Activation of Ca transport in cardiac microsomes enriches functional sets of ER and SR proteins

Abstract

Abstract The importance of sarcoplasmic reticulum (SR) Ca-handling in heart has led to detailed understanding of Ca-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Analyses of protein enrichments from the 3 membrane preparations (crude microsomes, MedSR, and HighSR), showed that only a third of microsomal proteins in heart, or 354 proteins, were enriched ≥2.0-fold in SR. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca leak (ryanodine receptor) co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins from other putative ER/SR subdomains also showed characteristic distributions among SR subpopulations. We conclude that active Ca loading of cardiac microsomes, reflecting the combined activities of Ca uptake by SERCA, and Ca leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.