Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor

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.