Increased hydrophobicity at the N-terminus/membrane interface impairs gating of the SCID-related ORAI1 mutant

Patients with severe combined immune deficiency (SCID)
suffer from defective T-cell Ca2 signaling. A loss of Ca2 entry
has been linked at the molecular level to single missense mutation
R91W in the store-operated Ca2 channel ORAI1. However,
the mechanistic impact of this mutation on ORAI1 function
remains unclear. Confocal Fo¨rster resonance energy
transfer microscopy revealed that dynamic store-operated coupling
of STIM1 to ORAI1 R91W was largely sustained similar to
wild-type ORAI1. Characterization of various point mutants at
position 91 by whole cell patch clamp recordings displayed that
neutral or even negatively charged amino acids did not abolish
ORAI1 function. However, substitution by hydrophobic leucine,
valine, or phenylalanine resulted in non-functional ORAI1 channels,
despite preserved STIM1 coupling. Besides conformational
constraints at the N terminus/membrane interface predicted for
the hydrophobic mutants, additional key factor(s) were suggested
to determine ORAI1 functionality. Calculation of the probability
for the 1sttransmembranedomainandits hydrophobicity revealed
a substantial increase for all hydrophobic substitutions that lead to
non-functional ORAI1 R91X mutants in contrast to those with
hydrophilic residues. Hence, increased hydrophobicity might lead
to disrupted permeation/gating, as an ORAI1 channel with
increased pore size and R91W mutation failed to recover activity.
In conclusion, the increase in hydrophobicity at the N terminus/
membrane interface represents the major cause for yielding nonfunctional
ORAI1 channels.