toxin-ii-(anemonia-sulcata) and Myotonia

The sea anemone toxin ATX II impairs skeletal muscle sodium channel inactivation, mimicking the persistent inward current observed in patients suffering from sodium channel myotonia. Mexiletine has beneficial effects on myotonia. To verify the efficiency of the drug on persistent inward current, we investigated the effect of 50 microM R(-)-mexiletine on sodium channels in cell-attached patches of rat skeletal muscle fibres, in the absence or presence of 2 microM ATX II. With the toxin, a proportion of channels displayed remarkable abnormal activity lasting the entire depolarisation, which resulted in a persistent inward current that represented up to 2.0% of the peak current. Mexiletine reduced by 75% the peak current elicited by depolarisation from -100 to -20 mV. This was due to the reduction by 60% of the maximal available peak current Imax and to the negative shift by -7 mV of steady-state inactivation. Mexiletine also greatly decreased the late current, but the effect was limited to 60% of reduction, comparable to that on Imax. Therefore mexiletine was able to block the ATX II-modified sodium channels, inhibiting the myotonia-producing persistent inward current.

Topics: Animals; Anti-Arrhythmia Agents; Cnidarian Venoms; Disease Models, Animal; In Vitro Techniques; Membrane Potentials; Mexiletine; Muscle Fibers, Skeletal; Muscle, Skeletal; Myotonia; Rats; Sodium Channels

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.

Topics: Animals; Butylamines; Carbon; Cnidarian Venoms; Membrane Potentials; Mexiletine; Muscle Fibers, Skeletal; Muscle, Skeletal; Myotonia; Rats; Sodium Channels; Structure-Activity Relationship

1. Mutations that impair inactivation of the sodium channel in skeletal muscle have recently been postulated to cause several heritable forms of myotonia in man. A peptide toxin from Anemonia sulcata (ATX II) selectively disrupts the inactivation mechanism of sodium channels in a way that mimics these mutations. We applied ATX II to rat skeletal muscle to test the hypothesis that myotonia is inducible by altered sodium channel function. 2. Single-channel sodium currents were measured in blebs of surface membrane that arose from the mechanically disrupted fibres. ATX II impaired inactivation as demonstrated by persistent reopenings of sodium channels at strongly depolarized test potentials. A channel failed to inactivate, however, in only a small proportion of the depolarizing steps. With micromolar amounts of ATX II, the ensemble average open probability at the steady state was 0.01-0.02. 3. Ten micromolar ATX II slowed the relaxation of tension after a single twitch by an order of magnitude. Delayed relaxation is the in vitro analogue of the stiffness experienced by patients with myotonia. However, peak twitch force was not affected within the range of 0-10 microM ATX II. 4. Intracellular injection of a long-duration, constant current pulse elicited a train of action potentials in ATX II-treated fibres. After-depolarizations and repetitive firing often persisted beyond the duration of the stimulus. Trains of action potentials varied spontaneously in amplitude and firing frequency in a similar way to the electromyogram of a myotonic muscle. Both the after-depolarization and the post-stimulus firing were abolished by detubulating the fibres with glycerol. 5. We conclude that a loss of sodium channel inactivation alone, without changes in resting membrane conductance, is sufficient to produce the electrical and mechanical features of myotonia. Furthermore, in support of previous studies on myotonic muscle from patients, this model provides direct evidence that only a small proportion of sodium channels needs to function abnormally to cause myotonia.

Topics: Action Potentials; Animals; Cnidarian Venoms; Disease Models, Animal; Female; Humans; Hyperkalemia; In Vitro Techniques; Isometric Contraction; Muscles; Mutation; Myotonia; Paralyses, Familial Periodic; Rats; Sea Anemones; Sodium Channel Blockers; Sodium Channels