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Mutation within the C-terminus of skeletal calsequestrin disrupts calcium binding

N.A. Beard1, L. Wei1, O.J. Fiebig2, M. Varsányi2, A.F. Dulhunty1, 1JCSMR, Australian National University, Acton, ACT, Australia, 2Ruhr Universitat, Bochum, Germany

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.