Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase
Experiments were performed to determine whether the organic Ca2+ channel blocker D-600 (gallopamil), which penetrates into muscle cells, affects sarcoplasmic reticulum (SR) Ca2+ uptake by directly inhibiting the light SR Ca2+-ATPase. We have previously shown that at 10 μM, D-600 inhibits LSR ATP-dependent Ca2+ uptake by 50% but has no effect on ATPase activity (21). These data suggest that the SR Ca2+-ATPase might be a potential target for D-600. The ATPase activity of the enzyme is associated with its hydrophilic cytoplasmic domain, whereas Ca2+ binding and translocation are associated with the transmembrane domain (18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermal inactivation experiments using the SR Ca2+-ATPase demonstrated that D-600 decreased the thermal stability of Ca2+ transport but had no effect on the stability of ATPase activity. In addition, D-600 at a concentration of 160 μM did not have any leaking effect of Ca2+ on the Ca2+-loaded SR. Thermal denaturation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the Ca2+-ATPase. No evidence for interaction with the nucleotide domain was obtained. We conclude that the Ca2+ blocker D-600 inhibits the SR Ca2+ pump specifically by interacting with the transmembrane Ca2+-binding domain of the Ca2+-ATPase.