Effects of High-Affinity Inhibitors on Partial Reactions, Charge Movements, and Conformational States of the Ca²⁺Transport ATPase (Sarco-Endoplasmic Reticulum Ca²⁺ATPase)

Abstract

The inhibitory effects of thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone, and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene on the Ca 2ϩ ATPase were characterized by comparative measurements of sequential reactions of the catalytic and transport cycle, including biochemical measurements and detection of charge movements within a single cycle. In addition, patterns of ATPase proteolytic digestion with proteinase K were derived to follow conformational changes through the cycle or after inhibitor binding. We find that thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone inhibit Ca 2ϩ binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca 2ϩ in the absence of ATP. It has been shown previously that binding of these inhibitors requires the E2 conformational state of the ATPase, obtained in the absence of Ca 2ϩ . We demonstrate here that E2 state conformational features are in fact induced by these inhibitors on the ATPase even in the presence of Ca 2ϩ . The resulting dead-end complex interferes with progress of the catalytic and transport cycle. Inhibition by 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene, on the other hand, is related to interference with a conformational transition of the phosphorylated intermediate (E1ϳP ⅐ 2Ca 2ϩ to E2-P ⅐ 2Ca 2ϩ transition), as demonstrated by increased phosphoenzyme levels and absence of bound Ca 2ϩ translocation upon addition of ATP. This transition includes large movements of ATPase headpiece domains and transmembrane segments, produced through utilization of ATP-free energy as the "conformational work" of the pump. We conclude that the mechanism of high-affinity Ca 2ϩ ATPase inhibitors is based on global effects on protein conformation that interfere with ATPase cycling.

Alterations in Ca2+-Buffering in Prion-Null Mice: Association with Reduced Afterhyperpolarizations in CA1 Hippocampal Neurons

“…The observation that the SERCA inhibitor, thapsigargin, abolished the increases in both buffering ( S ) and clearance (␥) in Prnp 0/0 neurons suggested that the differences were caused by changes in the ER. The role of SERCA is to pump Ca 2ϩ into the ER, resulting in Ca 2ϩ clearance, but it also acts as a Ca 2ϩ buffer (Higgins et al, 2006); both of these functions are inhibited by thapsigargin or CPA (Tadini-Buoninsegni et al, 2008). We conclude that changes in SERCA can explain increases in both buffering and clearance in the Prnp 0/0 neurons.…”

A diversity of SERCA Ca2+ pump inhibitors

“…Furthermore, of note are BHQ and CPA, which, like thapsigargin, have been used extensively as pharmacological tools by scientists to mobilize SERCA-loaded Ca 2+ stores [24]. Both inhibitors were shown previously to inhibit the Ca 2+ -ATPase by stabilizing it in one of the E2 conformational states [12,25] and, to date, many other inhibitors of this class have also been shown to cause this same effect. It was originally proposed that molecules such as CPA and BHQ bound to the same site as thapsigargin [26]; however, this was challenged by inhibitor competition studies [27], and then confirmed by crystal structures clearly showing them binding to different sites [28,29].…”

Computational Library Design for Increasing Haloalkane Dehalogenase Stability

“…21 The SSM technique is also well‐suited for the analysis of drug interactions with membrane transporters. In this respect, the effects of various inhibitors showing a variable affinity for SERCA were investigated using the SSM technique 22–24. It is worth mentioning that the SSM sensor combined with robotized instrumentation is expected to become an attractive platform technology for drug screening and development 25.…”