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

D. Gavriliouk,1 N.R. Scrimgeour,1 S. Grigoryev,2 L. Ma,1,3 F.H. Zhou,2 G.J. Barritt4 and G.Y. Rychkov,1,2 1School of Medical Sciences, University of Adelaide, Adelaide, SA 5005, Australia, 2School of Medicine, University of Adelaide, and South Australian Health and Medical Research Institute, Adelaide, SA 5005, Australia, 3Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia and 4School of Medicine, Flinders University of South Australia, Bedford Park, SA 5065, Australia.

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 ICRAC 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 ICRAC potentiation by alkaline pHi, whereas inhibition of ICRAC mediated by this mutant by acidic pHi remained largely unchanged (Tsujikawa et al., 2015), suggesting that some other mechanisms may be involved.

In this study we investigated dependence of ICRAC on pHi 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 ICRAC by ∼70%, compared to pHi 7.3. At the same time low pHi affected CRAC channel gating, promoting ICRAC activation at negative potentials. Similar increase in ICRAC activation could be achieved by increasing relative expression levels of Orai1 at neutral pHi, which suggested that intracellular acidification affects Orai1/STIM1 interactions. Intracellular alkalinisation strongly potentiated ICRAC amplitude, but only when EGTA was used as intracellular Ca2+ buffer. Replacing EGTA with BAPTA virtually abolished ICRAC potentiation by high pHi, suggesting that pH dependence of Ca2+ binding properties of intracellular Ca2+ buffers may contribute to CRAC channel dependence on pHi. To elucidate the mechanism of CRAC channel gating dependence on pHi we neutralised several negatively charged amino acids in STIM1 475DDVDDMDEE483 domain previously shown to modulate fast Ca2+ dependent inactivation (FCDI) of ICRAC. The amplitude of ICRAC mediated by Orai1 and either of two STIM1 double mutants, DD475/6AA or EE482/3AA, showed similar to WT CRAC channel dependence on pHi. However, Ca2+ dependent gating of these mutant channels showed a significantly weaker dependence on pHi and the relative expression levels of STIM1 and Orai1, compared to WT. These results suggest that the effects of pHi on ICRAC amplitude and gating are mediated through different mechanisms. The pHi dependence of ICRAC amplitude is likely to be mediated by protonatable residues in the Orai1 pore, whereas pHi 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