Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase

Experiments were performed to determine whether the organic Ca2+ channel bl ocker D-600 (gallopamil), which penetrates into muscle cells, affects sarco plasmic reticulum (SR) Ca2+ uptake by directly inhibiting the light SR Ca2-ATPase. We have previously shown that at 10 mu M, D-600 inhibits LSR ATP-d ependent Ca2+ uptake by 50% but has no effect on ATPase activity (21). Thes e data suggest that the SR Ca2+-ATPase might be a potential target for D-60 0. The ATPase activity of the enzyme is associated with its hydrophilic cyt oplasmic domain, whereas Ca2+ binding and translocation are associated with the transmembrane domain (18). In the present experiments, we determined w hich of the two domains of the ATPase is affected by D-600. Thermal inactiv ation experiments using the SR Ca2+-ATPase demonstrated that D-600 decrease d the thermal stability of Ca2+ transport but had no effect on the stabilit y of ATPase activity. In addition, D-600 at a concentration of 160 mu M did not have any leaking effect of Ca2+ on the Ca2+-loaded SR. Thermal denatur ation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the Ca2+-ATPase. No evidence for interaction wi th the nucleotide domain was obtained. We conclude that the Ca2+ blocker D- 600 inhibits the SR Ca2+ pump specifically by interacting with the transmem brane Ca2+-binding domain of the Ca2+-ATPase.