Cytosolic and luminal modulation of Ca(2+) signalling by parvalbumin and calreticulin in Xenopus oocytes

Many hormone-receptor signalling pathways trigger Ca2+ oscillations subsequent to inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Because of the Ca2+ dependency of the IP3 receptor, processes that affect Ca2+ removal from the vicinity of the IP3R Ca2+ release channel can be important modulators of hormonal signalling. This dissertation examines (1) the role of the ER luminal Ca2+ -binding chaperone calreticulin in the modulation of Ca2+ uptake by two SERCA isoforms, (2) cytosolic Ca2+ buffering by the EF-hand CaBP, parvalbumin and (3) the influence of thyroid hormone and its receptor on mitochondrial Ca2+ buffering. These goals are achieved by applying molecular biology techniques in conjunction with confocal imaging of IP3-induced Ca2+ wave activity in Xenopus laevis oocytes.;Spontaneous Ca2+ sparks evoked by parvalbumin. The EF hand CaBP, parvalbumin, buffers cytosolic Ca 2+ concentrations. Parvalbumin is reported to facilitate skeletal muscle relaxation and protects neuronal cells from high Ca2+ loads. Both Mg2+ and Ca2+ can bind to parvalbumin. At resting cytosolic concentrations, parvalbumin is bound to Mg2+. The kinetics of Ca2+ buffering by parvalbumin are determined by its slow Mg2+ dissociation rate. By injecting purified protein or by overexpressing parvalbumin in oocytes, the effects of Ca2+ buffering by parvalbumin on Ca2+ release events is examined.;Differential modulation of SERCA2 isoforms by calreticulin. In Xenopus oocytes, overexpression of SERCA isoforms increases IP3-induced Ca2+ wave frequency. Overexpression of the ER resident molecular chaperone, calreticulin (CRT), results in a sustained elevation of intracellular Ca2+ with a concomitant inhibition of repetitive Ca2+ waves following IP3R activation. This sustained elevation is also observed when CRT was co-expressed with SERCA 2b. CRT is a member of a novel class of ER chaperones with lectin activity. These chaperones bind to N-linked glycosylation residues to modulate protein folding. SERCA 2b has a luminal facing N-linked glycosylation site (N1036) that is not present in SERCA 2a. If the inhibition of Ca2+ wave activity is due to CRT interacting with the glycosylation site on SERCA 2b, then coexpression of CRT with SERCA 2a should not produce the inhibitory effect. Effects on IP3-induced Ca 2+ signalling are examined by coexpression of a CRT mutant with SERCA 2b, 2a and the mutant SERCA2b N1036A to determine whether CRT differentially modulates Ca2+ uptake by these ATPases.