Depolarisation of the sarcolemma triggers Ca2+ release through ryanodine receptor (RyR) calcium release channels in the sarcoplasmic reticulum (SR) of skeletal muscle. Calsequestrin (CSQ) is a highly acidic glycoprotein found in the sarcoplasmic reticulum of cardiac and skeletal muscle. CSQ is the major Ca2+ binding protein within the SR and also functions as an regulator of the RyR, by forming a quaternary complex with the RyR, triadin and junctin. CSQ binds Ca2+ with a high capacity and moderate affinity, with a major putative Ca2+ binding motive occurring in the negatively charged residue-rich C-terminal tail (CSQCTT, residues 354-367). The CSQCTT is also thought to be responsible for CSQ's interaction with triadin and junctin.
To examine the role of the CSQCTT in Ca2+ binding and the formation of the quaternary complex, point mutations within this region of CSQ were produced. After subcloning rabbit skeletal wildtype (WT) CSQ into a pGEX-5X-1 vector (containing a glutathione-S-transferase tag), two mutants were generated by PCR, E354A and E354A/D356A. Mutants and WT rabbit skeletal CSQ PCR products were transformed and expressed in Escherichia coli BL21(DE3).
We found that the Ca2+ binding capacity of CSQ was reduced as the numbers of negatively charged residues within the CSQCTT are decreased. Compared with WT CSQ (100%), maximal Ca2+ binding capacity of E354A, and E354A/D356A was 92% and 87 %, respectively. WT and mutant CSQ interactions with triadin and junctin were analysed utilizing a glutathione-S-transferase affinity column. Initial results suggest that like WT CSQ, E354A and E354A/D356A CSQ interact with both triadin and junctin under physiological conditions (150 mM salt, 1 mM Ca2+free).
Our results suggest that the CSQCTT forms a major Ca2+ binding motive in rabbit skeletal muscle, and that both resides E354 and D356 are required both for Ca2+ binding and for stabilizing the Ca2+ binding motif, but are less important residues for associated protein binding.