Ca2+ plays an essential role as a second messenger regulating many cellular functions including gene regulation and enzyme activity. Thus intracellular Ca2+ concentration is tightly regulated. Store operated Ca2+ channel (SOC) is a plasma membrane channel, which is activated upon depletion of endoplasmic reticulum (ER) Ca2+. This channel is compromised of two proteins STIM1, that is localized to the ER membrane and senses luminal Ca2+ concentration, and Orai1, that forms the channel at the plasma membrane. To date little is known about the synthesis and maturation of Orai1 protein. Calreticulin and calnexin are two lectin-like chaperones in the ER, which are involved in the synthesis and maturation of many membrane and secretory proteins. In addition to its chaperone function, calreticulin binds Ca2+ with high capacity and low affinity and regulates intracellular Ca2+ homeostasis. Previously, we showed that loss of calreticulin function activates ER stress pathways leading to the activation of the ubiquitin-proteasome pathway.

To determine the role of ER chaperones in the biogenesis of Orai1.

Wild type, calreticulin deficient (crt-/-), calnexin deficient (cnx-/-) and CHO cells were used for measurement of Store Operated Ca Channel activity. Cells were transfected with EGFP-HA-Orai1 cDNA to examine the localization of Orai1 using immunocytochemistry and live cell imaging using confocal microscopy.

Our data illustrates that loss of calreticulin function, but not calnexin, results in a significant decrease in the SOC activity. There was no significant change in the total Orai1 and STIM1 protein level in calreticulin deficient cells as was determined by Western blot analysis. Interestingly, loss of calreticulin function resulted in the accumulation of EGFP-HA-Orai1 protein intracellularly, which explains the decrease in SOC activity. We further demonstrate that inhibition of the ubiquitin-proteasome pathway enhances SOC activity in calreticulin deficient cells and show for the first time that Orai1 is ubiquitinated in different cell types.

Calreticulin, but not calnexin, function as the chaperone regulating proper maturation of Orai1 protein.


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