Ca2+ activation and Ca2+ inactivation of canine reconstituted cardiac sarcoplasmic reticulum Ca(2+)-release channels

1. Calcium-release channels (ryanodine receptors) of canine cardiac sarcoplasmic reticulum (SR) were incorporated into lipid bilayer membranes at the tip of a patch pipette. Using symmetrical 150 mM KCl solutions, [Ca2+] > 0.3 microM activated single channels of 627 pS conductance. The kinetics of Ca(2+)-mediated channel activation, deactivation and inactivation were studied by stepwise changes in pCa (-log[Ca2+]) and analysis of current means. 2. Steps of [Ca2+] activated the channel open probability (Po) along a time course which could be fitted by a single exponential. The activation time constant was dependent on [Ca2+], which decreased from 4.9 ms at pCa 6.5 to 0.2 ms at pCa 3. Subsequent rapid reduction in [Ca2+] decreased Po along a mono-exponential deactivation time course, the time constant of which was independent of the [Ca2+] during the preceding activation period. Further analysis yielded the rate constants kon of 2 x 10(8) (M s)-1 and koff of 2 x 10(2) s-1, an apparent dissociation constant (KD) of 1 microM, and a Hill coefficient of 1.05. 3. The open probability increased with [Ca2+], reaching a peak at about pCa 5.5. At pCa < or = 5.5, Po decreased time dependently, the time constants decreasing along with [Ca2+] from 1 s at 3 microM to 0.2 s at 1 mM. During the 0.5 s period at 3 microM Ca2+, Po fell by 13% due to an extension of the closed times. At 1 mM Ca2+, Po 'inactivated' by 72%, which was due mostly to long closures. These differences suggest that the Ca(2+)-mediated decay of Po was dependent on Ca2+ binding to an intermediate (KD, 3 microM) and a low affinity site (KD, 360 microM). On the return of pCa from 3 to > 8, the channels briefly re-opened. 4. A 'refractory' behaviour of the channel was not observed for 20 ms steps between < 10 nM and < 10 microM [Ca2+] (25 Hz). For steps between 10 nM and 1 mM, however, such behaviour was marked by infrequent and irregular channel openings. 5. The results are described by a three Ca2+ binding site model and compared with the literature.