Myoballs, i.e., spherical muscle cell regenerates, were cultured from the biopsied muscles of three patients with myotonic dystrophy, three patients with recessive generalized myotonia, and a patient with adynamia episodica. The membrane of these myoballs was voltage-clamped in the whole-cell mode for the recording of sodium currents (at 11, 24, and 37 degrees C). The voltage dependence of the steady-state activation and inactivation curves showed only minor abnormalities in all cases. The time constants of activation (tau m) and inactivation (tau h), when studied at the three temperatures, showed a characteristic pattern of abnormalities. In myotonic dystrophy, both tau m and tau h were larger than control; in recessive generalized myotonia and adynamia episodica both tau m and tau h were smaller than control. In the latter diseases, these time constants also showed a smaller than normal decrease with membrane depolarization. The changes seen for recessive generalized myotonia and adynamia episodica would favour the occurrence of myotonia, the opposite results for myotonic dystrophy would oppose myotonia.
The effects of both enantiomers of tocainide and of some of its chiral analogs on the inactivation of the sodium current in human myoballs were investigated with the whole-cell recording technique. Structure and electron densities of the applied compounds were calculated and compared to the results. Both the R(-) and the S(+) enantiomers had little effect on fast inactivation determined with short prepulses according to Hodgkin and Huxley (1952; h infinity curve). When the inactivating prepulses used in this pulse protocol were prolonged to 1024 ms, both tocainide enantiomers increased inactivation severely, suggesting that the drug binds to the channel when it is in the state of intermediate inactivation (Fakler et al. 1990). Tetrodotoxin-resistant "juvenile" sodium channels were more affected than tetrodotoxin-sensitive "adult" channels. The R form was four times as effective as the S form. The compound obtained by substitution of the methyl group on the chiral centre of tocainide with a benzyl group, although in the less potent S form, affected inactivation of the juvenile sodium channels much more than the potent (R)-tocainide. Two additional substitutions, performed on the aromatic ring of tocainide, gave a compound that was most potent in shifting the inactivation curves, but without any selectivity for juvenile or adult channels.
Intact muscle fibers or resealed fiber segments from 7 patients with recessive generalized myotonia were studied in vitro. All fibers had normal resting membrane potentials and normal resting [Ca2+]i several hours after removal. Contractions were characterized by slowed relaxation which was due to electrical after-activity. Often spontaneous depolarizations were recorded intracellularly. In all fibers, the steady state voltage-current relationship was abnormal, due to a reduced Cl- conductance. However, this conductance ranged from 0% to 66% of the total membrane conductance, whereas, in normal muscle, it was 80%. Theoretically, myotonic after-discharges would not appear until the Cl- conductance is below 20%. Thus, the membrane hyperexcitability must be due to another defect, at least in the preparations in which the Cl- conductance was only slightly reduced. In all patches from all patients investigated with the patch clamp technique, we observed reopenings of the Na+ channels throughout depolarizing pulses (such behavior was absent in normal muscle). If a patch was polarized to potentials less negative than the resting potential, the duration of the reopenings increased. We conclude that a combination of reduced Cl- conductance and the reopenings of Na+ channels underlie the electrical after-activity in recessive generalized myotonia.
The inactivation of the sodium channels in human medulloblastoma cells was investigated with the whole-cell recording technique. The potential dependence of inactivation ("inactivation curve") was determined by imposing a series of prepulses of varying amplitude on the membrane potential and measuring the maximum sodium current flowing after each prepulse at the test potential of -20 mV. The time dependence of inactivation was investigated by determining inactivation curves with prepulses of variable duration. A prolongation of the prepulse increased the degree of inactivation, even when the prepulse duration was much greater than the time constant for fast inactivation. This is explained by the existence of two additional states of "intermediate" inactivation of the sodium channel, the transition to which is slower than that to the state of fast inactivation and faster than that to the state of slow inactivation. The antiarrhythmic drug tocainide had no effect on fast inactivation, but a strong effect on intermediate inactivation. This explains the use dependence of this drug. The reaction model given by Chiu (1977) for the transitions from the open into the closed state of inactivation and vice versa is extended.
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