Activation of L-type calcium channel in twitch skeletal muscle fibres of the frog

1. The activation of the L-type calcium current (I-Ca) was studied in normally polarized (-100 mV) cut skeletal muscle fibres of the frog with the double Vaseline-gap voltage-clamp technique. Both external and internal solutions were Ca2+ buffered. Solutions were made in order to minimize all but the Ca2+ current. 2. The voltage-dependent components of the time course of activation were determined by two procedures: fast and slow components were evaluated by multiexponential fitting to current traces elicited by long voltage pulses (5 s) after removing inactivation; fast components were also determined by short voltage pulses having different duration (0.5-70 ms). 3. The components of deactivation were evaluated after removing the charge-movement current from the total tail current by the difference between two short (50 and 70 ms) voltage pulses to 10 mV, moving the same intramembrane charge. Two exponential components, fast and slow (time constants, 6 +/- 0.3 and 90 +/- 7 ms at -100 mV; n = 26), were found. 4. The time onset of I-Ca was evaluated either by multiexponential fitting to the I-Ca activation or by pulses of different duration to test the beginning of the 'on' and 'off' inequality. This was at about 2 ms, denoting that it was very early. 5. The time constants vs. voltage plots indicated the presence of four voltage-dependent components in the activation pathway. Various kinetic models are discussed. Models with independent transitions, like a Hodgkin-Huxley scheme, were excluded. Suitable models were a five-state sequential and a four-state cyclic with a branch scheme. The latter gave the best simulation of the data. 6. The steady-state activation curve saturated at high potentials. It had a half-voltage value of 1 +/- 0.02 mV and the opening probability was only 0.82 +/- 0.2 at 20 mV (n = 32). This result implies a larger number of functional calcium channels than mas previously supposed and is in agreement with the number of dihydropyridine (DHP) receptors calculated for the tubular system.