A single serine in the CC3 region of STIM1 is critical for STIM1-Orai1
binding and CRAC channel activation
Background and purpose: Store-operated Ca2+ entry (SOCE) is important
for the function of many cell types. It is controlled by the interaction
between ER Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel Orai1.
CAD of STIM1 is required for SOCE. It contains two putative coiled-coil
regions (CC2 and CC3). The role of CC3 remains to be elucidated.
Experimental approach: various plasmids carrying different fluorescent
protein genes were constructed for better understanding the influence of
S417G mutation in CC3 on SOCE activation; Confocal imaging system,
calcium imaging technique and FRET technique were employed to examine
the actions of 2-APB on the interaction between STIM1 C terminus and
Orai1. Key results: Single-point mutation of the residue (S417G)
abolishes STIM1 C-terminus interactions with Orai1. Mutation of S417
also abolished CAD-Orai1 binding and Orai1 channel activation,
eliminated STIM1 puncta formation and co-localization with Orai1 and
SOCE. 2-APB were found to restore the binding of STIM1 C-terminus
mutant(S417G) to Orai1 and dose-dependently activated Orai1 channel.
Both CBD and NBD of Orai1 is required for 2-APB-induced coupling between
Orai1 and STIM1 C terminus mutant(S417G) and CRAC channel activation. We
also demonstrated 2-APB lead to delayed activation of Orai1-K85E
channel, although Orai1-K85E obviously impair 2-APB-induced STIM1
C-terminus mutant(S417G)-Orai1 coupling. Conclusions and implications:
Our data suggest that S417 in the CC3 domain of STIM1 is critical for
STIM1-Orai1 binding and CRAC channel activation. We also proposed
experimental models of combined STM1 or Orai1 mutants with 2-APB to
better understand the activation mechanism of CRAC.