Beard, Nicole A.Casarotto, Marco G.Wei, LanVarsányi, MagdolnaLaver, Derek R.Dulhunty, Angela F.2016-03-282016-03-280006-3495http://hdl.handle.net/1885/100886Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which calsequestrin dissociates from the ryanodine receptor and the effect of calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of >/=4 mM dissociates calsequestrin from junctional face membrane, whereas in the range of 1-3 mM calsequestrin remains attached; 2), the association with calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.N.A.B. was supported by the Australian Research Council of Australia (project ID DP0344878), L.W. was supported by an Australian National University PhD Scholarship, and D.R.L. was supported by the National Health & Medical Research Council of Australia (grant No. 234420) and a Professorial Fellowship from the Australian Research Council.© 2005 by the Biophysical Societyacid phosphataseanimalscalciumcalcium-binding proteinscalsequestrincarrier proteinscasein kinase iichromatographydose-response relationship, drugelectrophoresis, polyacrylamide gelelectrophysiologyglutathioneimmunoblottinglipid bilayersmagnetic resonance spectroscopymembrane proteinsmixed function oxygenasesmuscle proteinsmuscle, skeletalmusclesphosphorylationprotein conformationrabbitsrecombinant fusion proteinsrecombinant proteinsryanodine receptor calcium release channelsarcoplasmic reticulumsignal transduction200510.1529/biophysj.104.0514412016-06-14