Stop refilling (Ca²⁺ stores)

Credit: CORBIS

Store-operated Ca²⁺ entry (SOCE) is the cellular response to loss of Ca²⁺ from the endoplasmic reticulum (ER), which allows ER Ca²⁺refilling following Ca²⁺ influx through the plasma membrane. SOCE is regulated to determine the magnitude and duration of Ca²⁺ influx. The mechanisms that inactivate SOCE are not entirely understood, and Palty et al. now identify SARAF (SOCE-associated regulatory factor) as a factor that promotes slow inactivation of SOCE and prevents excess Ca²⁺ refill.

Intracellular Ca²⁺ functions as a signal that regulates a wide range of cellular functions. Following stimulation, cells can rapidly release Ca²⁺ from the ER, the main intracellular Ca²⁺ storage site, and this is followed by a slower Ca²⁺ entry from outside the cell. Ca²⁺ entry through the plasma membrane is coupled to Ca²⁺ release from the ER by Ca²⁺ release-activated Ca²⁺ (CRAC) channels. STIM and ORAI proteins are essential components of CRAC channels, with ORAI forming a channel at the plasma membrane, and STIM residing at the ER membrane. STIM proteins detect changes in ER Ca²⁺ levels and, following Ca²⁺ depletion in the ER, they oligomerize and relocalize at ER–plasma membrane junctions, where they bind to ORAI and cause the opening of the plasma membrane channels, thus promoting SOCE.

Palty et al. performed a functional expression screen by transfecting a cDNA library into human embryonic kidney (HEK) cells, and identified a clone that alone could lower basal mitochondrial Ca²⁺ levels and greatly decrease ER and cytosolic Ca²⁺ levels. This clone was found to code for SARAF. Interestingly, fluorescently labelled SARAF colocalized with ER markers, indicating that the ER is the primary subcellular localization of SARAF.

SARAF function seems to prevent cell overloading with excessive Ca²⁺

To test whether SARAF regulates SOCE, the authors heterologously expressed STIM1 and ORAI1 in HEK cells and measured Ca²⁺ influx through CRAC channels in response to Ca²⁺ depletion from the ER in the presence or absence of SARAF. Although Ca²⁺ influx was detected in both cases, SOCE was more rapidly inactivated in the presence of SARAF. Importantly, the addition of Ca²⁺ chelators showed that CRAC channel inactivation required increased free cytosolic Ca²⁺levels, whereas the use of inhibitors of ER Ca²⁺ refilling indicated that store refilling contributes, in part, to SOCE inactivation. Thus, SARAF regulates Ca²⁺ entry through CRAC channels, and this depends on both cytosolic Ca²⁺levels and ER Ca²⁺ levels.

So how does SARAF regulate SOCE? Following ER Ca²⁺ depletion, SARAF translocated, in a STIM1-dependent manner, to the sites of STIM1–ORAI1 aggregation at the specialized ER–plasma membrane junctions, which suggests that SARAF directly inhibits SOCE. Human SARAF is predicted to have a single membrane-spanning domain, a cytosol-facing domain and an ER lumen-facing domain. Truncation experiments showed that the cytosolic domain is responsible for SOCE inactivation, whereas the luminal domain has an autoregulatory function, as its deletion led to strong inhibition of SOCE regardless of ER Ca²⁺ levels.

Using a FRET (fluorescence resonance energy transfer)-based approach, Palty et al. found that SARAF and STIM1 interacted during resting conditions, and that SOCE activation triggered molecular rearrangements between SARAF and STIM1. These rearrangements were ORAI-dependent and reduced the numbers of active STIM1 oligomers that underlie SOCE activation following Ca²⁺ store refilling. Moreover, when Ca²⁺ ER stores were refilled, the rearranged interaction between SARAF and STIM1 was reversed. Together, these results suggest that the association of SARAF with STIM1 facilitates the disaggregation of STIM1 oligomers to efficiently turn off ORAI1 when the ER lumen has been filled with Ca²⁺. However, further studies are required to elucidate the molecular mechanisms underlying the capacity of SARAF to sense Ca²⁺ levels and to directly inactivate SOCE.

SARAF function seems to prevent cell overloading with excessive Ca²⁺, and this may be therapeutically relevant for conditions that are accompanied by abnormal responses to intracellular Ca²⁺ levels such as Alzheimer's disease and prostate cancer.