Programme Contents

Regulation of Ca²⁺ release activated Ca²⁺ channels by intracellular pH

Depletion of intracellular Ca²⁺ stores in mammalian cells results in activation of Ca²⁺ entry pathway mediated primarily by Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Functional CRAC channel is composed of a hexamer of a protein called Orai1, which forms the channel pore, and a protein called STIM1, a Ca²⁺ binding protein that plays the role of Ca²⁺ sensor in the endoplasmic reticulum (Parekh & Putney, 2005). Previously it has been shown that activity of CRAC channels formed by Orai1 and STIM1 proteins strongly depend on both extracellular and intracellular pH (Beck et al., 2014). Structure-function studies have identified glutamate 106 in the Orai1 pore and aspartates 110 and 112 in the first extracellular loop as the main sites mediating I_CRAC dependence on extracellular pH (Scrimgeour et al., 2012; Beck et al., 2014). Recently histidine 155 in the intracellular loop of Orai1 has been suggested as CRAC channel intracellular pH sensor. However, H155F mutation of Orai1 was only shown to abolish I_CRAC potentiation by alkaline pH_i, whereas inhibition of I_CRAC mediated by this mutant by acidic pH_i remained largely unchanged (Tsujikawa et al., 2015), suggesting that some other mechanisms may be involved.

In this study we investigated dependence of I_CRAC on pH_i using HEK293T cells heterologously expressing different ratios of Orai1 and STIM1 proteins and whole-cell patch clamping. The results showed that intracellular acidification to pH 6.3 inhibited I_CRAC by ∼70%, compared to pH_i 7.3. At the same time low pH_i affected CRAC channel gating, promoting I_CRAC activation at negative potentials. Similar increase in I_CRAC activation could be achieved by increasing relative expression levels of Orai1 at neutral pH_i, which suggested that intracellular acidification affects Orai1/STIM1 interactions. Intracellular alkalinisation strongly potentiated I_CRAC amplitude, but only when EGTA was used as intracellular Ca²⁺ buffer. Replacing EGTA with BAPTA virtually abolished I_CRAC potentiation by high pH_i, suggesting that pH dependence of Ca²⁺ binding properties of intracellular Ca²⁺ buffers may contribute to CRAC channel dependence on pH_i. To elucidate the mechanism of CRAC channel gating dependence on pH_i we neutralised several negatively charged amino acids in STIM1 475DDVDDMDEE483 domain previously shown to modulate fast Ca²⁺ dependent inactivation (FCDI) of I_CRAC. The amplitude of I_CRAC mediated by Orai1 and either of two STIM1 double mutants, DD475/6AA or EE482/3AA, showed similar to WT CRAC channel dependence on pH_i. However, Ca²⁺ dependent gating of these mutant channels showed a significantly weaker dependence on pH_i and the relative expression levels of STIM1 and Orai1, compared to WT. These results suggest that the effects of pH_i on I_CRAC amplitude and gating are mediated through different mechanisms. The pH_i dependence of I_CRAC amplitude is likely to be mediated by protonatable residues in the Orai1 pore, whereas pH_i dependence of CRAC gating is likely to be mediated by the protonatable residues responsible for Orai1/STIM1 interactions. Considering almost ubiquitous expression of CRAC channels in mammalian cells, their regulation by pH may play an important role in pathophysiology of acidosis- and alkalosis-related conditions.

Beck A, Fleig A, Penner R, Peinelt C. (2014). Regulation of endogenous and heterologous Ca²⁺ release-activated Ca²⁺ currents by pH. Cell Calcium 56, 235-243

Parekh AB, Putney JW, Jr. (2005). Store-operated calcium channels. Physiol Rev 85, 757-810.

Scrimgeour NR, Wilson DP, Rychkov GY. (2012). Glutamate 106 in the Orai1 pore contributes to fast Ca²⁺-dependent inactivation and pH dependence of Ca²⁺ release-activated Ca²⁺ (CRAC) current. Biochem J 441, 743-753.

Tsujikawa H, Yu AS, Xie J, Yue Z, Yang W, He Y, Yue L. (2015). Identification of key amino acid residues responsible for internal and external pH sensitivity of Orai1/STIM1 channels. Sci Rep 5, 16747. DOI: 10.1038/srep16747