Biomedical subjects

Ging Kuo Wang

Publications and source records attributed to Ging Kuo Wang.

At least 19 recordsLinked to original sources

PubMed2007

Serine-401 as a batrachotoxin- and local anesthetic-sensing residue in the human cardiac Na+ channel.

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.

Amino Acid Sequence↗

PubMed2006

Preferential block of inactivation-deficient Na+ currents by capsaicin reveals a non-TRPV1 receptor within the Na+ channel.

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.

Animals↗

PubMed2006

Inhibition of Nav1.7 and Nav1.4 sodium channels by trifluoperazine involves the local anesthetic receptor.

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 Na(v)1.7 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 muM TFP induce major decreases in Na(v)1.7 and Na(v)1.4 current amplitudes and that 2 muM TFP causes hyperpolarizing shifts in the steady-state inactivation of Na(v)1.7 and Na(v)1.4. 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 Na(v)1.7 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.

Action Potentials↗

PubMed2006

Time-dependent block and resurgent tail currents induced by mouse beta4(154-167) peptide in cardiac Na+ channels.

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.

Amino Acid Substitution↗

PubMed2005

How batrachotoxin modifies the sodium channel permeation pathway: computer modeling and site-directed mutagenesis.

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.

Amino Acid Sequence↗

PubMed2005

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 alpha-subunit (hH1(alpha)) of human cardiac Na(+) channels (hNav1.5) plus beta(1)-subunits. Extracellular application of 5 microM 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 IC(50) values of 4.0 +/- 0.4 microM for I(Na) peak (I(Na peak)) and 0.9 +/- 0.1 microM 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 microM 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 microM 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(alpha) induce inactivation-deficient I(Na) and that n-3 PUFAs inhibit mutant I(Na).

Cell Line↗

PubMed2005

Block of inactivation-deficient cardiac Na(+) channels by acetyl-KIFMK-amide.

The Na(+) channel alpha-subunit contains an IFM motif that is critical for the fast inactivation process. In this study, we sought to determine whether an IFM-containing peptide, acetyl-KIFMK-amide, blocks open cardiac Na(+) channels via the inner cavity. Intracellular acetyl-KIFMK-amide at 2mM elicited a rapid time-dependent block (tau=0.24 ms) of inactivation-deficient human heart Na(+) channels (hNav1.5-L409C/A410W) at +50 mV. In addition, a peptide-induced tail current appeared conspicuously upon repolarization, suggesting that the activation gate cannot close until acetyl-KIFMK-amide is cleared from the open pore. Repetitive pulses (+50 mV for 20 ms at 1Hz) produced a substantial use-dependent block of both peak and tail currents by approximately 65%. A F1760K mutation (hNav1.5-L409C/A410W/F1760K) abolished the use-dependent block by acetyl-KIFMK-amide and hindered the time-dependent block. Competition experiments showed that acetyl-KIFMK-amide antagonized bupivacaine binding. These results are consistent with a model that two acetyl-KIFMK-amide receptors exist in proximity within the Na(+) channel inner cavity.

Amino Acid Substitution↗

PubMed2005

Tryptophan substitution of a putative D4S6 gating hinge alters slow inactivation in cardiac sodium channels.

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.

Amino Acid Sequence↗

PubMed2005

Ephedrine blocks rat sciatic nerve in vivo and sodium channels in vitro.

BACKGROUND: The sympathomimetic drug ephedrine has been used intrathecally as the sole local anesthetic for labor and delivery. Because ephedrine may be a useful adjuvant to local anesthetics, the authors investigated the local anesthetic properties of ephedrine in a rat sciatic nerve block model and the underlying mechanism in cultured cells stably expressing Na channels. METHODS: After approval of the animal protocol, the sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs, 0.2 ml ephedrine at 0.25, 1, 2.5, or 5% and/or bupivacaine at 0.125% was injected, and the wound was closed. Motor and sensory/nociceptive functions were evaluated by the force achieved by pushing against a balance and the reaction to pinch, respectively. The whole cell configuration of the patch clamp technique was used to record Na currents from human embryonal kidney cells stably transfected with Nav1.4 channels. RESULTS: The nociception blockade was significantly longer than the motor blockade at test doses of 2.5 and 5% of ephedrine, or when 1% ephedrine was combined with 0.125% bupivacaine (analysis of variance with repeated measures, P < 0.001, n = 8/group). In vitro, the 50% inhibitory concentrations of ephedrine at -150 and -60 mV were 1,043 +/- 70 and 473 +/- 13 mum, respectively. High-frequency stimulation revealed a use-dependent block of 18%, similar to most local anesthetics. CONCLUSIONS: Because ephedrine's properties are at least partly due to Na channel blockade, detailed histopathologic investigations are justified to determine the potential of ephedrine as an adjuvant to clinically used local anesthetics.

Adrenergic alpha-Agonists↗

PubMed2004

Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway.

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 were >12-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.

Anesthetics, Local↗

PubMed2004

Peripherally administered amitriptyline derivatives have differential anti-allodynic effects in a rat model of neuropathic pain.

We assessed if derivatives of amitriptyline could alleviate mechanical allodynia in a rat model of neuropathic pain. N-methyl amitriptyline (NMA), and amitriptyline HCl (AHC) were compared to evaluate the antiallodynic effects produced by systemic and peripheral administration. Under general anesthesia with halothane, neuropathic injury was produced in rats by tightly ligating the left L5 and L6 spinal nerves. All animals developed neuropathic pain behaviors within 7 days after the surgery. For systemic delivery, three groups of rats were injected intraperitoneally with NMA (15 mg/kg), AHC (12.5 mg/kg), or saline. For peripheral routes of injection, rats were divided into 3 x 3 groups and injected with 100, 330, and 1000 nmol/kg doses of each NMA, AHC and 0.9% normal saline. NMA and AHC suppressed mechanical allodynia following peripheral administration but not following intraperitoneal injection. NMA was less potent but showed a longer-acting response than AHC. This study shows that NMA can be used to relieve mechanical allodynia and is a longer-acting agent than AHC.

Amitriptyline↗

PubMed2004

Electrophysiologic properties of lidocaine, cocaine, and n-3 fatty-acids block of cardiac Na+ channels.

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.

Animals↗

PubMed2004

State-dependent block of voltage-gated Na+ channels by amitriptyline via the local anesthetic receptor and its implication for neuropathic pain.

Amitriptyline is a tricyclic antidepressant, which also alleviates various pain syndromes at its therapeutic plasma concentration (0.36-0.90 microM). Accumulated evidence suggests that such efficacy may be due to block of voltage-gated Na(+) channels. The Na(+) channel alpha-subunit protein consists of four homologous domains (D1-D4), each with six transmembrane segments (S1-S6). The aims of this study were to locate the amitriptyline receptor in the Na(+) channel alpha-subunit and to compare the amitriptyline affinity in open, inactivated, and resting states of the Na(+) channel. Wild-type and mutant rat skeletal muscle alpha-subunit Na(+) channels were expressed in human embryonic kidney cells and assayed under whole-cell voltage clamp conditions. Our results indicate that the amitriptyline receptor overlaps with the local anesthetic receptor to a great extent in Na(+) channels. Residues N434 (at D1-S6), L1280 (D3-S6), and F1579 (D4-S6) may jointly form parts of the amitriptyline/local anesthetic receptor, with residue L1280 being most critical for amitriptyline binding. Open-channel block by amitriptyline was assessed in inactivation-deficient Na(+) channels and compared with the resting- and inactivated-channel block in wild-type channels. The open-channel block by amitriptyline has the highest affinity, with a 50% inhibitory concentration (IC(50)) of 0.26 microM. The inactivated-channel block by amitriptyline had a weaker affinity (0.51 microM), whereas the resting-channel displayed the weakest affinity (33 microM). We hypothesize that selective block of both persistent late openings and the inactivated state of neuronal Na(+) channel isoforms by amitriptyline also occurs at its therapeutic concentration and likely contributes to its efficacy in pain syndromes.

Amitriptyline↗

PubMed2004

Phase Ia and Ib study of amitriptyline for ulnar nerve block in humans: side effects and efficacy.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Adult↗

PubMed2004

Local anesthetic properties of a novel derivative, N-methyl doxepin, versus doxepin and bupivacaine.

UNLABELLED: Among various tricyclic antidepressants, doxepin and amitriptyline are also long-acting local anesthetics. We synthesized a new compound, N-methyl doxepin, and investigated whether this derivative possesses local anesthetic properties. N-methyl doxepin and doxepin were tested in a rat sciatic nerve model at 2.5, 5.0, and 10 mM. Proprioceptive, motor, and nociceptive blockade were evaluated and compared with those induced by 0.5% bupivacaine. Block of Na(+) channels by N-methyl doxepin and doxepin was assessed in cultured pituitary tumor cells under voltage clamp conditions. N-methyl doxepin elicited complete nociceptive blockade that generally lasted longer than that caused by doxepin (e.g., approximately 7.4 h versus 5.3 h at 10 mM). Significant differences were observed for full recovery of function at all concentrations and for the duration of complete blockade except at 2.5 mM. Bupivacaine at 0.5% (15.4 mM) was less effective in producing complete blockade (approximately 1.5 h) than N-methyl doxepin and doxepin. Both doxepin and N-methyl doxepin were potent Na(+) channel blockers, although N-methyl doxepin displayed a slower wash-in rate. No morphological alterations were detected in cross-sectioned sciatic nerve specimens with these three drugs. We conclude that N-methyl doxepin is a potent Na(+) channel blocker and a long-acting local anesthetic for rat sciatic nerve blockade. IMPLICATIONS: N-methyl doxepin and doxepin are both potent Na(+) channel blockers; they elicit rat sciatic nerve block lasting longer than that induced by bupivacaine and seem to be nontoxic to peripheral nerves at concentrations up to 10 mM.

Anesthetics, Local↗

PubMed2003

Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels.

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.

Anti-Arrhythmia Agents↗

PubMed2003

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.

Anti-Arrhythmia Agents↗

PubMed2003

Veratridine block of rat skeletal muscle Nav1.4 sodium channels in the inner vestibule.

Veratridine (VTD) is an alkaloid toxin found in Liliaceae plants. VTD causes persistent opening of the voltage-gated Na+ channel and reduces its single-channel conductance by 75 %. The mechanisms for these different VTD actions are unknown. Recent reports indicate that the VTD receptor aligns closely with the local anaesthetic (LA) receptor, which resides at D1S6, D3S6 and D4S6 of the Na+ channel alpha-subunit. To study this alignment, we created a mutant with cysteine substitutions at three S6 residues (rNav1.4-N434C/L1280C/F1579C). Under voltage-clamp conditions, amitriptyline and bupivacaine remained as potent blockers of this mutant channel when expressed in human embryonic kidney cells, whereas VTD completely failed to cause persistent opening. Unexpectedly, VTD at 100 microM progressively blocked mutant currents by 90.4 +/- 1.6 % (n = 5), as assayed at 0.1 Hz for 15 min. This VTD block was reversed little during wash-off: approximately 70 % of mutant currents did not return in 30 min. An increase in channel opening either by repetitive pulses at 1 Hz or by the inhibition of the fast inactivation hastened the VTD block. Co-application of amitriptyline or bupivacaine, which targeted the LA receptor, prevented this VTD block. Our data suggest that (a) the VTD receptor and the LA receptor overlap extensively, (b) receptor-bound VTD lies in the inner vestibule, and (c) VTD blocks this mutant channel as a bona fide Na+ channel blocker. We propose that VTD likewise blocks the wild-type open Na+ channel, albeit partially, to decrease the unitary conductance and to stabilize the open conformation for persistent opening.

Amino Acid Sequence↗