Plasticity in Ca2+ selectivity of Orai1/Orai3 heteromeric channel

Abstract

A general cellular response following depletion of intracellular calcium stores involves activation of store-operated channels (SOCs). While Orai1 forms the native Ca(2+) release-activated Ca(2+) (CRAC) channel in mast and T cells, the molecular architecture of less Ca(2+) selective SOCs is insufficiently defined. Here we present evidence that diminished Ca(2+) selectivity and robust Cs(+) permeation together with a reduced fast inactivation are characteristics of heteromeric Orai1 and Orai3 channels in contrast to their homomeric forms. The first extracellular loop of these Orai isoforms differs by two aspartates replacing glutamates that affect the selectivity. Co-expression of an Orai3 mutant that mimicked the first loop of Orai1 with either Orai1 or Orai3 recovered or decreased Ca(2+) selectivity, respectively. Heteromeric Orai1/3 protein assembly provides a concept for less Ca(2+)-selective SOCs.

Fig. 1.

Orai1 and Orai3 co-expression exhibits reduced Ca²⁺ selectivity. (A) Fluorescence images from a representative cell co-expressing CFP-Orai1 and YFP-Orai3 or (B) CFP-/YFP-Orai3, overlay and calculated FRET. (C) Average FRET values determined for the constructs shown in (A and B) and CFP-/YFP-Orai1. (D) Time course of whole-cell currents at −86 mV and +62 mV of HEK cells overexpressing Stim1, Orai1, and Orai3 or (F) Stim1 and Orai1 are monitored upon passive store-depletion by 10 mM EGTA in a 10 mM Ca²⁺ bath solution followed by perfusion of a Cs⁺ based divalent free solution (Cs⁺-DVF) after 200 s. (E) Corresponding representative current voltage relationships were taken from voltage ramps in both a Ca²⁺ and Cs⁺ containing bath solution for Stim1, Orai1, and Orai3 expressing cells or (G) Stim1 and Orai1 co-expression. (H) IV characteristic for Na⁺-DVF solution for Stim1 and Orai1 or (I) Stim1, Orai1, and Orai3 overexpression.

Fig. 2.

Permeation properties of Orai3-Orai1 tandem. (A) Localization, overlay and FRET of CFP-/YFP-Orai3-Orai1. (B) Time course of Stim1 and Orai3-Orai1 co-expression depicting activation of Ca²⁺ currents upon passive store-depletion, followed by perfusion of a Cs⁺-DVF solution after 200 s. Corresponding representative current voltage relationships were taken from voltage ramps in a (C) 10 mM Ca²⁺, (D) Cs⁺-DVF, or (F) Na⁺–DVF containing bath solution for Stim1 and Orai3-Orai1 expressing cells. (E) Fully activated Ca²⁺ currents of Stim1 and Orai3-Orai1 co-expression are followed by Na⁺-DVF solution. (G) Ca²⁺ current of Stim1 co-expressed with either Orai1, Orai3 or Orai3-Orai1 were activation by store-depletion and Na⁺ and Cs⁺ was substituted by TEA⁺ in the bath solution after 200 s. (H) Time course of currents and (I) current-voltage relationship of a co-expression of STIM1 with either Orai1, Orai3, or Orai3-Orai1 activated by a 10 mM EGTA and K⁺-based pipette solution.

Fig. 3.

Unique Ca²⁺ feedback regulation and 2-APB response of Orai3-Orai1 tandem. (A–C) Voltage steps from a holding potential of 0mV to −100, −80, −60, and −40 mV are depicted for a co-expression of Stim1 with either (A) Orai1, (B) Orai3, or (C) Orai3-Orai1 upon full store-depletion. (D and E) Normalized voltage step to −80 mV are shown for a co-expression of Stim1 with Orai1, Orai3, or Orai3-Orai1 over (D) 1,800 ms or (E) 100 ms. (F) Time course of fully activated Ca²⁺ currents of Stim1 and Orai3-Orai1 co-expression or solely expressing Orai3-Orai1 cells (in red) followed by addition of 75 μM 2-APB at 200 s. (G) IV characteristic for Stim1 and Orai3-Orai1 co-expression at depicted time-points (1) for maximum store-operated Ca²⁺ currents, (2) 5 s, and (3) 80 s after addition of 2-APB (75 μM). (H) Time course of of fully activated Ca²⁺ currents of Stim1, Orai1, and Orai3 expression following addition of 75 μM 2-APB at 200 s.

Fig. 4.

Asymmetry of aspartates and glutamates in Orai1 and Orai3 first loop determines Ca²⁺ selectivity and Cs⁺ permeability. Upper panel, amino acid sequence of the first and third transmembrane domain and the first loop of both Orai1 and Orai3 including glutamates (red) and aspartates (green) that affect permeability. Time course of HEK cells co-expressing (A) Stim1 and Orai3-E85D-E89D, (C) Stim1, Orai1 and Orai3-E85D-E89D, or (E) Stim1, Orai3, and Orai3-E85D-E89D showing activation of Ca²⁺ currents upon passive store-depletion, followed by perfusion of a Cs⁺-DVF solution after 200 s. (B, D, and F) Corresponding representative current-voltage relationships were taken from voltage ramps for a 10 mM Ca²⁺ containing bath solutions as well as a Cs⁺-DVF solution for (A, C, and E). (G) Representative current-voltage relationships in a Na⁺-DVF solution for Stim1 and Orai3-E85D-E89D, as well as a co-expression with Orai1 or Orai3. (H) Summary of reversal potentials in a Ca²⁺ and Na⁺-DVF solution, decrease (↓) or increase (↑) of inward currents when switching from Ca²⁺ to Cs⁺-DVF solution, and calculated permeability ratios P_Cs/Na for Stim1 and Orai3-E85D-E89D as well as a co-expressed with Orai1 or Orai3.