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)

Abstract

The secretory pathway Ca2+ ATPase (SPCA) provides the Golgi apparatus with a Ca2+ supply essential for Ca2+-dependent enzymes involved in the post-translational modification of proteins in transit through the secretory pathway. Ca2+ in the Golgi apparatus is also agonist-releasable and plays a role in hormone-induced Ca2+ transients. Although the Ca2+ ATPase inhibitors thapsigargin is more selective for the sarcoplasmic–endoplasmic reticulum Ca2+ 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 Ca2+-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 IC50 values of 0.13μM and 0.18mM, respectively), which were of 62- and 8.3-fold greater potency than the values for hSERCA2b (IC50 values; 8.1μM and 1.5mM, respectively). Other inhibitors tested such as bis-phenol-A, tetrabromobisphenol-A and trifluoperazine inhibited both Ca2+ ATPases similarly. Furthermore, bis-phenol was able to mobilize Ca2+ 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.