Functional Interaction of an Orai1 Pore Residue with the Inactivation Domain of STIM1

Abstract

The Inactivation Domain of STIM1 (IDSTIM: aa 470-491) was described as necessary for Ca2+-dependent inactivation (CDI) of CRAC channels in three early reports (Mullins et al. 2009, Derler et al. 2009, Lee et al. 2009). We revisited the role of IDSTIM in light of the observation that STIM1:Orai1 ratio is a critical determinant of CDI (Scrimgeour et al. 2009, Hoover et al. 2011).We measured CRAC currents activated by a truncated STIM1 (aa 1-469), which lacks IDSTIM, in HEK cells transfected at higher STIM1:Orai1 ratios than were used previously. We observed limited but significant inactivation for STIM1(1-469) + Orai1 under these conditions (∼1/3 of the extent seen with WT STIM1 + Orai1). The inactivation supported by STIM1(1-469) was sensitive to extracellular [Ca2+] and to intracellular BAPTA but not EGTA.How does IDSTIM triple the extent of CDI? We first considered the possibility that IDSTIM increases local [Ca2+]. Open probability and single-channel conductance determined by analysis of monovalent current noise were identical for STIM1(1-469) and WT STIM1, arguing against a model in which IDSTIM enhances inactivation indirectly by increasing local [Ca2+] near the inner pore mouth.We next looked for a functional interaction between IDSTIM and the Orai1 pore. Like STIM1(1-469), the Orai1 inner pore mutation W76A also supported inactivation to ∼1/3 of the WT extent when studied at a high STIM1:Orai1 ratio. While the mutations STIM1(1-469) and W76A reduced CDI to similar extents when studied individually, no additional reduction of inactivation was seen when STIM1(1-469) was co-expressed with Orai1 W76A. Together, these data suggest that IDSTIM and Orai1 residue W76 act in concert to boost CDI to a maximal level for a given local [Ca2+].