Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum

Abstract

1. Calcium-release channels of sheep cardiac junctional sarcoplasmic reticulum were incorporated into planar phospholipid bilayers. Single-channel current fluctuations were recorded under voltage clamp conditions. 2. Channels incorporate into the bilayer with a fixed orientation and channel open probability is regulated by the calcium concentration at the cytosolic face of the membrane. 3. Addition of caffeine (0.5-2.0 mM) to the cytosolic side of the membrane increased the open probability of the calcium-activated calcium-release channel by increasing the frequency of opening without significant alteration to the durations of open events. This effect was observed at both 0.1 and 10 microM-activating cytosolic calcium. 4. Caffeine (0.5-2.0 mM) did not activate the channel at a subactivating cytosolic calcium concentration (80 pM). 5. At subactivating calcium concentrations, channels could be activated by higher concentrations of caffeine (greater than 5.0 mM) revealing a second, calcium-independent, mechanism for channel activation. Channel openings induced by these high concentrations of caffeine at subactivating calcium concentrations displayed different kinetics from those observed with calcium as the sole activating ligand or with combinations of calcium and low concentrations of caffeine. 6. Activation of channel opening by caffeine in the presence of calcium did not affect single-channel conductance. Channel openings produced by caffeine at subactivating cytosolic calcium concentrations had identical conductance and relative permeability to those seen on calcium activation. 7. Channels activated by caffeine at both activating and subactivating calcium concentrations were characteristically modified by ryanodine, Ruthenium Red, ATP and magnesium, implying that the same channel is involved under both conditions.