Regulation of Ca2+ release through inositol 1,4,5-trisphosphate receptors (InsP3R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca2+ signals. In this study, regulation of Ca2+ release by phosphorylation of type 1 InsP3R (InsP3R-1 was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP3R-1 splice variants. Ca2+ release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP3R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP3R-1,InsP3-induced Ca2+ release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/ S1755A mutants. Ca2+ release through the S2- S1589E/ S1755E InsP3R-1 was enhanced ∼8-fold over wild type and ∼50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2-InsP3R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca2+ release was enhanced ∼3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/ Glu mutation failed to further enhance Ca2+ release. The sensitivity of Ca2+ release in cells expressing S2+ S1755E InsP3R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP3R-1. In contrast, mutation of S2+ S1589E InsP 3R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2-S1589E/S1755E InsP3R-1 resulted in robust Ca2+ oscillations when cells were stimulated with concentrations of α-IgM antibody that were threshold for stimulation in S2- wild type InsP3R-1-expressing cells. However, at higher concentrations of α-IgM antibody, Ca2+ oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP3R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.
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