Long-term follow-up of a patient with type 2 Timothy syndrome and the partial efficacy of mexiletine.

We report a detailed case of type 2 TS due to a p.(Gly402Ser) mutation in exon 8 of the CACNA1C gene. The patient shows a marked prolongation of repolarization with a mean QTc of 540 ms. He shows no structural heart disease, syndactyly, or cranio-facial abnormalities. However, he shows developmental delays, without autism, and dental abnormalities. The cardiac phenotype is very severe, with a resuscitated cardiac arrest at 2.5 years of age, followed by 26 appropriate shocks during nine years of follow-up. Adding mexiletine to nadolol resulted in a reduction of the QTc and a slight decrease in the number of appropriate shocks.

Discovery of N-Aryloxypropylbenzylamines as Voltage-Gated Sodium Channel NaV 1.2-Subtype-Selective Inhibitors.

We previously reported that a lipophilic N-(4'-hydroxy-3',5'-di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give (2)) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human NaV 1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective NaV 1.2 inhibitors and >500 times less potent in inhibiting NaV 1.6 channels. On the other hand, a derivative (compound 4) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC50 value of 49.9±1.6 mg kg-1 . As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.

Discovery of a new mexiletine-derived agonist of the hERG K+ channel.

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in the rapid component (IKr) of cardiac action potential repolarization phase. A large number of structurally different compounds block hERG and cause a high risk of arrhythmias. Among the drugs that block hERG channel, a few compounds have been identified as hERG channel activators. Such compounds may be useful, at least in theory, for the treatment of long term QT syndrome. Here we describe a new activator of hERG channel, named MC450. This compound is a symmetric urea, derived from (R)-mexiletine. Using patch-clamp recordings, we found that MC450 increased the activation current of hERG channel, with an EC50 of 41±4µM. Moreover MC450 caused a depolarizing shift in the voltage dependence of inactivation from -64.1±1.2mV (control), to -35.9±1.4mV, whereas it had no effect on the voltage dependence of activation. Furthermore, MC450 slowed current inactivation and the effect of MC450 was attenuated by the inactivation-impaired double mutant G628C/S631C.

Antiarrhythmic Mexiletine: A Review on Synthetic Routes to Racemic and Homochiral Mexiletine and its Enantioseparation.

Mexiletine is an oral class IB antiarrhythmic agent. Although it was primarily studied for the treatment of ventricular arrhythmias, it has been demonstrated to be useful also for the treatment of chronic painful diabetic neuropathy, neuropathic pain, skeletal muscle channelopathies, and recently amyotrophic lateral sclerosis. This review presents a detailed report on the different synthetic routes to racemic and homochiral mexiletine developed in the last decades, as well as analytical studies regarding enantioseparation methods and enantiomeric excess determination. Finally, some analogues of mexiletine reported in the literature, most of which along with pharmacological studies, have been mentioned.

Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism.

Semi-rational Directed Evolution of Monoamine Oxidase for Kinetic Resolution of rac-Mexiletine.

Semi-rational directed evolution was applied to the D5 variant of monoamine oxidase from Aspergillus niger (MAO-N-D5) with the aim of deriving the more desirable (R)-mexiletine through the kinetic resolution of mexiletine enantiomers. Although MAO-N-D5 shows no activity towards rac-mexiletine, theoretical molecular docking studies revealed the potential binding conformations of both mexiletine enantiomers and MAO-N-D5. The key factors affecting the catalytic activity and specificity were identified. Based on the docking results, six residues in the binding pocket and along the binding pathway were selected as key sites for saturation mutagenesis of MAO-N-D5. Through several rounds of screening and combinatorial experiments, two active MAO variants with high enantioselectivities towards (S)-mexiletine evolved, namely A-1 (F210V/L213C, E = 101) and AC-1 (F210V/I367T, E = 69). Molecular simulation experiments indicated that the introduced activity of these variants may be due to the reduced steric hindrance in the binding pocket of the relatively small-sized amino acid residues, a synergetic effect of the entrance residue mutation, and the formation of a new disulfide bond.

Mexiletine metabolites: a review.

Mexiletine belongs to class IB antiarrhythmic drugs and it is still considered a drug of choice for treating myotonias. However some patients do not respond to mexiletine or have significant side effects limiting its use; thus, alternatives to this drug should be envisaged. Mexiletine is extensive metabolized in humans via phase I and phase II reactions. Only a small fraction (about 10%) of the dose of mexiletine administered is recovered without modifications in urine. Although in the past decades Mex metabolites were reported to be devoid of biological activity, recent studies seem to deny this assertion. Actually, several hydroxylated metabolites showed pharmacological activity similar to that of Mex, thus contributing to its clinical profile. Purpose of this review is to summarize all the studies proposed till now about mexiletine metabolites, regarding structureactivity relationship studies as well as synthetic strategies. Biological and analytical studies will be also reported.

Bisphenol A binds to the local anesthetic receptor site to block the human cardiac sodium channel.

Bisphenol A (BPA) has attracted considerable public attention as it leaches from plastic used in food containers, is detectable in human fluids and recent epidemiologic studies link BPA exposure with diseases including cardiovascular disorders. As heart-toxicity may derive from modified cardiac electrophysiology, we investigated the interaction between BPA and hNav1.5, the predominant voltage-gated sodium channel subtype expressed in the human heart. Electrophysiology studies of heterologously-expressed hNav1.5 determined that BPA blocks the channel with a K(d) of 25.4±1.3 µM. By comparing the effects of BPA and the local anesthetic mexiletine on wild type hNav1.5 and the F1760A mutant, we demonstrate that both compounds share an overlapping binding site. With a key binding determinant thus identified, an homology model of hNav1.5 was generated based on the recently-reported crystal structure of the bacterial voltage-gated sodium channel NavAb. Docking predictions position both ligands in a cavity delimited by F1760 and contiguous with the DIII-IV pore fenestration. Steered molecular dynamics simulations used to assess routes of ligand ingress indicate that the DIII-IV pore fenestration is a viable access pathway. Therefore BPA block of the human heart sodium channel involves the local anesthetic receptor and both BPA and mexiletine may enter the closed-state pore via membrane-located side fenestrations.

Enantioselective drug-protein interaction between mexiletine and plasma protein.

OBJECTIVES: This study examined the interaction of mexiletine enantiomers with human plasma, human serum albumin (HSA), and human α1-acid glycoprotein (hAGP), and characterized the binding modes of mexiletine enantiomers with hAGP in the molecular level. METHODS: Enantiomer separation of mexiletine was performed using precolumn derivatization chiral HPLC. The ultrafiltration technique was used to separate the free mexiletine in plasma matrix. Molecular dynamics simulations and free energy calculations were assessed using molecular mechanics and the generalized Born surface area method. KEY FINDINGS: Significant differences in enantioselective binding to human plasma were observed (R>S). The hAGP-mexiletine binding profile exhibited similar enantioselectivity (R>S) to that in human plasma, whereas HSA-mexiletine interaction was S>R at pH 7.4. Moreover, the results of comparative studies indicated that mexiletine had the highest binding affinity for F1-S, a variant of hAGP. Based on the computational studies, residues such as Arg90, Leu79, Ser89 and Phe89 showed an energy difference of more than -0.35 kcal/mol between the enantiomers. CONCLUSIONS: hAGP may be one of the key proteins leading to the enantioselective protein bindings of mexiletine in human plasma (R>S). The residues Arg90, Leu79, Ser89 and Phe89 of hAGP may have important roles in the observed enantioselectivity.

Stereoselective binding of mexiletine and ketoprofen enantiomers with human serum albumin domains.

AIM: To investigate the stereoselective binding of mexiletine or ketoprofen enantiomers with different recombinant domains of human serum albumin (HSA). METHODS: Three domains (HSA DOM I, II and III) were expressed in Pichia pastoris GS115 cells. Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains. The binding properties of the standard ligands, digitoxin, phenylbutazone and diazepam, and the chiral drugs to HSA domains were investigated using ultrafiltration. The concentrations of the standard ligands, ketoprofen and mexiletine were analyzed with HPLC. RESULTS: The recombinant HSA domains were highly purified as shown by SDS-PAGE and Western blotting analyses. The standard HSA ligands digitoxin, phenylbutazone and diazepam selectively binds to DOM I, DOM II and DOM III, respectively. For the chiral drugs, R-ketoprofen showed a higher binding affinity toward DOM III than S-ketoprofen, whereas S-mexiletine bound to DOM II with a greater affinity than R-mexiletine. CONCLUSION: The results demonstrate that HSA DOM III possesses the chiral recognition ability for the ketoprofen enantiomers, whereas HSA DOM II possesses that for the mexiletine enantiomers.

Synthesis and toxicopharmacological evaluation of m-hydroxymexiletine, the first metabolite of mexiletine more potent than the parent compound on voltage-gated sodium channels.

The first synthesis of m-hydroxymexiletine (MHM) has been accomplished. MHM displayed hNav1.5 sodium channel blocking activity, and tests indicate it to be ∼2-fold more potent than the parent mexiletine and to have more favorable toxicological properties than mexiletine. Thus, MHM and possible related prodrugs might be studied as agents for the treatment of arrhythmias, neuropathic pain, and myotonias in substitution of mexiletine (metabolite switch), which has turned out to be tainted with common toxicity.

Enantioselective determination of mexiletine and its metabolites p-hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal-phase liquid chromatography-tandem mass spectrometry: application to pharmacokinetics.

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.

Evaluation of the pharmacological activity of the major mexiletine metabolites on skeletal muscle sodium currents.

BACKGROUND AND PURPOSE: Mexiletine (Mex), an orally effective antiarrhythmic agent used to treat ventricular arrhythmias, has also been found to be effective for myotonia and neuropathic pain. It is extensively metabolized in humans but little information exists about the pharmacodynamic properties of its metabolites. EXPERIMENTAL APPROACH: To determine their contribution to the clinical activity of Mex, p-hydroxy-mexiletine (PHM), hydroxy-methyl-mexiletine (HMM), N-hydroxy-mexiletine (NHM) (phase I reaction products) and N-carbonyloxy beta-D-glucuronide (NMG) (phase II reaction product) were tested on sodium currents (I(Na)) of frog skeletal muscle fibres. Sodium currents were elicited with depolarizing pulses from different holding potentials (HP=-140, -100, -70 mV) and stimulation frequencies (0.25, 0.5, 1, 2, 5, 10 Hz) using the vaseline-gap voltage-clamp method. KEY RESULTS: All the hydroxylated derivatives blocked the sodium channel in a voltage- and use-dependent manner. The PHM, HMM and NHM metabolites were up to 10-fold less effective than the parent compound. However, HMM showed a greater use-dependent behaviour (10 Hz), compared to Mex and the other metabolites. Similar to Mex, these products behaved as inactivating channel blockers. Conjugation with glucuronic acid (NMG) resulted in almost complete abolition of the pharmacological activity of the parent compound. CONCLUSIONS AND IMPLICATIONS: Thus, although less potent, the phase I metabolites tested demonstrated similar pharmacological behaviour to Mex and might contribute to its clinical profile.

Catalytic roles of CYP2D6.10 and CYP2D6.36 enzymes in mexiletine metabolism: in vitro functional analysis of recombinant proteins expressed in Saccharomyces cerevisiae.

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one-third of the medicines in current clinical use and exhibits genetic polymorphism with interindividual differences in metabolic activity. To precisely investigate the effect of CYP2D6*10B and CYP2D6*36 frequently found in Oriental populations on mexiletine metabolism in vitro, CYP2D6 proteins of wild-type (CYP2D6.1) and variants (CYP2D6.10 and CYP2D6.36) were heterologously expressed in yeast cells and their mexiletine p- and 2-methyl hydroxylation activities were determined. Both variant CYP2D6 enzymes showed a drastic reduction of CYP2D6 holo- and apoproteins compared with those of CYP2D6.1. Mexiletine p- and 2-methyl hydroxylation activities on the basis of the microsomal protein level at the single substrate concentration (100 microM) of variant CYP2D6s were less than 6% for CYP2D6.10 and 1% for CYP2D6.36 of those of CYP2D6.1. Kinetic analysis for mexiletine hydroxylation revealed that the affinity toward mexiletine of CYP2D6.10 and CYP2D6.36 was reduced by amino acid substitutions. The Vmax and Vmax/Km values of CYP2D6.10 on the basis of the microsomal protein level were reduced to less than 10% of those of CYP2D6.1, whereas the values on the basis of functional CYP2D6 level were comparable to those of CYP2D6.1. Although it was impossible to estimate the kinetic parameters for the mexiletine hydroxylation of CYP2D6.36, the metabolic ability toward mexiletine was considered to be poorer not only than that of CYP2D6.1 but also than that of CYP2D6.10. The same tendency was also observed in kinetic analysis for bufuralol 1''-hydroxylation as a representative CYP2D6 probe. These findings suggest that CYP2D6*36 has a more drastic impact on mexiletine metabolism than CYP2D6*10.

Inhibitory effects of psychotropic drugs on mexiletine metabolism in human liver microsomes: prediction of in vivo drug interactions.

Mexiletine, an anti-arrhythmic agent, is used for the control of ventricular arrhythmias and for neuropathic pain from cancer or diabetes mellitus. It is sometimes used together with psychotropic drugs in patients with depression, schizophrenia or sleep disorder. It is metabolized mainly by cytochrome P450 (CYP) 2 D 6 and, to a minor extent, by CYP1A2. To predict possible drug interactions between mexiletine and psychotropic drugs, the inhibitory effects of 14 psychotropic drugs (phenytoin, carbamazepine, fluvoxamine, paroxetine, fluoxetine, citalopram, sertraline, imipramine, desipramine, haloperidol, thioridazine, olanzapine, etizolam, and quazepam) on mexiletine metabolism in human liver microsomes were determined. Fluoxetine (Ki=0.6+/- 0.1 microM), sertraline (Ki=7.6+/- 0.8 microM) and desipramine (Ki=3.2+/- 0.5 microM) competitively inhibited the mexiletine p-hydroxylation in human liver microsomes. Thioridazine (Kis=0.5+/- 0.2 microM; Kii =3.6+/-1.6 microM) and paroxetine (Kis=1.7+/- 0.7 microM; Kii=3.6+/- 0.9 microM) exhibited a mixed-type inhibition (competitive and non-competitive) toward mexiletine p-hydroxylation in human liver microsomes. The changes of the in vivo clearance of mexiletine by the psychotropic drugs were predicted by 1+(I/Ki) using the in vitro Ki and unbound inhibitor concentrations in liver. The values were calculated as 2.4 for paroxetine, 5.5 for fluoxetine, 1.1 for sertraline, 2.8 for desipramine and 2.2 for thioridazine. In addition, paroxetine exhibited a mechanism-based inactivation with Ki=0.7 microM and Kinact=0.15 min(-1). The present study predicted the possibility of drug interactions between mexiletine and paroxetine, fluoxetine, desipramine, and thioridazine in clinical use.

A multifunctional aromatic residue in the external pore vestibule of Na+ channels contributes to the local anesthetic receptor.

Voltage-gated Na(+) (Na(v)) channels are responsible for initiating action potentials in excitable cells and are the targets of local anesthetics (LA). The LA receptor is localized to the cytoplasmic pore mouth formed by the S6 segments from all four domains (DI-DIV) but several outer pore-lining residues have also been shown to influence LA block (albeit somewhat modestly). Many of the reported amino acid substitutions, however, also disrupt the inactivated conformations that favor LA binding, complicating the interpretation of their specific effects on drug block. In this article, we report that an externally accessible aromatic residue in the Na(v) channel pore, DIV-Trp1531, when substituted with cysteine, completely abolished LA block (e.g., 300 microM mexiletine induced a use-dependent block with 65.0 +/- 2.9% remaining current and -11.0 +/- 0.6 mV of steady-state inactivation shift of wild-type (WT) channels versus 97.4 +/- 0.7% and -2.4 +/- 2.1 mV of W1531C, respectively; p < 0.05) without destabilizing fast inactivation (complete inactivation at 20 ms at -20 mV; V(1/2) = -70.0 +/- 1.6 mV versus -48.6 +/- 0.5 mV of WT). W1531C also abolished internal QX-222 block (200 microM; 98.4 +/- 3.4% versus 54.0 +/- 3.2% of WT) without altering drug access. It is interesting that W1531Y restored WT blocking behavior, whereas W1531A channels exhibited an intermediate phenotype. Together, our results provide novel insights into the mechanism of drug action, and the structural relationship between the LA receptor and the outer pore vestibule.

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.

Mexiletine carbonyloxy beta-D-glucuronide: a novel metabolite in human urine.

1. The study was performed to isolate and characterize a glucuronic acid conjugate of mexiletine that releases mexiletine on acid hydrolysis from urine samples obtained from healthy volunteers following a single oral dose of mexiletine. 2. The [M-H]- ion of the isolated metabolite was observed at m/z 398 in the negative electrospray ionization mass spectrum. This mass number was 44 higher than that of the product generated when mexiletine is subjected to direct glucuronidation. In positive-ion mode, collision-induced dissociation of the quasimolecular ion [M+NH4]+, m/z 417, gave product ions at m/z 224, 180 and 58. These mass spectral data indicated that the metabolite contained a carbonyloxy moiety in its structure in addition to mexiletine and a glucuronic acid moiety. 3. The presence of this carbonyloxy moiety was further supported by the following chemical reactions. When the metabolite was hydrolysed with an aqueous solution of 1 M sodium hydroxide at room temperature, mexiletine was released, whereas the N-methoxycarbonyl derivative of mexiletine was obtained after treatment of the metabolite with methanolic sodium hydroxide solution. 4. The results indicated that the structure of the isolated metabolite was the N-carbonyloxy beta-D-glucuronic acid conjugate of mexiletine.

Role of specific cytochrome P450 enzymes in the N-oxidation of the antiarrhythmic agent mexiletine.

1. Mexiletine is extensively metabolized in man by C- and N-oxidation and the aim of the present study was to characterize major cytochrome P450 enzyme(s) involved in the formation of N-hydroxymexiletine. 2. Incubations with genetically engineered microsomes indicated that the formation rate of N-hydroxymexiletine was highest in the presence of microsomes expressing high levels of either CYP1A2 or CYP2E1 and the formation of N-hydroxymexiletine by human liver microsomes was inhibited about 40% by antibodies directed against CYP1A1/1A2 or CYP2E1. Additional incubations demonstrated that formation of N-hydroxymexiletine was decreased 47 and 51% by furafylline, 40 microm and 120 microm, respectively, and decreased 55 and 67% by alpha-naphthoflavone, 1 microm and 3 microm, respectively (all p < 0.05 versus control). 3. The formation rate of N-hydroxymexiletine in human liver microsomes was highly correlated with CYP2B6 (RS-mexiletine, r = 0.7827; R-(-)-enantiomer, r = 0.7034; S-(+)-enantiomer, r = 0.7495), CYP2E1 (S-(+)-enantiomer, r = 0.7057) and CYP1A2 (RS-mexiletine, r = 0.5334; S-(+)-enantiomer, r = 0.6035). 4. In conclusion, we have demonstrated that CYP1A2 is a major human cytochrome P450 enzyme involved in the formation of N-hydroxymexiletine. However, other cytochrome P450 enzymes (CYP2E1 and CYP2B6) also appear to play a role in the N-oxidation of this drug.

Mexiletine block of disease-associated mutations in S6 segments of the human skeletal muscle Na(+) channel.

1. Over twenty different missense mutations in the alpha-subunit of the adult skeletal muscle Na(+) channel (hSkM1) have been identified as a cause of myotonia or periodic paralysis. We examined state-dependent mexiletine block for mutations involving the putative binding site in S6 segments (V445M, S804F, V1293I, V1589M and M1592V). Whole-cell Na(+) currents were measured from wild-type (WT) and mutant channels transiently expressed in HEK cells. 2. Use-dependent block (10 ms pulses to -10 mV, at 20 Hz) in 100 microM mexiletine was reduced modestly by mutations in IVS6 (V1589M, M1592V) and enhanced by the mutation in IS6 (V445M). For mutations in IIS6 (S804F) and IIIS6 (V1293I) use-dependent block was not statistically different from that of wild-type channels. 3. Resting-state block (10 ms pulses to -10 mV from -150 mV, at 0.1 Hz) of S6 mutants was comparable to that of WT (dissociation constant for resting channels, K(R) = 650 +/- 40 microM, n = 9). The S6 mutant with the greatest change in K(R) was V445M (K(R) = 794 +/- 45 microM, n = 5), but this difference was only marginally significant (P = 0.047). 4. A modified technique for estimating local anaesthetic affinity of inactivated channels was developed to reduce errors due to slow inactivation and to failure of drug binding to reach equilibrium. Mexiletine affinity for inactivated channels was reduced by mutations in IVS6 (V1589M: dissociation constant for the inactivated state (K(I)) = 44.7 microM; M1592V: K(I) = 40.0 microM) and increased by the mutation in IS6 (V445M: K(I) = 15.0 microM), compared to wild-type channels (K(I) = 28.3 microM). 5. We conclude that the disease-associated S6 mutations in domains I-IV cause at most a 2-fold change in inactivated state affinity and have even less of an effect on resting block. Model simulations show that the reduced use-dependent block of IVS6 mutants derives primarily from an increased off-rate at hyperpolarized potentials, whereas the enhanced use-dependent block of the IS6 mutant was due to a higher affinity for inactivated V445M channels.