The gating of the sheep skeletal sarcoplasmic reticulum Ca(2+)-release channel is regulated by luminal Ca2+

Abstract

The effects of changes in luminal [Ca2+] have been investigated in sheep skeletal sarcoplasmic reticulum (SR) Ca(2+)-release channels after activation of the channels by different ligands from the cytosolic side of the channel. Native heavy SR membrane vesicles were incorporated into planar phospholipid bilayers under voltage-clamp conditions. Experiments were carried out in symmetrical 250 mM Cs+. Lifetime analysis indicates that channels activated solely by cytosolic Ca2+ exhibit at least two open and five closed states. The open events are very brief and are close to the minimum resolvable duration. When channels are activated solely by cytosolic Ca2+, luminal Ca2+ does not appear to exert any regulatory effect. The Po and duration of the open and closed lifetimes are unchanged. However, if channels are activated by ATP alone or by ATP plus cytosolic Ca2+, increases in luminal [Ca2+] produce marked increases in Po and in the duration of the open lifetimes. Our results demonstrate that maximum activation of the skeletal SR Ca(2+)-release channel by ATP cannot be obtained in the absence of millimolar luminal [Ca2+].