Evidence for Ca ²⁺ Activation and Inactivation Sites on the Luminal Side of the Cardiac Ryanodine Receptor Complex

We have used tryptic digestion to determine whether Ca(2+) can regulate cardiac ryanodine receptor (RyR) channel gating from within the lumen of the sarcoplasmic reticulum (SR) or whether Ca(2+) must first flow through the channel and act via cytosolically located binding sites. Cardiac RyRs were incorporated into bilayers, and trypsin was applied to the luminal side of the bilayer. We found that before exposure to luminal trypsin, the open probability of RyR was increased by raising the luminal [Ca(2+)] from 10 micromol/L to 1 mmol/L, whereas after luminal trypsin exposure, increasing the luminal [Ca(2+)] reduced the open probability. The modification in the response of RyRs to luminal Ca(2+) was not observed with heat-inactivated trypsin, indicating that digestion of luminal sites on the RyR channel complex was responsible. Our results provide strong evidence for the presence of luminally located Ca(2+) activation and inhibition sites and indicate that trypsin digestion leads to selective damage to luminal Ca(2+) activation sites without affecting luminal Ca(2+) inactivation sites. We suggest that changes in luminal [Ca(2+)] will be able to regulate RyR channel gating from within the SR lumen, therefore providing a second Ca(2+)-regulatory effect on RyR channel gating in addition to that of cytosolic Ca(2+). This luminal Ca(2+)-regulatory mechanism is likely to be an important contributing factor in the potentiation of SR Ca(2+) release that is observed in cardiac cells in response to increases in intra-SR [Ca(2+)].