ACTIVATION AND INACTIVATION OF EXCITATION CONTRACTION COUPLING IN RAT SOLEUS MUSCLE

1. Potassium (K+) contractures have been used to characterize the processes of activation and inactivation of excitation-contraction coupling during prolonged depolarization of fibres in small bundles dissected from rat soleus muscles at 23-degrees-C. 2. The smallest measurable K+ contracture tension was recorded with depolarization to -40 mV in 30 mM-K+ and maximum tension was achieved between -26 mV in 80 mM-K+ and -19 mV in 120 mM-K+. 3. The rate of inactivation of K+ contracture tension was voltage dependent. Tension decayed from 80 to 20% of the peak amplitude within 44.0 +/- 2.2 s at -26 mV (in 80 mM-K+), compared with 66.7 +/- 4.8 s at -35 mV (in 40 mM-K+). Results are given as mean +/- 1 S.E.M. 4. The effect of inactivation on maximum tension was determined using a two pulse protocol in which a 'conditioning' depolarization in solutions containing 20-120 mM-K+ was applied for 0.5-10 min before a 'test' depolarization to -8 mV in 200 mM-K+. The amplitude of the test contracture was compared with the mean amplitude of 'control' 200 mM-K+ contractures elicited in normally polarized fibres immediately before and after the two pulse protocol. Conditioning depolarization to -47 mV (in 20 mM-K+) did not reduce test 200 mM-K+ contracture tension. Significant inactivation was seen with further conditioning depolarization to more positive potentials: after 10 min at -40 mV (in 30 mM-K+), or -35 mV (in 40 mM-K+), test 200 mM-K+ contracture tension was reduced by 33 and 70% respectively. 5. In contrast to amphibian muscle, where maximum tension falls to zero within a few minutes of depolarization to potentials positive to -50 mV, test 200 mM-K+ contracture tension in rat soleus fibres fell initially rapidly and then slowly, but was not reduced to zero, even after 10 min at -19 mV in 120 mM-K+. 6. The fast phase of inactivation of test 200 mM-K+ contracture tension occurred during the decay of the conditioning K+ contracture. The slow phase of inactivation reached completion after 10 min of conditioning depolarization and occurred during the period when conditioning tension was reduced to zero or to a plateau level. Both phases of inactivation in rat soleus fibres are slow compared with fast and slow inactivation times of 5-100 s respectively reported for amphibian muscle. 7. When repolarized after prolonged depolarization, the muscle fibres were initially refractory, i.e. unable to produce tension in response to electrical stimulation. Repriming was observed after the refractory period: twitch and tetanic tension slowly recovered to final values that were within 90-100% of the peak tension recorded before the two pulse depolarization. The rate of repriming was measured as the time taken for tetanic tension to reach 90% of its final value and depended (a) on the depolarizing potassium concentration and (b) on the period of exposure to the high-potassium solution. Repriming times were 3-5 min after brief (30-50 s) exposures to solutions containing 30-60 mM-K+. The longest repriming times recorded were 15-20 min. Repriming slowed to this rate after 3 min at -26 mV in 80 mM-K+ and did not slow further with depolarization to more positive membrane potentials, or with prolonged depolarization. 8. The biphasic time course of inactivation and repriming suggest that, as in amphibia, there are two inactivated states in the excitation-contraction coupling process in mammalian skeletal muscle. These may be located in the 'voltage-sensor', or in the coupling mechanism between the voltage sensor and the calcium release channel.