The inhibition of digitoxin on Store Operated Calcium Entry is dependent on the phosphorylation sites and pore region of Orai1.

BACKGROUND: Mostly mediated by STIM1 and Orai1, Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway that has been linked with many types of diseases like myopathy, epilepsy, immune deficient diseases, and cancer. Currently many details about SOCE process have been revealed. However, the dissection of the molecular mechanisms underlying SOCE activation and the development possible treatments of SOCE related diseases are hindered, partly due to the lack of more specific pharmacological tools and poor understandings of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin.

OBJECTIVE AND METHODS: Using a combination of Ca2+ and förster resonance energy transfer (FRET) imaging, we aimed to systemically delineate the sites or domains needed for the inhibition of digitoxin on SOCE.

RESULTS: We revealed that the inhibitory effects of digitoxin are exerted on Orai1. If digitoxin were applied to resting Orai1, then its inhibition was dependent on S27-S30 residues of Orai1. While 8h-incubation of digitoxin with STIM1-prebound Orai1 or its constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27/S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1.

CONCLUSIONS: Our study identified two sites critical for the inhibition on Orai1 channels, providing valuable targets for future design of SOCE inhibitors.