Store Operated Calcium Entry Controls Intracellular Calcium Waves In Xenopus Oocytes

Abstract

In many non-excitable cells such as glial cells, oocytes or beta-cells, fast transmission and integration of information are encoded in the variations of the intracellular Ca2+ concentration. These variations range from a single calcium peak (wave) to a repetitive pattern (oscillations). Intracellular Ca2+ oscillations are made of highly spatially and timely controlled cytoplasmic waves of Ca2+ fueled by two main Ca2+ sources: the extracellular compartment and the endoplasmic reticulum (ER). Specific and highly regulated ion channels and pumps located on the plasma and ER membranes control the cytoplasmic Ca2+ levels as well as the refilling of the ER stores. When the ER Ca2+ stores are depleted, a mechanism termed Store Operated Calcium Entry (SOCE) drives Ca2+ inside the cell to refill them. In the oocytes of the frog Xenopus laevis intracellular Ca2+ variations are converted into chloride currents by Ca2+-Activated Chloride Channels (CACC) located in the plasma membrane, allowing real time monitoring of cytoplasmic Ca2+ levels. We recently showed that SOCE and IP3 receptors (IP3R) define a functional complex allowing mid-range Ca2+ signaling to CACC in those oocytes (Courjaret and Machaca, Nat. Commun. 2014). Here we aimed at understanding how SOCE influences intracellular Ca2+ release by IP3R and oscillations. Injecting an IP3 analog induced a Ca2+ transient oscillation and triggered SOCE. When SOCE influence was increased using hyperpolarizing pulses or reduced by removing extracellular Ca2+, the duration of the Ca2+ oscillation was respectively reduced or increased. Intracellular Ca2+ imaging also revealed that increasing SOCE was reducing the amplitude and increasing the frequency of the individual Ca2+ waves that constitute the oscillation. In addition, removing extracellular Ca2+ more than 30 minutes after the end of the oscillation restored the Ca2+ release, indicating that the ER stores are not fully depleted during the process. Surprisingly, while a short SOCE was able to inhibit intracellular Ca2+ release, increasing the duration/amplitude of the SOCE event was able to trigger a new Ca2+ wave at a very precise timing. This indicated that a strong reloading of the ER stores could overcome the inability of IP3R receptors to release Ca2+ and suggested a luminal regulation of the receptor. To further understand how SOCE was modulating Ca2+ release we therefore monitored Ca2+ levels in the ER lumen by expressing the FRET sensor D1ER. After injection of IP3, D1ER imaging revealed the depletion/reloading of the ER stores by SOCE as a function of the membrane potential. Finally, during the long voltage jumps that are able to induce a Ca2+ wave, the D1ER signal revealed that the high Ca2+ in the ER outlasts the cytoplasmic Ca2+ event leaving the IP3R facing a low cytoplasmic Ca2+ concentration and a high luminal one. Together these findings shed a new light on the complex regulation of IP3R and their tuning by Ca2+ on both sides of the ER membrane.