Background: Store-operated calcium entry (SOCE), primarily mediated by
Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that
has been linked to human diseases including myopathy, epilepsy, immunodeficiency,
and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms
underlying SOCE activation, the development of therapies against dysfunctional SOCE
significantly lags behind, partly due to the lack of more specific pharmacological tools
and poor understanding of currently available SOCE modifiers, including the a newly
identified SOCE inhibitor, digitoxin.
Objective and methods: Capitalizing on Ca2+ imaging and pharmacological tools, we
aimed to systemically delineate the mechanism of action of digitoxin by defining how it
impinges on Orai1 to exert its suppressive effect on SOCE.
Results: The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues
of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a
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
Conclusions: Our study identified two regions that are critical for the inhibition on Orai1
channels, providing valuable hotspots for future design of SOCE inhibitors.