Received for publication November 26, 2007.
Revised January 22, 2008.
Accepted for publication January 22, 2008.

Effects of high affinity inhibitors on partial reactions, charge movements and conformational states of the Ca²⁺ transport ATPase (SERCA)

Abstract

The inhibitory effects of thapsigargin, cyclopiazonic acid, 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 following 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 was previously shown 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.

Key words: Ion transporters (SERCA, Na/K ATPase, CFTR), Structure-activity relationships and modeling, Prediction of structure-function/proteomics, Protein-binding

This article has been cited by other articles:

				G. Bartolommei, N. Devaux, F. Tadini-Buoninsegni, M. Moncelli, and H.-J. Apell Effect of Clotrimazole on the Pump Cycle of the Na,K-ATPase Biophys. J., August 15, 2008; 95(4): 1813 - 1825. [Abstract] [Full Text] [PDF]

				A. D. Powell, E. C. Toescu, J. Collinge, and J. G. R. Jefferys Alterations in Ca2+-Buffering in Prion-Null Mice: Association with Reduced Afterhyperpolarizations in CA1 Hippocampal Neurons J. Neurosci., April 9, 2008; 28(15): 3877 - 3886. [Abstract] [Full Text] [PDF]