To investigate the role of phosphorylation at T-930 on Ins(1,4,5)P3 receptor function, we engineered two mutants one with an alanine substitution at T-930 (T930A), and the other with a phosphomimetic mutation where the threonine is replaced by a glutamic acid (T930E). We chose glutamic acid as its side chain closely resembles that of a phosphorylated negatively charged threonine side chain. As such the T930E mutant is expected to mimic the behavior of the Ins(1,4,5)P3 receptor phosphorylated at T-930. In contrast, the alanine substitution at T930 serves as a control and provides a mutant that is not phosphorylatable at this residue. Furthermore, it also controls for the structural need for a threonine at this position.
The ubiquitous distribution of the Ins(1,4,5)P3 receptor makes it challenging to study the effect of different mutants on Ins(1,4,5)P3 receptor function because of the background signal due to endogenous Ins(1,4,5)P3 receptors in most cells. Therefore to study the functional consequences of phosphorylation at T-930, we used the DT40 chicken lymphocyte cell line because of the existence of a DT40 line (3KO), where all three Ins(1,4,5)P3 receptor genes (type 1, 2, and 3) have been knocked out. This provides a clean background to analyze the function of different expressed Ins(1,4,5)P3 receptor mutants. Using the 3KO line as the parental line, we generated stable DT40 cell lines expressing the wild-type rat Ins(1,4,5)P3 receptor, the T930A or the T930E mutants. Following the establishment of the cell lines we amplified and sequence confirmed that they are in fact expressing the relevant Ins(1,4,5)P3 receptor mutant.
Western blot analysis shows that the three different cell lines express the Ins(1,4,5)P3 receptor at equivalent levels, whereas no immuno-reactivity is detected in the 3KO line (). The growth rate of the different DT40 cell lines were equivalent, arguing that expression of the different mutants did not have a dramatic effect on cell survival or ability to replicate (). Finally, both resting Ca2+ levels and Ca2+ store content were similar among the different cell lines (), showing that expression of the T930E or T930A mutant does not alter Ca2+ homeostasis significantly at rest.
To test the sensitivity of the different Ins(1,4,5)P3 receptor mutants, we developed an assay to measure Ca2+ release from intracellular stores in situ in response to an Ins(1,4,5)P3 dose response. For these experiments DT40 cells were loaded with caged-Ins(1,4,5)P3 and Ins(1,4,5)P3 was uncaged for different durations as indicated in . Cells expressing wild-type (WT) Ins(1,4,5)P3 receptor respond with an exponential increase in Ca2+ release along the Ins(1,4,5)P3 uncaging dose response (). This assay provides a direct test of Ins(1,4,5)P3 receptor function because Ins(1,4,5)P3 produced following uncaging binds to and gates the Ins(1,4,5)P3 receptor without invoking additional intermediaries.
Figure 2. The T930E mutant exhibits decrease Ins(1,4,5)P3-dependent Ca2+ release sensitivity. DT40 cells were loaded with a Ca2+ dye and caged-Ins(1,4,5)P3 and exposed to different UV uncaging pulse durations to produce a dose response of Ins(1,4,5) (more ...)
Surprisingly in cells expressing the T930A mutant Ins(1,4,5)P3-dependent Ca2+ release is sensitized compared with the wild-type receptor (). The T930A mutant receptor responds with increased Ca2+ release at lower Ins(1,4,5)P3 concentrations although the maximum amount of Ca2+ release is comparable to the wild-type receptor (). This sensitization is unlikely to be due to higher expression levels of the T930A mutant as compared with WT, because the different cell lines express equivalent levels of Ins(1,4,5)P3 receptors () and because the maximal Ca2+ release is similar between the two cell lines ().
In contrast, cells expressing the T930E mutant exhibit the opposite phenotype of decreased Ca2+ release in response to Ins(1,4,5)P3 uncaging (). Interestingly, even at high levels of Ins(1,4,5)P3 these cells release significantly smaller levels of Ca2+ (). This is not due to decreased store Ca2+ content, since T930E expressing cells have a similar store Ca2+ load as cells expressing the wild-type receptor or the T930A mutant (). In contrast cells expressing the T930A mutant or the wild-type receptor produce significantly higher Ca2+ release levels at long uncaging durations (). Because the T930E cell line expresses equivalent amount of Ins(1,4,5)P3 receptors, these results argue that either the affinity of the T930E mutant to Ins(1,4,5)P3 is low or that gating/permeation or Ca2+ dependence of the receptor are affected by the T930E mutation leading to short lived or small Ca2+ release.
To further investigate which of these mechanisms is affected in the T930E mutant, we directly measured the Ins(1,4,5)P3 binding affinity of the different mutants in microsomes derived from the stable DT40 cell lines (). Microsomes from brain, which is enriched in Ins(1,4,5)P3 receptors, were used as a positive control and showed significant Ins(1,4,5)P3-binding that was competed effectively by cold Ins(1,4,5)P3 (). Ins(1,4,5)P3 binding to microsomes isolated for the DT40 cell lines expressing the wild-type Ins(1,4,5)P3 receptor showed lower binding than brain microsomes, which was also effectively competed away using cold Ins(1,4,5)P3 (). Consistent with the Ins(1,4,5)P3 uncaging data (), microsomes from cells expressing the T930A mutant exhibited higher Ins(1,4,5)P3 binding affinity than their wild-type counterparts, whereas Ins(1,4,5)P3 binding to T930E microsomes was hardly detectable and only slightly above the background binding observed in the 3KO cells (). These data show that the behavior of the T930A and T930E mutants in situ can be explained solely based on their Ins(1,4,5)P3 binding affinity without the need to invoke alterations to Ca2+ dependence, gating or permeation of the receptor. Our results further show that phosphorylation at T930 leads to a significant decrease in Ins(1,4,5)P3 binding affinity.
Figure 3. The T930E mutation decreases Ins(1,4,5)P3 binding affinity. Microsomal preparations from brain as a positive control and from the different DT40 cell lines as indicated were subjected to the Ins(1,4,5)P3 binding assay to measure the (more ...)