Cellular and ionic basis for T-wave alternans under long-QT conditions. (1/271)

BACKGROUND: T-wave alternans (TWA), an ECG phenomenon characterized by beat-to-beat alternation of the morphology, amplitude, and/or polarity of the T wave, is commonly observed in the acquired and congenital long-QT syndromes (LQTS). This study examines the cellular and ionic basis for TWA induced by rapid pacing under conditions mimicking the LQT3 form of the congenital LQTS in an arterially perfused canine left ventricular wedge preparation. METHODS AND RESULTS: Transmembrane action potentials from epicardial, M, and endocardial cells and 6 to 8 intramural unipolar electrograms were simultaneously recorded together with a transmural ECG and isometric tension development. In the presence of sea anemone toxin (ATX-II; 20 nmol/L), an increase in pacing rate (from a cycle length [CL] of 500 to 400 to 250 ms) produced a wide spectrum of T-wave and mechanical alternans. Acceleration to CLs of 400 to 300 ms produced mild to moderate TWA principally due to beat-to-beat alternation of repolarization of cells in the M region. Transmural dispersion of repolarization during alternans was exaggerated during alternate beats. Acceleration to CLs of 300 to 250 ms caused more pronounced beat-to-beat alternation of action potential duration (APD) of the M cell, resulting in a reversal of repolarization sequence across the ventricular wall, leading to alternation in the polarity of the T wave. The peak of the negative T waves coincided with repolarization of the M region, whereas the end of the negative T wave coincided with the repolarization of epicardium. In almost all cases, electrical alternans was concordant with mechanical alternans. Torsade de pointes occurred after an abrupt acceleration of CL, which was associated with marked TWA. Both ryanodine and low [Ca2+]o completely suppressed alternans of the T wave, APD, and contraction, suggesting a critical role for intracellular Ca2+ cycling in the maintenance of TWA. CONCLUSIONS: Our results suggest that TWA observed at rapid rates under long-QT conditions is largely the result of alternation of the M-cell APD, leading to exaggeration of transmural dispersion of repolarization during alternate beats, and thus the potential for development of torsade de pointes. Our data also suggest that unlike transient forms of TWA that damp out quickly and depend on electrical restitution factors, the steady-state electrical and mechanical alternans demonstrated in this study appears to be largely the result of beat-to-beat alternans of [Ca2+]i.  (+info)

ATX II, a sodium channel toxin, sensitizes skeletal muscle to halothane, caffeine, and ryanodine. (2/271)

BACKGROUND: The function or expression of subtypes of the sodium ion (Na+) channel is altered in biopsies or cultures of skeletal muscle from many persons who are susceptible to malignant hyperthermia (MH). ATX II, a specific Na+ channel toxin from a sea anemone, causes delayed inactivation of the channel similar to that seen in cell cultures of MH muscle. ATX II was added to skeletal muscle to determine whether altered Na+ channel function could increase the sensitivity of normal skeletal muscle to agents (halothane, caffeine, ryanodine) to which MH muscle is hypersensitive. METHODS: Studies were performed of fiber bundles from the vastus lateralis muscle of persons who were deemed not MH susceptible (MH-) or MH susceptible (MH+) according to the MH diagnostic test and of strips of diaphragm muscle from rats. Preparations in a tissue bath containing Krebs solution were connected to a force transducer. ATX II was introduced 5 min before halothane, caffeine, or ryanodine. RESULTS: ATX II increased the magnitude of contracture to halothane in preparations from most MH-, but not MH+, human participants. After ATX II treatment, preparations from 9 of 24 MH- participants generated contractures to halothane, 3%, that were of the same magnitude as those from MH+ participants. Preparations from four of six ATX II-treated healthy participants also gave responses of the same magnitude as those of MH-susceptible participants to a graded halothane challenge (0.5-3%). The contractures to bolus doses of halothane in specimens from male participants were more than three times larger than the contractures in specimens from female participants. In rat muscle, ATX II increased the magnitude of contracture to caffeine (2 mM) and decreased the time to produce a 1-g contracture to ryanodine (1 microM). CONCLUSIONS: ATX II, which causes delayed inactivation of the Na+ channel in cell cultures similar to that reported in cultures of MH+ skeletal muscle, increased the sensitivity of normal muscle to three agents to which MH+ muscle is hypersensitive. The increased sensitivity to halothane, 3%, occurred in most (79%), but not all, MH- participants, and this effect was most evident in male participants. Therefore, abnormal function of the Na+ channel, even if it is a secondary event in MH, may contribute to a positive contracture test result for MH.  (+info)

Structural conservation of the pores of calcium-activated and voltage-gated potassium channels determined by a sea anemone toxin. (3/271)

The structurally defined sea anemone peptide toxins ShK and BgK potently block the intermediate conductance, Ca(2+)-activated potassium channel IKCa1, a well recognized therapeutic target present in erythrocytes, human T-lymphocytes, and the colon. The well characterized voltage-gated Kv1.3 channel in human T-lymphocytes is also blocked by both peptides, although ShK has a approximately 1,000-fold greater affinity for Kv1.3 than IKCa1. To gain insight into the architecture of the toxin receptor in IKCa1, we used alanine-scanning in combination with mutant cycle analyses to map the ShK-IKCa1 interface, and compared it with the ShK-Kv1.3 interaction surface. ShK uses the same five core residues, all clustered around the critical Lys(22), to interact with IKCa1 and Kv1.3, although it relies on a larger number of contacts to stabilize its weaker interactions with IKCa1 than with Kv1.3. The toxin binds to IKCa1 in a region corresponding to the external vestibule of Kv1.3, and the turret and outer pore of the structurally defined bacterial potassium channel, KcsA. Based on the NMR structure of ShK, we deduce the toxin receptor in IKCa1 to have x-y dimensions of approximately 22 A, a diameter of approximately 31 A, and a depth of approximately 8 A; we estimate that the ion selectivity lies approximately 13 A below the outer lip of the toxin receptor. These dimensions are in good agreement with those of the KcsA channel determined from its crystal structure, and the inferred structure of Kv1.3 based on mapping with scorpion toxins. Thus, these distantly related channels exhibit architectural similarities in the outer pore region. This information could facilitate development of specific and potent modulators of the therapeutically important IKCa1 channel.  (+info)

Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes. (4/271)

Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13-20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105-120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.  (+info)

Increased hindrance on the chiral carbon atom of mexiletine enhances the block of rat skeletal muscle Na+ channels in a model of myotonia induced by ATX. (5/271)

1 The antiarrhythmic drug mexiletine (Mex) is also used against myotonia. Searching for a more efficient drug, a new compound (Me5) was synthesized substituting the methyl group on the chiral carbon atom of Mex by an isopropyl group. Effects of Me5 on Na+ channels were compared to those of Mex in rat skeletal muscle fibres using the cell-attached patch clamp method. 2 Me5 (10 microM) reduced the maximal sodium current (INa) by 29.7+/-4.4 % (n=6) at a frequency of stimulation of 0.3 Hz and 65.7+/-4.4 % (n=6) at 1 Hz. At same concentration (10 microM), Mex was incapable of producing any effect (n=3). Me5 also shifted the steady-state inactivation curves by -7. 9+/-0.9 mV (n=6) at 0.3 Hz and -12.2+/-1.0 mV (n=6) at 1 Hz. 3 In the presence of sea anemone toxin II (ATX; 5 microM), INa decayed more slowly and no longer to zero, providing a model of sodium channel myotonia. The effects of Me5 on peak INa were similar whatever ATX was present or not. Interestingly, Me5 did not modify the INa decay time constant nor the steady-state INa to peak INa ratio. 4 Analysis of ATX-induced late Na+ channel activity shows that Me5 did not affect mean open times and single-channel conductance, thus excluding open channel block property. 5 These results indicate that increasing hindrance on the chiral atom of Mex increases drug potency on wild-type and ATX-induced noninactivating INa and that Me5 might improve the prophylaxis of myotonia.  (+info)

Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity. (6/271)

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K(D) 50- and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.  (+info)

Structure-function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein. (7/271)

Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp(45), EqtII Trp(116/117) and EqtII Trp(149), were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp(116/117) resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp(45) and EqtII Trp(149), was altered to a certain extent. EqtII Trp(149) was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with (2)H(2)O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp(45) and EqtII Trp(149) were more exposed to water, and also remained as such in the membrane-bound form.  (+info)

Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. (8/271)

OBJECTIVES: To define the cellular mechanisms responsible for the development of life-threatening arrhythmias in response to sympathetic activity in the congenital and acquired long QT syndromes (LCQTS). METHODS: Transmembrane action potentials (AP) from epicardial (EPI), M and endocardial (ENDO) cells and a transmural electrocardiogram were simultaneously recorded from an arterially perfused wedge of canine left ventricle. We examined the effect of beta-adrenergic agonists and antagonists on action potential duration (APD90), transmural dispersion of repolarization (TDR) and the development of Torsade de Pointes (TdP) in models of LQT1, LQT2 and LQT3 forms of LQTS. RESULTS: I(Ks) block with chromanol 293B (LQT1) homogeneously prolonged APD90 of the three cell types without increasing TDR. Addition of isoproterenol prolonged QT and APD90 of M but abbreviated that of EPI and ENDO, causing a persistent increase in TDR; Torsade de Pointes developed or could be induced only in the presence of isoproterenol. I(Kr) block with d-sotalol (LQT2) and augmentation of late I(Na) with ATX-II (LQT3) prolonged APD90 of M more than EPI and ENDO, causing increases in QT and TDR. TdP developed in the absence of isoproterenol. In LQT2 isoproterenol initially prolonged, then abbreviated, the APD90 of M but always abbreviated EPI, thus transiently increasing TDR and the incidence of TdP. In LQT3, isoproterenol always abbreviated APD90 of the three cell types, causing a persistent decrease in TDR and suppression of TdP. The arrhythmogenic as well as protective actions of isoproterenol were reversed by propranolol. CONCLUSIONS: Our data suggest that beta-adrenergic stimulation induces TdP by increasing transmural dispersion of repolarization in LQT1 and LQT2 but suppresses TdP by decreasing dispersion in LQT3. The data indicate that beta-blockers are protective in LQT1 and LQT2 but may facilitate TdP in LQT3.  (+info)

• 1
• 2
• 3
• 4
• 5
• 6
• 7
• 8
• 9
• 10