TY - JOUR T1 - Effects of High-Affinity Inhibitors on Partial Reactions, Charge Movements, and Conformational States of the Ca<sup>2+</sup> Transport ATPase (Sarco-Endoplasmic Reticulum Ca<sup>2+</sup> ATPase) JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1134 LP - 1140 DO - 10.1124/mol.107.043745 VL - 73 IS - 4 AU - Francesco Tadini-Buoninsegni AU - Gianluca Bartolommei AU - Maria Rosa Moncelli AU - Daniel M. Tal AU - David Lewis AU - Giuseppe Inesi Y1 - 2008/04/01 UR - http://molpharm.aspetjournals.org/content/73/4/1134.abstract N2 - 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 Ca2+ 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 Ca2+ binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca2+ 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 Ca2+. We demonstrate here that E2 state conformational features are in fact induced by these inhibitors on the ATPase even in the presence of Ca2+. 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 (E1∼P · 2Ca2+ to E2-P · 2Ca2+ transition), as demonstrated by increased phosphoenzyme levels and absence of bound Ca2+ 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 Ca2+ ATPase inhibitors is based on global effects on protein conformation that interfere with ATPase cycling. The American Society for Pharmacology and Experimental Therapeutics ER -