Ca2+-induced folding and aggregation of skeletal muscle sarcoplasmic reticulum calsequestrin. The involvement of the trifluoperazine-binding site

Z. He, A. K. Dunker, C. R. Wesson, W. R. Trumble

Research output: Contribution to journalArticle

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Abstract

Calsequestrin is an intermediate affinity, high capacity Ca2+-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca2+-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca2+-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca2+-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca2+-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca2+- binding by calsequestrin.

Original languageEnglish (US)
Pages (from-to)24635-24641
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number33
StatePublished - Jan 1 1993

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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