Bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)-methane (bis-phenol) is a potent and selective inhibitor of the secretory pathway Ca²⁺ ATPase (SPCA1).
The secretory pathway Ca(2+) ATPase (SPCA) provides the Golgi apparatus with a Ca(2+) supply essential for Ca(2+)-dependent enzymes involved in the post-translational modification of proteins in transit through the secretory pathway. Ca(2+) in the Golgi apparatus is also agonist-releasable and plays a role in hormone-induced Ca(2+) transients. Although the Ca(2+) ATPase inhibitors thapsigargin is more selective for the sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA) than for SPCA, no inhibitor has been characterised that selectively inhibits SPCA. A number of inhibitors were assessed for their selectivity to the human SPCA1d compared to the more ubiquitous human SERCA2b. Each isoform was over-expressed in COS-7 cells and the Ca(2+)-dependent ATPase activity measured in their microsomal membranes. Both bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane(bis-phenol) and 2-aminoethoxydiphenylborate (2-APB) selectively inhibited SPCA1d (with IC(50) values of 0.13 μM and 0.18 mM, respectively), which were of 62- and 8.3-fold greater potency than the values for hSERCA2b (IC(50) values; 8.1 μM and 1.5mM, respectively). Other inhibitors tested such as bis-phenol-A, tetrabromobisphenol-A and trifluoperazine inhibited both Ca(2+) ATPases similarly. Furthermore, bis-phenol was able to mobilize Ca(2+) in cells that had been pre-treated with thapsigargin. Therefore we conclude that given the potency and selectivity of bis-phenol it may prove a valuable tool in further understanding the role of SPCA in cellular processes.
School of Biosciences, The University of Birmingham Edgbaston, Birmingham B15 2TT, United Kingdom.
Pei
P
Lai
Francesco
F
Michelangeli
D000818:Animals; D002084:Butylated Hydroxytoluene / Q000031:analogs & derivatives* / Q000494:pharmacology; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000235:genetics; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D014162:Transfection
D004791:Enzyme Inhibitors; D002084:Butylated Hydroxytoluene; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; C008437:2,2'-methylenebis(4-methyl-6-tert-butylphenol)