Expression and inhibitor studies of Ca2+-ATPases

Many compounds inhibit the function of the Ca'^-ATPase of the sarcoplasmic reticulum (SERCA). These are generally hydrophobic compounds containing -OH groups. Curcumin is shown to be an inhibitor of the Ca^'-ATPase. Studies of the effects of mixtures of inhibitors show that 2,5-di(propyI)-l,4-benzohydroquinone and 2,5-di(fg/7- butyl)-] ,4-benzohydroquinone (BHQ) bind to the same site on the ATPase, but that the binding site for BHQ is separate lErom that for butylatedhydroxy toluene, ellagic acid, diethylstilbestrol (DBS), curcumin and nonylphenol, and that the binding site for curcumin is separate fi-om that 6)r ellagic acid and DES. The presence of BHQ, DES, ellagic acid and nonylphenol increase the a@inity of the ATPase for curcumin, suggesting that binding of one inhibitor results in a change in the ATPase to a conformation with higher affinity for the second inhibitor. In QT-6 cells curcumin prevents the release of calcium normally seen on addition of the Ca'-ATPase inhibitor trilobilide, suggesting that curcumin blocks the inositol sensitive calcium release channel (IP3R). In sarcoplasmic reticulum it was shown that curcumin reduces the rate of slippage on the Ca"-ATPase and so increases calcium accumulation into SR vesicles, despite being an inhibitor of ATPase function. SERCA and SERCA-GFP have been expressed in Cos-7 cells. A chimera between SERCA and a Ca"-ATPase from Heliothis virescens, HVSERCA, showed higher levels of expression in Cos-7 cells than HVSERCA. SERCA and HVSERCA have been expressed in S.cerexnsiae and comparative inhibitor studies using this system have shown that trilobilide inhibits SERCA but not HVSERCA, whereas BHQ inhibits both of these pumps.