Biophysical phenotypes of SCN5A mutations causing long QT and Brugada syndromes.

Long QT and Brugada syndromes are two hereditary cardiac diseases. Brugada syndrome has so far been associated with only one gene, SCN5A, which encodes the cardiac sodium channel. However, in long QT syndrome (LQTS) at least six genes, including the SCN5A, are implicated.

The distinct HERG missense mutation L564P causes long QT syndrome in one French Canadian family.

Background: Long QT syndrome is a congenital abnormality of cardiac repolarization causing syncope and sudden death from ventricular tachyarrhythmias known as torsades de pointes. This hereditary cardiac disorder often shows an increase of the value of the QT interval corrected for heart rate over 0.45 s in a 12-lead electrocardiogram.

Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes.

Unlabelled: Familial long QT syndrome (LQTS) and Brugada syndrome are two distinct human hereditary cardiac diseases known to cause ventricular tachyarrhythmias (torsade de pointes) and idiopathic ventricular fibrillation, respectively, which can both lead to sudden death.

Objective: In this study we have identified and electrophysiologically characterized, in patients having either LQTS or Brugada syndrome, three mutations in SCN5A (a cardiac sodium channel gene).

Method: The mutant channels were expressed in a mammalian expression system and studied by means of the patch clamp technique.

SCN5A mutation (T1620M) causing Brugada syndrome exhibits different phenotypes when expressed in Xenopus oocytes and mammalian cells.

Brugada syndrome is a hereditary cardiac disease causing abnormal ST segment elevation in the ECG, right bundle branch block, ventricular fibrillation and sudden death. In this study we characterized a new mutation in the SCN5A gene (T1620M), causing the Brugada syndrome. The mutated channels were expressed in both Xenopus leavis oocytes and in mammalian tsA201 cells with and without the beta-subunit and studied using the patch clamp technique.

Cysteine scanning analysis of the IFM cluster in the inactivation gate of a human heart sodium channel.

Unlabelled: The conserved isoleucine-phenylalanine-methionine (IFM) hydrophobic cluster located in the III-IV linker of voltage-gated sodium channels has been identified as a major component of the fast inactivation gate in these channels.

Objectives: The aim of our study was to probe the contribution of each amino acids of the IFM cluster to the inactivation.

Methods: A combination of site-directed mutagenesis, cysteine covalent modification and electrophysiological recording techniques were used to elucidate the role of isoleucine1485 and methionine1487 on hH1 sodium channels expressed in tsA201 cells.

Modulation of HERG potassium channel properties by external pH.

We expressed the human eag-related gene (HERG), known to encode for the cardiac potassium channel IKr, in Chinese hamster ovary (CHO) cells. This study investigated effects of external pH (pHo) on HERG current properties using the whole-cell patch-clamp technique. We observed that current amplitude was decreased and kinetics of activation and deactivation were faster when pHo was lowered from 7.

Thioridazine lengthens repolarization of cardiac ventricular myocytes by blocking the delayed rectifier potassium current.

Proarrhythmia has been observed with the antipsychotic agent thioridazine (THIO). The mechanisms underlying these effects are unknown. The objectives of this study were 1) to characterize the effects of THIO on cardiac repolarization and 2) to determine whether lengthening of the Q-T interval could be explained by blocking major K+-repolarizing currents.

Extrapore residues of the S5-S6 loop of domain 2 of the voltage-gated skeletal muscle sodium channel (rSkM1) contribute to the mu-conotoxin GIIIA binding site.

The tetradomain voltage-gated sodium channels from rat skeletal muscle (rSkM1) and from human heart (hH1) possess different sensitivities to the 22-amino-acid peptide toxin, mu-conotoxin GIIIA (mu-CTX). rSkM1 is sensitive (IC50 = 51.4 nM) whereas hH1 is relatively resistant (IC50 = 5700 nM) to the action of the toxin, a difference in sensitivity of >100-fold.

Electrophysiological study of chimeric sodium channels from heart and skeletal muscle.

The alpha-subunit cDNAs encoding voltage-sensitive sodium channels of human heart (hH1) and rat skeletal muscle (rSkM1) have been expressed in the tsA201 mammalian cell line, in which inactivation properties appear to be normal in contrast to Xenopus oocytes. A series of rSkM1/hH1 chimeric sodium channels has been evaluated to identify the domains of the alpha-subunits that are responsible for a set of electrophysiological differences between hH1 and rSkM1, namely, midpoints and slope factors of steady-state activation and inactivation, inactivation kinetics and recovery from inactivation kinetics and their voltage-dependence. The phenotype of chimeric channels in which each hH1 domain was successively introduced into a rSkM1 alpha-subunit framework confirmed the following conclusions.

Myotonic dystrophy kinase modulates skeletal muscle but not cardiac voltage-gated sodium channels.

Altered modulation of skeletal muscle voltage-gated sodium channels by myotonic dystrophy kinase (DMPK) has been proposed as a possible mechanism underlying myotonia in this disease. We examined the effect of a recombinant mouse DMPK on the functional properties of human skeletal muscle (hSkM1) and cardiac (hH1) voltage-gated sodium channels in the Xenopus oocyte expression system. Co-expression of DMPK with hSkM1 in oocytes resulted in significantly lower peak sodium current amplitude as compared to cells expressing hSkM1 alone in agreement with a previous report.

Restoration of fast inactivation in an inactivation-defective human heart sodium channel by the cysteine modifying reagent benzyl-MTS: analysis of IFM-ICM mutation.

It has been suggested that the region linking domain III and IV of voltage-gated sodium channels forms the inactivation gate. A combination of site-directed mutagenesis, cysteine covalent modification, and electrophysiological recording techniques was used to identify the role of the Phe1486, a conserved phenylalanine residue located in the III-IV linker of Na+ channels. This Phe1486 is part of a hydrophobic amino acid cluster (IFM) that was proposed to play an essential role in the fast inactivation of voltage-gated sodium channels.

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control.

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter.

Effects of Tityus serrulatus scorpion toxin gamma on voltage-gated Na+ channels.

The effects of Brazilian scorpion Tityus serrulatus toxin gamma (TiTx gamma) were studied on voltage-gated Na+ channels from human heart (hHl) and rat skeletal muscle (rSkM1). The Na+ channels were expressed in Xenopus laevis oocytes, and Na+ currents were recorded using two-microelectrode voltage-clamp techniques. In control experiments, the threshold of activation of hH1 is more negative than that of rSkM1 by approximately 20 mV.

Okadaic acid enhances prepulse facilitation of cardiac alpha 1-subunit but not endogenous calcium channel currents in Xenopus laevis oocytes.

Xenopus laevis oocytes can be selected to express relatively high levels of endogenous Ca currents. These currents are facilitated by prepulses. Facilitated endogenous Ca currents are unaffected by okadaic acid, RpcAMPS or the dihydropyridine (DHP) antagonist (+) PN 200-110.

Electrophysiological characteristics of cloned skeletal and cardiac muscle sodium channels.

The alpha-subunit encoding for voltage-gated sodium channels rSkM1 (rat skeletal muscle subtype 1) and hH1 (human heart subtype 1) has been cloned and expressed by various groups under various conditions in Xenopus oocytes and the tsA201 (HEK 293) mammalian cell line derived from human embryonic kidney cells. In this study, we have expressed hH1 and rSkM1 in tsA201 cells for comparison under the same conditions using patch-clamp methods. Our results show significant differences in the current-voltage (I-V) relationship, kinetics of current decay, voltage dependence of steady-state inactivation, and the time constant for recovery from inactivation.

Sea anemone toxin (ATX II) modulation of heart and skeletal muscle sodium channel alpha-subunits expressed in tsA201 cells.

We have expressed recombinant alpha-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, taufasth) and toxin-bound (slow component, tauslowh) channels.

A cellular automata model of the heart and its coupling with a qualitative model.

Cellular Automata (CA) models offer a good compromise between computational complexity and biological plausibility while qualitative models have expressive power for explicitly describing dynamic processes. In this paper we present a 2D CA model and its coupling with a qualitative model. The CA model includes elements characterizing muscle, nodal tissue, and bypass conduction.

An interactive qualitative model in cardiology.

Qualitative modeling is a generic term that involves explicit and qualitative representations of the physical world. It can extend the realm of pure mathematical modeling in the sense that qualitative descriptions can, on one hand, simulate complex physical systems and processes and, on the other, produce linguistic descriptions and summaries of simulated system behavior. These summaries should be an essential element of the human/machine interface if truly interactive computational environments are to be developed.

Characterizing the mu-conotoxin binding site on voltage-sensitive sodium channels with toxin analogs and channel mutations.

The three-dimensional organization of the domains of the rat skeletal muscle sodium channel subtype 1 (rSkM1) and the toxin-channel interaction surface have been explored by a complementary mutagenesis approach. This method involves probing mutant channels with analogs of the peptide toxin, mu-conotoxin (mu-CTX), for which the tertiary structure has been determined. mu-CTX has an overall net charge of +5.

Deep knowledge and computer-assisted instruction in cardiology.

In this paper, we describe a qualitative heart model that is part of a computing environment, CARDIOLAB, and whose role includes the diagnosis and Computer Assisted Instruction (CAI) in cardiology. The model is based on a "deep knowledge" approach to diagnosis. Deep knowledge representations model the inner works of complex physical systems.

Functional expression and properties of the human skeletal muscle sodium channel.

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the alpha-subunit of the adult human skeletal muscle Na+ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injected Xenopus oocytes and from transiently transfected tsA201 cells. The Na+ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line.

Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation.

Mutations in the adult human skeletal muscle Na+ channel alpha subunit cause the disease paramyotonia congenita. Two paramyotonia congenita mutations, R1448H and R1448C, substitute histidine and cysteine for arginine in the S4 segment of domain 4. These mutations, expressed in a cell line, have only small effects on the activation of Na+ currents, but mutant channels inactivate more slowly with less voltage dependence than wild-type channels and exhibit an enhanced rate of recovery from inactivation.

Lidocaine block of human heart sodium channels expressed in Xenopus oocytes.

The tertiary amine lidocaine is used clinically for preventing cardiac arrhythmias, and has been widely studied on mammalian tissue. Xenopus oocytes were used as an expression system to study the effect of lidocaine on a sodium (Na) channel, derived from a full-length human heart (hH1) cDNA clone. The concentration dependence of the lidocaine block of hH1 Na current was consistent with a binding stoichiometry of 1:1.

Chimeric study of sodium channels from rat skeletal and cardiac muscle.

Two isoforms of voltage-dependent Na channels, cloned from rat skeletal muscle, were expressed in Xenopus oocytes. The currents of rSkM1 and rSkM2 differ functionally in 4 properties: (i) tetrodotoxin (TTX) sensitivity, (ii) mu-conotoxin (mu-CTX) sensitivity, (iii) amplitude of single channel currents, and (iv) rate of inactivation. rSkM1 is sensitive to both TTX and mu-CTX.