Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca 2+ release activated Ca 2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca 2+ sensor required for activation of CRAC current (I CRAC ) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca 2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I CRAC . In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I CRAC . The results show that cells expressing low Orai1 : STIM1 ratios produce I CRAC with strong fast Ca 2+ -dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I CRAC with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca 2+ , Ba 2+ and Sr 2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca 2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

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Ca2+ release activated Ca2+ (CRAC) channels composed of two cellular proteins, Ca2+-sensing stromal interaction molecule 1 (STIM1) and pore-forming Orai1, are the main mediators of the Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. Previously it has been shown that the amplitude of CRAC current (ICRAC) strongly depends on extracellular and intracellular pH. Here we investigate the intracellular pH (pHi) dependence of ICRAC mediated by Orai1 and STIM1ectopically expressed in HEK293 cells. The results indicate that pHi affects not only the amplitude of the current, but also Ca2+ dependent gating of CRAC channels. Intracellular acidification changes the kinetics of ICRAC, introducing prominent re-activation component in the currents recorded in response to voltage steps to strongly negative potentials. ICRAC with similar kinetics can be observed at normal pHi if the expression levels of Orai1 are increased, relative to the expression levels of STIM1. Mutations in the STIM1 inactivation domain significantly diminish the dependence of ICRAC kinetics on pHi, but have no effect on pHi dependence of ICRAC amplitude, implying that more than one mechanism is involved in CRAC channel regulation by intracellular pH.

FCDI (fast Ca²⁺-dependent inactivation) is a mechanism that limits Ca²⁺ entry through Ca²⁺ channels, including CRAC (Ca²⁺ release-activated Ca²⁺) channels. This phenomenon occurs when the Ca²⁺ concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca²⁺ sensor that communicates the Ca²⁺ load of the intracellular stores to Orai1, have been shown to regulate fast Ca²⁺-dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca²⁺-dependent inactivation in this channel are not well understood. We have identified that a pore mutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca²⁺-binding site that regulates fast Ca²⁺-dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca²⁺-dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC. Unlike WT ICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na⁺ permeation through the E106D Orai1 pore by Ca²⁺ is diminished. These results suggest that Glu¹⁰⁶ inside the CRAC channel pore is involved in co-ordinating the Ca²⁺-binding site that mediates fast Ca²⁺-dependent inactivation.

Lipid accumulation in hepatocytes can lead to non-alcoholic fatty liver disease (NAFLD), which can progress to non-alcoholic steatohepatitis (NASH) and Type 2 diabetes (T2D). Hormone-initiated release of Ca²⁺ from the endoplasmic reticulum (ER) stores and subsequent replenishment of these stores by Ca²⁺ entry through SOCs (store-operated Ca²⁺ channels; SOCE) plays a critical role in the regulation of liver metabolism. ER Ca²⁺ homoeostasis is known to be altered in steatotic hepatocytes. Whether store-operated Ca²⁺ entry is altered in steatotic hepatocytes and the mechanisms involved were investigated. Lipid accumulation in vitro was induced in cultured liver cells by amiodarone or palmitate and in vivo in hepatocytes isolated from obese Zucker rats. Rates of Ca²⁺ entry and release were substantially reduced in lipid-loaded cells. Inhibition of Ca²⁺ entry was associated with reduced hormone-initiated intracellular Ca²⁺ signalling and enhanced lipid accumulation. Impaired Ca²⁺ entry was not associated with altered expression of stromal interaction molecule 1 (STIM1) or Orai1. Inhibition of protein kinase C (PKC) reversed the impairment of Ca²⁺ entry in lipid-loaded cells. It is concluded that steatosis leads to a substantial inhibition of SOCE through a PKC-dependent mechanism. This enhances lipid accumulation by positive feedback and may contribute to the development of NASH and insulin resistance.