 24 h. Full recovery occurred after approximately 6 days. Skin histology including peripheral nerve fibers appeared unaffected by BLA.BLA inhibits Nav1.7 and Nav1.8 Na+ currents in a use-dependent manner. Co-injection of BLA ator=0.125 mM with lidocaine and epinephrine elicits complete cutaneous analgesia that lasts for up to 24 h without adverse effects.

Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na(+) channels. However, ranolazine block of muscle and neuronal Na(+) channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na(+) currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na(+) channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC(50)) >/= 60 microM. Use-dependent block of Na(+) channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 microM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na(+) currents by ranolazine with estimated IC(50) values of 2.4, 6.2, and 1.7 microM, respectively. On- and off-rates of ranolazine were 8.2 microM(-1) s(-1) and 22 s(-1), respectively, for Nav1.4 open channels and 7.1 microM(-1) s(-1) and 14 s(-1), respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na(+) channels reduced the potency of ranolazine approximately 17-fold. We conclude that: 1) both muscle and neuronal Na(+) channels are as sensitive to ranolazine block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na(+) channels; and 3) ranolazine block of open Na(+) channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.

Antidepressants, such as traditional tricyclic antidepressants (TCAs), are the first-line treatment for various pain syndromes. Available evidence indicates that TCAs may target Na+ channels for their analgesic action. In this report, we examined the effects of contemporary antidepressants sertraline and paroxetine on (1) neuronal Na+ channels expressed in GH3 cells and (2) muscle rNav1.4 Na+ channels heterologously expressed in Hek293t cells. Our results showed that both antidepressants blocked Na+ channels in a highly state-dependent manner. The 50% inhibitory concentrations (IC50) for sertraline and paroxetine ranged approximately 18-28 microM: for resting block and approximately 2-8 microM: for inactivated block of neuronal and rNav1.4 Na+ channels. Surprisingly, the IC50 values for both drugs were about 0.6-0.7 microM: for the open channel block of persistent late Na+ currents generated through inactivation-deficient rNav1.4 mutant Na+ channels. For comparison, the open channel block in neuronal hNav1.7 counterparts yielded IC50 values around 0.3-0.4 microM: for both drugs. Receptor mapping using fast inactivation-deficient rNav1.4-F1579A/K mutants with reduced affinities toward local anesthetics (LAs) and TCAs indicated that the F1579 residue is not involved in the binding of sertraline and paroxetine. Thus, sertraline and paroxetine are potent open channel blockers that target persistent late Na+ currents preferentially, but their block is not mediated via the phenylalanine residue at the known LA/TCA receptor site.

Capsaicin elicits burning pain via the activation of the vanilloid receptor (TRPV1). Intriguingly, several reports showed that capsaicin also inhibits Na+ currents but the mechanisms remain unclear. To explore this non-TRPV1 action we applied capsaicin to HEK293 cells stably expressing inactivation-deficient rat skeletal muscle Na+ mutant channels (rNav1.4-WCW). Capsaicin elicited a conspicuous time-dependent block of inactivation-deficient Na+ currents. The 50% inhibitory concentration (IC50) of capsaicin for open Na+ channels at +30 mV was measured 6.8+/-0.6 microM (n=5), a value that is 10-30 times lower than those for resting (218 microM) and inactivated (74 microM) wild-type Na+ channels. On-rate and off-rate constants for capsaicin open-channel block at +30 mV were estimated to be 6.37 microM(-1) s(-1) and 34.4 s(-1), respectively, with a calculated dissociation constant (KD) of 5.4 microM. Capsaicin at 30 microM produced approximately 70% additional use-dependent block of remaining rNav1.4-WCW Na+ currents during repetitive pulses at 1 Hz. Site-directed mutagenesis showed that the local anesthetic receptor was not responsible for the capsaicin block of the inactivation-deficient Na+ channel. Interestingly, capsaicin elicited little time-dependent block of batrachotoxin-modified rNav1.4-WCW Na+ currents, indicating that batrachotoxin prevents capsaicin binding. Finally, neuronal open Na+ channels endogenously expressed in GH3 cells were as sensitive to capsaicin block as rNav1.4 counterparts. We conclude that capsaicin preferentially blocks persistent late Na+ currents, probably via a receptor that overlaps the batrachotoxin receptor but not the local anesthetic receptor. Drugs that target such a non-TRPV1 receptor could be beneficial for patients with neuropathic pain.

The calmodulin (CaM) inhibitor trifluoperazine (TFP) can produce analgesia when given intrathecally to rats; however, the mechanism is not known. We asked whether TFP could modulate the Nav1.7 sodium channel, which is highly expressed in the peripheral nervous system and plays an important role in nociception. We show that 500 nM and 2 μM TFP induce major decreases in Nav1.7 and Nav1.4 current amplitudes and that 2 μM TFP causes hyperpolarizing shifts in the steady-state inactivation of Nav1.7 and Nav1.4. CaM can bind to the C-termini of voltage-gated sodium channels and modulate their functional properties; therefore we investigated if TFP modulation of sodium channels was due to CaM inhibition. However, the TFP inhibition was not replicated by whole cell dialysis of a calmodulin inhibitory peptide, indicating that major effects of TFP do not involve a disruption of CaM-channel interactions. Rather, our data show that TFP inhibition is state dependent and that the majority of the TFP inhibition depends on specific amino-acid residues in the local anesthetic receptor site in sodium channels. TFP was also effective in vivo in causing motor and sensory blockade after subfascial injection to the rat sciatic nerve. The state-dependent block of Nav1.7 channels with nanomolar concentrations of TFP raises the possibility that TFP, or TFP analogues, might be useful for regional anesthesia and pain management and could be more potent than traditional local anesthetics.

A structural model of the rNav1.4 Na+ channel with batrachotoxin (BTX) bound within the inner cavity suggested that the BTX pyrrole moiety is located between a lysine residue at the DEKA selectivity filter (Lys1237) and an adjacent phenylalanine residue (Phe1236). We tested this pyrrole-binding model by site-directed mutagenesis of Phe1236 at D3/P-loop with 11 amino acids. Mutants F1236D and F1236E expressed poorly, whereas nine other mutants either expressed robust Na+ currents, like the wild-type (F1236Y/Q/K), or somewhat reduced current (F1236G/A/C/N/W/R). Gating properties were altered modestly in most mutant channels, with F1236G displaying the greatest shift in activation and steady-state fast inactivation (-10.1 and -7.5 mV, respectively). Mutants F1236K and F1236R were severely resistant to BTX after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz), whereas seven other mutants were sensitive but with reduced magnitudes compared with the wild type. It is noteworthy that rNav1.4-F1236K mutant Na+ channels remained highly sensitive to block by the local anesthetic bupivacaine, unlike several other BTX-resistant mutant channels. Our data thus support a model in which BTX, when bound within the inner cavity, interacts with the D3/P-loop directly. Such a direct interaction provides clues on how BTX alters the Na+ channel selectivity and conductance.

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 (IC(50)) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 +/- 3.3 microM (n = 5) and 81.7 +/- 10.6 microM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC(50) values for resting-channel block at -140 mV were12-fold higher than those for open-channel block. With 300 microM benzocaine, rapid time-dependent block (tau approximately 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.

The antidepressant amitriptyline is used as an adjuvant in the treatment of chronic pain conditions. Among its many actions, this drug also blocks ion channels, such as Na channels. Preliminary animal studies suggested that amitripty-line would be a longer-lasting local anesthetic than bupivacaine, with potentially fewer side effects. Therefore, the authors investigated the adverse effects and effectiveness of this drug when given for ulnar nerve blockade in human volunteers.After obtaining written institutional review board approval and informed consent, a typical phase Ia trial was conducted by administration to the ulnar nerve at the level of the wrist in an open-label, dose-escalating fashion. Amitripty-line hydrochloride, 4 ml, at concentrations of 5, 10, and 20 mM (n = 4-9/group) was used for each volunteer. If no major side effects and nerve block were encountered, comparison in a randomized, double-blinded trial of amitriptyline (20 mM) to placebo and bupivacaine (4 mM) (n = 4-9/group), was to follow. A blunt needle was used to grade the pain, and motor blockade was assessed by the Froment test.There was no significant statistical difference in terms of side effects (pain, swelling, erythema, and sedation) among any groups. The analgesic effects of 20 mM amitriptyline and 4 mm bupivacaine solution were significantly higher than those of the placebo solution.Because of the lack of evidence that amitripty-line provides better nerve blockade than current local anesthetics and the potential for neurotoxicity, its use for peripheral nerve blockade in humans seems limited.

Lidocaine and cocaine, two local anesthetics, and n-3 polyunsaturated fatty acids in fish oils, inhibit the voltage-gated Na(+) channels of cardiomyocytes. This inhibition by lidocaine and n-3 fish oil is associated with antiarrhythmic effects, whereas with cocaine lethal arrhythmias may occur. These electrophysiologic studies show that at the concentrations tested, the n-3 fish oil fatty acids and lidocaine share three actions on I(Na): a potent inhibition of I(Na); a strong voltage-dependence of this inhibition; and a large shift of the steady-state inactivation to hyperpolarized potentials. By contrast cocaine shares only the potent inhibition of I(Na). The voltage-dependence of the inhibition is much decreased with cocaine, which produces only a very small leftward shift of the voltage-dependence of inactivation. The large leftward shift of the steady-state inactivation seems very important in the prevention of fatal arrhythmias by the n-3 fatty acids. Thus, we suggest that it is lack of this effect by cocaine, which is one factor, that eliminates its ability to prevent fatal cardiac arrhythmias. Further we report that in cultured neonatal rat cardiomyocytes n-3 fish oil fatty acids terminate the tachycardia induced by the alpha(1) adrenergic agonist, phenylephrine, whereas cocaine accelerates the tachycardia and causes bouts of tachyarrhythmias.

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium-aggravated myotonia, long QT-3 syndrome, and neuropathic pain. This drug elicits tonic block of Na(+) channels when cells are stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation-deficient mutant Na(+) channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a beta1 subunit. The 50% inhibitory concentrations (IC(50)) for the inactivated-state block and the resting-state block of wild-type Na(+) channels by mexiletine were measured as 67.8 +/- 7.0 microm and 431.2 +/- 9.4 microm, respectively (n= 5). In contrast, the IC(50) for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 +/- 0.1 microm (n= 5), which was within the therapeutic plasma concentration range (2.8-11 microm). Estimated on- and off-rates for the open-state block by mexiletine at +30 mV were 10.4 microm(-1) s(-1) and 54.4 s(-1), respectively. Use-dependent block by mexiletine was greater in inactivation-deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC(50) values for the block of persistent late hNav1.4 currents in chloramine-T-pretreated cells by mexiletine was 7.5 +/- 0.8 microm (n= 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open-channel block of persistent late Na(+) currents, which may arise during various pathological conditions.

Background and Objectives The antidepressant amitriptyline is used as an adjuvant in the treatment of a variety of chronic pain conditions. This drug interacts with many receptors and ion channels, such as Na+ channels. In a randomized, double-blinded, and placebo-controlled trial, we investigated whether amitriptyline also is capable of providing cutaneous analgesia when applied topically in 14 healthy volunteers. Methods Amitriptyline hydrochloride was prepared as a 45% water/45% isopropanol/10% glycerin solution and titrated to pH 8.5 with sodium hydroxide. Four areas, 2 on each arm, of approximately 1 cm2 each were marked on the ventral aspect of the upper arm. A piece of gauze, placed on each of the marked areas and affixed to the arm with an occlusive plastic dressing, was saturated via syringe with placebo and amitriptyline solutions (10 mmol/L, 50 mmol/L, and 100 mmol/L). After 1 hour, the dressings and gauze were removed. A 16-G blunt needle was used to grade the pain at the marked area once per hour (1 = complete analgesia, 10 = normal pain sensation). Results The analgesic effects of 50 mmol/L and 100 mmol/L solutions of amitriptyline were significantly higher than those of the placebo or the 10 mmol/L solution. However, no significant difference was found between the analgesia provided by the placebo solution versus the 10 mmol/L solution or between the 50 mmol/L versus the 100 mmol/L solution. The only side effect observed was a concentration-dependent redness of the skin. Conclusions Topically applied amitriptyline is effective as an analgesic in humans. Different vehicles may improve its efficacy and decrease the skin redness observed.

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.

Voltage-gated Na(+) channels are heteromeric membrane glycoproteins responsible for the generation of action potentials. A number of diverse lipid-soluble neurotoxins, such as batrachotoxin, veratridine, aconitine, grayanotoxins, pyrethroid insecticides, brevetoxins and ciguatoxin, target voltage-gated Na(+) channels for their primary actions. These toxins promote Na(+) channel opening, induce depolarization of the resting membrane potential, and thus drastically affect the excitability of nerve, muscle and cardiac tissues. Poisoning by these lipid-soluble neurotoxins causes hyperexcitability of excitable tissues, followed by convulsions, paralysis and death in animals. How these lipid-soluble neurotoxins alter Na(+) channel gating mechanistically remains unknown. Recent mapping of receptor sites within the Na(+) channel protein for these neurotoxins using site-directed mutagenesis has provided important clues on this subject. Paradoxically, the receptor site for batrachotoxin and veratridine on the voltage-gated Na(+) channel alpha-subunit appears to be adjacent to or overlap with that for therapeutic drugs such as local anaesthetics (LAs), antidepressants and anticonvulsants. This article reviews the physiological actions of lipid-soluble neurotoxins on voltage-gated Na(+) channels, their receptor sites on the S6 segments of the Na(+) channel alpha-subunit and a possible linkage between their receptors and the gating function of Na(+) channels.

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.

Biochemical evidence indicates that veratridine (VTD) and batrachotoxin (BTX) share a common binding site in Na+ channels. Under whole-cell voltage-clamp conditions, we examined this single receptor hypothesis by studying the VTD phenotype in BTX-resistant muscle Na+ channels, microl-I433K, N434K, L437K, F1579K, and N1584K. Derived from point mutations at segments D1-S6 and D4-S6, these mutant Na+ channels are resistant to 5 microM BTX when expressed in human embryonic kidney cells. In contrast to the wild-type phenotype, VTD at 200 microM elicits little or no maintained current during a test pulse at +50 mV, and little or no "tail" current after the test pulse in all BTX-resistant mutant channels. Paradoxically, VTD retains its ability to inhibit the peak Na+ current in BTX-resistant mutant Na+ channels. To explain these mutant phenotypes, we propose a two-step binding reaction scheme. An initial VTD-binding interaction with the Na+ channel results in the inhibition of peak current amplitude, and a second binding reaction results in the trapping of VTD within the D1-S6 and D4-S6 domain interface. The failure of BTX-resistant mutant Na+ channels to trap VTD suggests that segments of D1-S6 and D4-S6 form a common receptor for VTD and BTX.

1. Voltage-gated Na+ channels undergo two types of inactivation in response to depolarization. One type, fast inactivation, occurs with a time scale of milliseconds. The other, slow inactivation, occurs over seconds to minutes. In addition, these two processes appear to be distinct at the molecular level. However, the molecular mechanism of Na+ channel slow inactivation is unknown. 2. We used patch clamp techniques to study slow inactivation, activation and fast inactivation in alpha-subunit cDNA clones for wild-type human heart Na+ channels (hH1) and rat skeletal muscle Na+ channels (mu1) transiently expressed in human embryonic kidney (HEK) cells. Our experiments showed that the Na+ channel slow inactivation phenotype (development, steady state and recovery) differed dramatically between hH1 and mu1. Slow inactivation in mu1 had a faster onset, a steeper voltage dependence, and was more complete compared with hH1. In addition, recovery from slow inactivation was much slower for mu1 than for hH1. Activation and fast inactivation kinetics were also different in hH1 and mu1. In hH1, fast inactivation was slower and V values of activation and steady-state fast inactivation (hthorn ) were more negative than in mu1. 3. To better understand the molecular basis of Na+ channel slow inactivation, Na+ channel chimaeras were constructed with domains from hH1 and mu1. The slow inactivation phenotype in the chimaeras (domains denoted by subscripts) mu1(1)hH1(2,3,4), mu1(1,2)hH1(3,4) and mu1(1,2,3)hH1(4) was intermediate compared with that of wild-type. However, the chimaera mu1(1)hH1(2,3,4) was more like wild-type hH1, while the chimaeras mu1(1,2)hH1(3,4) and mu1(1,2,3)hH1(4) were more similar to wild-type mu1. In the chimaeras, activation resembled that of mu1, fast inactivation resembled that of hH1, and steady-state fast inactivation fell between that of hH1 and mu1. 4. The data demonstrate that all four domains can modulate the Na+ channel slow inactivation phenotype. However, domains D1 and D2 may play a more prominent role in determining Na+ channel slow inactivation phenotype than D3 and D4. The results also support previous conclusions that D3 and D4 (and the D3-D4 linker) play an important role in Na+ channel fast inactivation, and that activation may require non-equivalent contributions from all four domains.

Cocaine and lidocaine are local anesthetics (LAs) that block Na currents in excitable tissues. Cocaine is also a cardiotoxic agent and can induce cardiac arrhythmia and ventricular fibrillation. Lidocaine is commonly used as a postinfarction antiarrhythmic agent. These LAs exert clinically relevant effects at concentrations that do not obviously affect the normal function of either nerve or skeletal muscle. We compared the cocaine and lidocaine affinities of human cardiac (hH1) and rat skeletal (mu 1) muscle Na channels that were transiently expressed in HEK 293t cells. The affinities of resting mu 1 and hH1 channels were similar for cocaine (269 and 235 microM, respectively) and for lidocaine (491 and 440 microM, respectively). In addition, the affinities of inactivated mu 1 and hH1 channels were also similar for cocaine (12 and 10 microM, respectively) and for lidocaine (19 and 12 microM, respectively). In contrast to previous studies, our results indicate that the greater sensitivity of cardiac tissue to cocaine or lidocaine is not due to a higher affinity of the LA receptor in cardiac Na channels, but that at physiological resting potentials (-100 to -90 mV), a greater percentage of hH1 channels than mu 1 channels are in the inactivated (i.e., high-affinity) state.

Local anesthetics (LAs) block voltage-gated Na+ channels in excitable cells, whereas batrachotoxin (BTX) keeps these channels open persistently. Previous work delimited the LA receptor within the D4-S6 segment of the Na+ channel alpha-subunit, whereas the putative BTX receptor was found within the D1-S6. We mutated residues at D4-S6 critical for LA binding to determine whether such mutations modulate the BTX phenotype in rat skeletal muscle Na+ channels (mu1/rSkm1). We show that mu1-F1579K and mu1-N1584K channels become completely resistant to 5 microM BTX. In contrast, mu1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization. Furthermore, we demonstrate that cocaine elicits a profound time-dependent block after channel activation, consistent with preferential LA binding to BTX-modified open channels. We propose that channel opening promotes better exposure of receptor sites for binding with BTX and LAs, possibly by widening the bordering area around D1-S6, D4-S6, and the pore region. The BTX receptor is probably located at the interface of D1-S6 and D4-S6 segments adjacent to the LA receptor. These two S6 segments may appose too closely to bind BTX and LAs simultaneously when the channel is in its resting closed state.

We compared wild-type rat skeletal muscle NaChs ( μ 1) and a mutant NaCh (Y1586K) that has a single amino acid substitution, lysine (K) for tyrosine (Y), at position 1586 in the S6 transmembrane segment of domain 4. In Y1586K, macroscopic current decay is faster, the V 1/2 of the activation curve is shifted in the depolarized direction, and the fast-inactivation curve is less steep compared with μ 1. After an 8-ms depolarization pulse, Y1586K recovers from inactivation much more slowly than μ 1. The recovery is double exponential, suggesting recovery from two inactivation states. Varying the depolarization protocols isolates entry into an additional, "atypical" inactivation state in Y1586K that is distinct from typical fast or slow inactivation. Substitution of positively charged arginine (R) at Y1586 produces an inactivation phenotype similar to that of Y1586K. Substitution by negatively charged aspartic acid (D) or uncharged alanine (A) at Y1586 produces an inactivation phenotype similar to μ 1. Our results suggest that the positive charge of lysine (K) produces the atypical inactivation state in Y1586K. We propose that a conformational change during depolarization alters the relative position of the 1586K residue in the D4-S6 segment and that atypical inactivation in Y1586K occurs via an electrostatic interaction in or near the inner pore region.

Cocaine block of human cardiac (hH1) and rat skeletal (mu1) muscle sodium channels was examined under whole-cell voltage clamp in transiently transfected HEK293t cells. Low affinity block of resting mu1 and hH1 channels at -180 mV was the same, and high affinity block of inactivated channels at -70 mV was the same. Cocaine block of hH1 channels was greater than block of mu1 channels at voltages between -120 mV and -90 mV, suggesting that greater steady-state inactivation of hH1 channels in this voltage range makes them more susceptible to cocaine block. We induced shifts in the voltage dependence of steady-state inactivation at mu1 and hH1 channels by constructing mu1/hH1 channel chimeras or by coexpressing the wild-type channels with the rat brain beta1 subunit. In contrast to several previous reports, coexpression of the rat brain beta1 subunit with mu1 or hH1 produced large positive shifts in steady-state inactivation. Shifts in the voltage dependence of steady-state inactivation elicited linear shifts in steady-state cocaine block, yet these manipulations did not affect the cocaine affinity of resting or inactivated channels. These data, as well as simulations used to predict block, indicate that state-dependent cocaine block depends on both steady-state inactivation and channel activation, although inactivation appears to have the predominant role.