We investigated inactivation of the slow calcium current (ICa) at very positive potentials (over 30-40 mV) and recovery from inactivation in cut twitch skeletal muscle fibres of the frog, using the double-vaseline-gap technique. External solutions were buffered against changes in [Ca2+] (Ca(2+)-buffered) with malate. Internal solutions were Ca(2+)-buffered with high concentrations of either EGTA (60 mM) or BAPTA (30 mM). 2. ICa decayed to a steady-state level somewhat less than zero. Inactivation was most rapid at a potential 10 mV more negative than that which elicited the maximal ICa. 3. Involvement of current-dependent processes (i.e. tubular Ca2+ depletion and Ca2+ entry-dependent inactivation) in determining the decay of ICa was excluded, since inactivation was not affected by replacing Ca2+ with Ba2+ or when the size of ICa was reduced by decreasing the [Ca2+]o. Partial block of Ca2+ channels with nifedipine slowed inactivation. This was, however, independent of the size of ICa. Furthermore, neither the peak of ICa nor its time constant of decay nor the time course of ICa recovery from inactivation were affected by changing the [Ca2+]i from pCa 10 to 6. 4. ICa was potentiated during a post-pulse preceded by a pre-pulse at potentials ranging from -60 to -30 mV, whereas a U-shaped inactivation curve was observed at pre-pulse potentials more positive than -30 mV. This curve was asymmetric, since the ascending branch stabilized at a level less than unity. The U-shaped form of the curve depended on post-pulse voltage: it became more pronounced when the post-pulse depolarization increased. Moreover, the activation and inactivation kinetics of ICa during the post-pulse differed from control values. Similar results were found when Ca2+ was replaced with Ba2+. 5. The ICa recovery from inactivation was voltage dependent from -50 to -80 mV; it was voltage independent at more negative potentials, proving that recovery includes a voltage-independent step. 6. The asymmetric U-shaped inactivation curve can be reproduced by a four-state cyclic model without assuming a Ca(2+)-dependent step. Taking into account that recovery from inactivation includes a voltage-independent step which becomes rate limiting at extreme negative potentials, and that during the post-pulse the activation kinetics is faster, we propose a model which has six states, two closed, one open and three inactivated.
