Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletalmuscle

Ca2+-handling proteins are important regulators of the excitation-contracti on-relaxation cycle in skeletal muscle fibres. Although domain binding stud ies suggest protein coupling between various Ca2+-regulatory elements of tr iad junctions, no direct biochemical evidence exists demonstrating high-mol ecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca2+ reservoir and thereby occupies a central position in Ca2+ homeostasis: we therefore used calseque strin blot overlay assays in order to determine complex formation between s arcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clear ly labelled four major protein bands in one-dimensional (1D) and 2D electro phoretically separated membrane preparations from adult skeletal muscle. Im munoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the r yanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca2+ concentration, as well as antibody and purified calsequestrin binding. Importantly, comple x formation as determined by blot overlay assays was confirmed by different ial co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca2+-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associate d junctional sarcoplasmic reticulum complex seems to underlie the physiolog ical regulation of skeletal muscle contraction and relaxation. which suppor ts the biochemical concept that Ca2+ homeostasis is regulated by direct pro tein-protein interactions. (C) 2001 Elsevier Science B.V. All rights reserv ed.