CA2-INDUCED FOLDING AND AGGREGATION OF SKELETAL-MUSCLE SARCOPLASMIC-RETICULUM CALSEQUESTRIN - THE INVOLVEMENT OF THE TRIFLUOPERAZINE-BINDING SITE()

Calsequestrin is an intermediate affinity, high capacity Ca2+-binding protein found in the lumen of the sarcoplasmic reticulum of both skele tal and cardiac muscle cells. Previous sequence analysis suggested tha t calsequestrin may contain a hydrophobic binding site for the drug tr ifluoperazine, a site shared by the calmodulin family and shown to pla y a role in calmodulin/calmodulin receptor interaction. Previous studi es showed that, upon Ca2+ binding, calsequestrin undergoes a conformat ional 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 s ite in the Ca2+-induced conformational change of calsequestrin are fur ther studied using the calmodulin antagonists trifluoperazine and meli ttin. If trifluoperazine or melittin is added to calsequestrin prior t o Ca2+ addition, then Ca2+-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity t o trypsin digestion. If, however, Ca2+ is added prior to trifluoperazi ne 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 calsequestri n that exhibit high Ca2+ binding capacity have previously been shown t o 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 max imal Ca2+ binding by calsequestrin. These data suggest that the triflu operazine-binding site is critically involved in the Ca2+-induced intr amolecular folding step required for the intermolecular interactions l eading to high capacity Ca2+-binding by calsequestrin.