The protein dystrophin is absent from patients with Duchenne muscular dystrophy and from the muscles of mdx mice. Recent studies have shown that dystrophin is located at the surface membrane and at the triadic junction, where it is associated with the transverse tubular membrane. Since the triadic junction is the site of excitation-contraction (EC) coupling, we have investigated whether intramembrane charge movement, a step in EC coupling, is modified by the absence of dystrophin. Charge movements are thought to arise from the transverse tubular membrane and to underlie the dependence of sarcoplasmic reticulum Ca2+ release on transverse tubular membrane potential. We find no differences between intramembrane charge movements or passive membrane electrical properties measured in muscles from mdx mice compared with normal mice. If dystrophin does play a role in EC coupling, that role is likely to be subsequent to the charge movement step.
SUMMARY1. The effects of tetracaine, a local anaesthetic that inhibits muscle contraction, on membrane potential and intramembrane charge movements were investigated in fast twitch rat muscle fibres (extensor digitorum longus).2. The resting membrane potentials of surface fibres from muscles bathed in isotonic Ringer solution containing 2 mM-tetracaine were well maintained, but higher concentrations of tetracaine caused a time-dependent fall of potential. Muscle fibres bathed in hypertonic solutions containing 2 mm-tetracaine were rapidly depolarized. In both isotonic and hypertonic solutions, the depolarizing effect of tetracaine could not be reversed.3. Charge movement measurements were made using the middle-of-the-fibre voltage clamp technique. The voltage dependence of charge movements measured in cold isotonic solutions was well fitted by a Boltzmann distribution (Q( V) = Qmax/(1 +exp(-( V"-V)/k)) where Qmax = 373+28 nC ,uF', V =-179+ 12 mV and k = 12-6+08 mV (n = 6, 2°C; means+ s.E. of means). Similar values were obtained when 2 mM-tetracaine was added to the isotonic bathing fluid (Qmax = 406+2-3 nCjuF-, V= -14 1+1 3 mV, k = 15f3+08 mV; n = 8, 2°C).4. Charge movements measured around mechanical threshold in muscle fibres bathed in hypertonic solutions were reduced when 2 mM-tetracaine was added to the bathing fluid. The tetracaine-sensitive component of charge was well fitted with an unconstrained Boltzmann distribution which gave: Qmax = 7-5 nC /tF-1, V = -46-5 mV, k = 5-5 mV. The e-fold rise of the foot of the curve was 9 3 mV.
SUMMARY1. The middle of the fibre voltage-clamp technique was used to measure ionic currents and non-linear charge movements in intact, organ-cultured (in vitro denervated) mammalian fast-twitch (rat extensor digitorum longus) muscle fibres.2. Muscle fibres organ cultured for 4 days can be used as electrophysiological and morphological models for muscles in vivo denervated for the same length of time.3. Sodium currents in organ-cultured muscle fibres are similar to innervated fibres except that in the temperature range 0-20 0C (a) in the steady state, the voltage distribution of inactivation in cultured fibres is shifted negatively some 20 mV; (b) at the same temperature and membrane potential, the time constant of inactivation in cultured fibres is about twice that of innervated fibres.4. Potassium currents in innervated and cultured fibres at 15 0C can be fitted with the Hodgkin-Huxley n variable rasied to the second power. Despite the large rang we would estimate that the maximum value ofthe steady-state potassium conductance of cultured fibres is about one-half that of innervated fibres.5. The estimated maximum amount of charge moved in cultured fibre is about one-third that in innervated fibres. Compared to innervated fibres, culturing doubles the kinetics of the decay phase of charge movement. The possibility of a negative shift of the voltage distribution of charge movements in cultured fibres is discussed.
A study was made of charge movements and the transverse tubular systems in rat EDL and soleus muscle fibres maintained for up to five days in organ culture. In the cultured EDL muscle the maximum amount of charge moved was about one third of that in innervated muscle. Charge movements in innervated soleus fibres are small, less than 10 nC/microF, and difficult to resolve. They remain small following organ culturing. The ultrastructural study examined the concentration of junctional feet because of their proposed key role in excitation-contraction coupling. The general architecture of the triads and the spacing of the feet in both muscle types was largely unchanged by culturing. In cultured EDL muscles the small changes in feet concentration did not parallel the large fall in charge movement. The results reported here support a previous conclusion that, in mammalian muscle, there is not a simple relation between charge and feet. The stimulation of cultured soleus muscles with a fast twitch pattern of electrical activity produced no observable changes in morphology.
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