A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the 
main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of 
skeletal muscle, seems well supported. Opinions are still divided, however, 
concerning the triadin domain involved, either the cytoplasmic or the lumenal 
domain, and the exact role played by Ca(2+), in the protein-to-protein 
interaction. Further support for colocalization of HRC with triadin cytoplasmic 
domain is provided here by experiments of mild tryptic digestion of tightly 
sealed TC vesicles. Accordingly, we show that HRC is preferentially 
phosphorylated by endogenous CaM K II, anchored to SR membrane on the 
cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate 
that HRC can be isolated as a complex with triadin, following equilibrium 
sucrose-density centrifugation in the presence of mM Ca(2+). Here, we 
characterized the COOH-terminal portion of rabbit HRC, expressed and purified as 
a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and 
to the interaction with triadin on blots, as a function of the concentration of 
Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as 
the Ca(2+)-binding site responsible, both for the acceleration in mobility of 
HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for 
the enhancement by high Ca(2+) of the interaction between HRC and triadin 
cytoplasmic segment. (c)2001 Elsevier Science.