Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP3R) serve to discharge Ca2+ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP3R isoforms and protect cells from apoptosis, reducing ER Ca2+ release. However, it has not been elucidated which IP3R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP3R I, strongly suppresses IP3-mediated Ca2+ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca2+ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca2+ release. Moreover, regulating Ca2+ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP3R III, leading to diminished Ca2+ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt

Regulation of ryanodine receptor-dependent calcium signaling by polycystin-2

Mutations in polycystin-2 (PC2) cause autosomal dominant polycystic kidney disease. A function for PC2 in the heart has not been described. Here, we show that PC2 coimmunoprecipitates with the cardiac ryanodine receptor (RyR2) from mouse heart. Biochemical assays showed that the N terminus of PC2 binds the RyR2, whereas the C terminus only binds to RyR2 in its open state. Lipid bilayer electrophysiological experiments indicated that the C terminus of PC2 functionally inhibited RyR2 channel activity in the presence of calcium (Ca(2+)). Pkd2(−/−) cardiomyocytes had a higher frequency of spontaneous Ca(2+) oscillations, reduced Ca(2+) release from the sarcoplasmic reticulum stores, and reduced Ca(2+) content compared with Pkd2(+/+) cardiomyocytes. In the presence of caffeine, Pkd2(−/−) cardiomyocytes exhibited decreased peak fluorescence, a slower rate of rise, and a longer duration of Ca(2+) transients compared with Pkd2(+/+). These data suggest that PC2 is important for regulation of RyR2 function and that loss of this regulation of RyR2, as occurs when PC2 is mutated, results in altered Ca(2+) signaling in the heart

Syntaxin 5 regulates the endoplasmic reticulum channel-release properties of polycystin-2

Polycystin-2 (PC2), the gene product of one of two genes mutated in dominant polycystic kidney disease, is a member of the transient receptor potential cation channel family and can function as intracellular calcium (Ca2+) release channel. We performed a yeast two-hybrid screen by using the NH2 terminus of PC2 and identified syntaxin-5 (Stx5) as a putative interacting partner. Coimmunoprecipitation studies in cell lines and kidney tissues confirmed interaction of PC2 with Stx5 in vivo. In vitro binding assays showed that the interaction between Stx5 and PC2 is direct and defined the respective interaction domains as the t-SNARE region of Stx5 and amino acids 5 to 72 of PC2. Single channel studies showed that interaction with Stx5 specifically reduces PC2 channel activity. Epithelial cells overexpressing mutant PC2 that does not bind Stx5 had increased baseline cytosolic Ca2+ levels, decreased endoplasmic reticulum (ER) Ca2+ stores, and reduced Ca2+ release from ER stores in response to vasopressin stimulation. Cells lacking PC2 altogether had reduced cytosolic Ca2+ levels. Our data suggest that PC2 in the ER plays a role in cellular Ca2+ homeostasis and that Stx5 functions to inactivate PC2 and prevent leaking of Ca2+ from ER stores. Modulation of the PC2/Stx5 interaction may be a useful target for impacting dysregulated intracellular Ca2+ signaling associated with polycystic kidney disease

A recently identified member of the glutathione transferase structural family modifies cardiac RyR2 substate activity, coupled gating and activation by Ca(2+) and ATP

The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [(3)H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca(2+). CLIC-2 (0.2–10 μM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10–100 μM) or sub-activating (100 nM) cytoplasmic Ca(2+) concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5′-[β,γ-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 μM cytoplasmic Ca(2+) than with 100 nM or 10 μM Ca(2+). Further, CLIC-2 prevented the usual ∼50-fold increase in activity when the cytoplasmic Ca(2+) concentration was increased from 100 nM to 100 μM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels

Polycystin-1 Interacts with Inositol 1,4,5-Trisphosphate Receptor to Modulate Intracellular Ca2+ Signaling with Implications for Polycystic Kidney Disease*

The PKD1 or PKD2 genes encode polycystins (PC) 1 and 2, which are associated with polycystic kidney disease. Previously we demonstrated that PC2 interacts with the inositol 1,4,5-trisphosphate receptor (IP3R) to modulate Ca2+ signaling. Here, we investigate whether PC1 also regulates IP3R. We generated a fragment encoding the last six transmembrane (TM) domains of PC1 and the C-terminal tail (QIF38), a section with the highest homology to PC2. Using a Xenopus oocyte Ca2+ imaging system, we observed that expression of QIF38 significantly reduced the initial amplitude of IP3-induced Ca2+ transients, whereas a mutation lacking the C-terminal tail did not. Thus, the C terminus is essential to QIF38 function. Co-immunoprecipitation assays demonstrated that through its C terminus, QIF38 associates with the IP3-binding domain of IP3R. A shorter PC1 fragment spanning only the last TM and the C-terminal tail also reduced IP3-induced Ca2+ release, whereas another C-terminal fragment lacking any TM domain did not. Thus, only endoplasmic reticulum-localized PC1 can modulate IP3R. Finally, we show that in the polarized Madin-Darby canine kidney cells, heterologous expression of full-length PC1 resulted in a smaller IP3-induced Ca2+ response. Overexpression of the IP3-binding domain of IP3R reversed the inhibitory effect of PC1, suggesting interaction of full-length PC1 (or its cleavage forms) with endogenous IP3R in Madin-Darby canine kidney cells. These results indicate that the behavior of full-length PC1 in mammalian cells is congruent with that of PC1 C-terminal fragments in the oocyte system. These data demonstrate that PC1 inhibits Ca2+ release, perhaps opposing the effect of PC2, which facilitates Ca2+ release through the IP3R