Abstract
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.
Highlights
Ca2+ release activated Ca2+ (CRAC) channels composed of two cellular proteins, Ca2+-sensing stromal interaction molecule 1 (STIM1) and pore-forming Orai[1], are the main mediators of the Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores
We investigate the intracellular pH dependence of ICRAC mediated by Orai[1] and STIM1ectopically expressed in HEK293 cells
It has been shown that the current amplitude, fast Ca2+ dependent inactivation (FCDI), re-activation, potentiation by 2-APB, and selectivity of CRAC channels for divalent cations strongly depend on the relative amounts of Orai[1] and STIM1 proteins in the cell[15, 16]
Summary
Ca2+ release activated Ca2+ (CRAC) channels composed of two cellular proteins, Ca2+-sensing stromal interaction molecule 1 (STIM1) and pore-forming Orai[1], are the main mediators of the Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. It has been shown that the amplitude of CRAC current (ICRAC) strongly depends on extracellular and intracellular pH. Under normal physiological conditions extracellular pH (pHo) in healthy tissues is maintained within a narrow range between 7.3 and 7.4, while intracellular pH (pHi) is kept between 7.1–7.21. Activity of almost ubiquitously expressed Ca2+ release activated Ca2+ (CRAC) channels, formed by Orai[1] and STIM1 proteins, has been shown to strongly depend on both extracellular and intracellular pH8–10. Extracellular acidification inhibits, whereas alkalinisation increases CRAC current (ICRAC) amplitude with pKa of about 8. This has been consistently shown in several publications using both, heterologous expression of Orai[1] and STIM1 and cells expressing endogenous ICRAC8–11. Intracellular acidification has been shown to functionally uncouple STIM1 and Orai[1] without causing a complete dissociation of STIM1/Orai[1] complex, suggesting www.nature.com/scientificreports/
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