Differential role of STIM1 and STIM2 during transient inward (Tin) current generation and the maturation process in the Xenopus oocyte

Abstract

BackgroundThe Xenopus oocyte is a useful cell model to study Ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. Here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule (STIM) proteins that are fundamental elements of the store-operated calcium-entry (SOCE) phenomenon, as they are both sensors for Ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow Ca2+ influx.ResultsEndogenous STIM1 and STIM2 expression was demonstrated, and their synthesis was knocked down 48–72 h after injecting oocytes with specific antisense sequences. Selective elimination of their mRNA and protein expression was confirmed by PCR and Western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of SOC channels elicited by G protein-coupled receptor (GPCR)-activated Ca2+ release, and the process of maturation stimulated by progesterone. Activation of SOC channels was monitored electrically by measuring the Tin response, a Ca2+-influx-dependent Cl− current, while maturation was assessed by germinal vesicle breakdown (GVBD) scoring and electrophysiology.ConclusionsIt was found that STIM2, but not STIM1, was essential in both responses, and Tin currents and GVBD were strongly reduced or eliminated in cells devoid of STIM2; STIM1 knockdown had no effect on the maturation process, but it reduced the Tin response by 15 to 70%. Thus, the endogenous SOCE response in Xenopus oocytes depended mainly on STIM2, and its expression was necessary for entry into meiosis induced by progesterone.Electronic supplementary materialThe online version of this article (doi:10.1186/s12899-014-0009-x) contains supplementary material, which is available to authorized users.

Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry

“…Some studies have indicated that STIM2 does not play a role in SOCE, while others have found it has a minor contribution to SOCE [ 2 , 21 , 22 , 24 , 36 , 37 , 38 , 39 , 40 ]. In contrast, STIM2 has been found to be the primary regulator of SOCE in mouse cortical neurons and Xenopus oocytes [ 41 , 42 ]. However, in rat cortical neurons, STIM1 was critical for SOCE, while STIM2 was important for regulation of resting [Ca 2+ ] ER [ 43 ].…”

“…Silencing of STIM2 in HEK293, neutrophil-like HL-60 cells, rat pulmonary arterial smooth muscle, airway myocytes, and CD4 T + cells did not affect SOCE [ 2 , 21 , 23 , 37 , 38 , 39 ]. In contrast, knockdown of STIM2 in HeLa cells, B cells, MDA-MB-231 cells, and mouse embryonic fibroblasts (MEFs) resulted in a small reduction of SOCE, while a more significant reduction in SOCE was seen when STIM2 was silenced in murine T cells, cortical neurons, mast cells, and Xenopus oocytes [ 3 , 21 , 22 , 40 , 41 , 42 , 53 , 54 ]. Even more surprising was the finding that STIM2 inhibited SOCE when stably overexpressed in HEK293, PC12, A7r5 and Jurkat T cells [ 23 , 25 ].…”

“…Differences between our work and previously published data regarding a threshold of activation for STIM2 recruitment of STIM1 to ER-PM junctions is likely explained by differences in experimental approach used to silence Stim2 expression. Many of the early studies used siRNA knockdown of STIM2 [ 2 , 21 , 23 , 37 , 42 ], yielding varying levels of efficacy, which could significantly impact the interpretation of the data. It is likely that partial knockdown of STIM2 expression may not be sufficient to reveal a role of STIM2 in SOCE.…”

Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation

“…Oocyte maturation was stimulated with progesterone and validated by pERK2/ERK2 immunoblot analysis and GVBD counting. Batches of 40 defolliculated oocytes (stage VI) noninjected and injected with GIRK5K13AR14A and/or GIRK5 siRNA were incubated in 2 mL of ND96 with 10 μ m progesterone for 1, 3, 5, 7, and 10 h according to Serrano‐Flores [41]. To study the effect of barium on GVBD (Fig.…”

Molecular physiology and pathophysiology of stromal interaction molecules

“…3,103,150,151 In contrast, in Xenopus oocytes, STIM2, but not STIM1, played a predominant role in SOCE. 152 Alternatively, silencing of STIM2 expression in HEK293, neutrophil-like HL-60 cells, rat pulmonary arterial smooth muscle, airway myocytes, and CD4 T þ cells did not affect SOCE. 1,103,148,[153][154][155] In mouse neurons, where STIM2 is the dominant isoform, there was a significant reduction of SOCE following knockout of STIM2.…”