Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase.
Experiments were performed to determine whether the organic Ca(2+) channel blocker D-600 (gallopamil), which penetrates into muscle cells, affects sarcoplasmic reticulum (SR) Ca(2+) uptake by directly inhibiting the light SR Ca(2+)-ATPase. We have previously shown that at 10 microM, D-600 inhibits LSR ATP-dependent Ca(2+) uptake by 50% but has no effect on ATPase activity (21). These data suggest that the SR Ca(2+)-ATPase might be a potential target for D-600. The ATPase activity of the enzyme is associated with its hydrophilic cytoplasmic domain, whereas Ca(2+) 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 Ca(2+)-ATPase demonstrated that D-600 decreased the thermal stability of Ca(2+) transport but had no effect on the stability of ATPase activity. In addition, D-600 at a concentration of 160 microM did not have any leaking effect of Ca(2+) on the Ca(2+)-loaded SR. Thermal denaturation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the Ca(2+)-ATPase. No evidence for interaction with the nucleotide domain was obtained. We conclude that the Ca(2+) blocker D-600 inhibits the SR Ca(2+) pump specifically by interacting with the transmembrane Ca(2+)-binding domain of the Ca(2+)-ATPase.
Ortega
A
A
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City 04510, México. aortega@servidor.unam.mx
Becker
V M
VM
Alvarez
R
R
Lepock
J R
JR
Gonzalez-Serratos
H
H
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D002151:Calorimetry; D005711:Gallopamil / Q000494:pharmacology*; D006358:Hot Temperature; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D002121:Calcium Channel Blockers; D005711:Gallopamil; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D002118:Calcium