Gated regulation of CRAC channel ion selectivity by STIM1

Abstract

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states, typically without changes in ion selectivity. In store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, stromal interaction molecule 1 (STIM1), remains unknown. CRAC channels are distinguished by a very high Ca(2+) selectivity and are instrumental in generating sustained intracellular calcium concentration elevations that are necessary for gene expression and effector function in many eukaryotic cells. Here we probe the central features of the STIM1 gating mechanism in the human CRAC channel protein, ORAI1, and identify V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produce constitutively active CRAC channels that are open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels are not Ca(2+)-selective, their Ca(2+) selectivity is dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca(2+) selectivity is also seen in wild-type ORAI1 channels by increasing the number of STIM1 activation domains that are directly tethered to ORAI1 channels, or by increasing the relative expression of full-length STIM1. Thus, exquisite Ca(2+) selectivity is not an intrinsic property of CRAC channels but rather a tuneable feature that is bestowed on otherwise non-selective ORAI1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism in which permeation and gating are closely coupled.

Figure 1. State-dependent accessibility of pore-lining residues localizes the activation gate to the extracellular TM1 region

a, Schematic representation of the key pore-lining residues in TM1 . b, MTSEA modification of G98C is protected by La³⁺. A HEK293 cell co-expressing G98C E106D Orai1 and STIM1 was exposed to two applications of MTSEA (100 μM), the first in the presence of La³⁺ (100 μM), and the second following washout of La³⁺. Periodic applications of a divalent free (DVF) solution facilitated washout of La³⁺. MTSEA inhibition was quantified by the relief of block induced by BMS (5 mM) (arrows). c, State-dependent modification of G98C. MTSEA (200 μM) was applied for 120 s to resting cells, then washed off. Following whole-cell break in, I_CRAC was activated by passive store depletion by dialyzing in BAPTA. BMS was applied to examine relief from MTSEA blockade (arrows). A second application of MTSEA and BMS provide a measure of blockade in open channels. A DVF solution was periodically applied to monitor Na⁺-I_CRAC. d, Summary of MTSEA blockade of open G98C E106D Orai1 in the presence and absence of La³⁺. e, Summary of blockade of G98C E106D and D110C Orai1 by MTSEA in closed and open channels. Values are mean ± s.e.m.

Figure 2. Mutations at V102 cause STIM1-independent constitutive Orai1 activation

a, Time course of the development of I_CRAC in cells expressing WT or V102C Orai1 and STIM1 following whole-cell break-in. Intracellular Ca²⁺ stores were depleted by dialyzing cells with 8 mM BAPTA. b, V102C Orai1 currents are constitutively active in the absence of STIM1 co-expression. c, [Ca²⁺]_i measurements in HEK293 cells expressing the indicated Orai1 constructs in the absence of STIM1. WT: wild-type. UT: untransfected. d, Localization of V102C Orai1-CFP before, and following ER Ca²⁺ store depletion in the absence (left) or presence (right) of STIM1-YFP. e, Mutational analysis of V102. Normalized current densities of V102 substitutions plotted against the solvation energies of the substituted amino acids in the presence or absence of STIM1 co-expression. Currents were normalized to the mutant yielding maximal current density for each condition (Ala for STIM1-free cells and Ile in STIM1-co-expressing cells). Green points in the top graph highlight residues yielding large constitutively active currents in the absence of STIM1. Red points in the lower graph highlight residues that are not constitutively active, but require STIM1 for activation.

Figure 3. STIM1 regulates ion selectivity of constitutively active V102C Orai1 channels

a, Current-voltage (I-V) relationships of V102C Orai1 currents in 20 mM Ca²⁺ and DVF Ringer's solutions. Arrows emphasize the reversal potential (V_rev) in each case. The bar graphs (right) summarize (mean±sem) V_rev of V102C Orai1 currents in the presence or absence of STIM1. b, Effects of substituting extracellular Na⁺ with NMDG⁺ on V102C Orai1 currents in the absence or presence of STIM1. c, effects of replacing the standard extracellular Ringer's solution with Na⁺- or Cs⁺-based DVF solutions. In the absence of STIM1, large Cs⁺ currents are seen in V102C Orai1 channels. By contrast, no Cs⁺ conduction is observed in the presence of STIM1. d, I-V relationship of currents in the V102C L276D Orai1 double mutant in the presence or absence of STIM1. The bar graphs summarize the V_rev values (mean±sem) of this mutant in the presence or absence of STIM1. e, Relative permeabilities of V102C Orai1 channels to different organic monovalent cations is plotted against the size of each cation in the presence or absence of STIM1. Dotted lines are fits to the hydrodynamic relationship. Values of d_pore estimated from the fits are 4.9 Å for V102C Orai1 + STIM1 channels and 6.9 Å for V102C Orai1 channels.

Figure 4. STIM1 dose-dependently modulates the ion selectivity of WT Orai1 channels

a, The addition of S domains to WT Orai1 produces a rightward shift in the V_rev of I_CRAC. I-V relationships of WT Orai1 channels tagged to either one or two S domains in the 20 mM Ca²⁺ and DVF Ringer's solutions are shown. The bar graphs summarize the V_rev (mean±SEM) in each solution. The top and bottom traces for each condition are from the same cell. b, Effects of substituting extracellular Na⁺ with an impermeant ion, NMDG⁺. Removal of Na⁺ diminishes the inward current in Orai1-S channels, but not Orai1-SS channels. c, I-V relationships and reversal potentials (mean±SEM) of V102C, V102C-S and V102C-SS channels. Increasing the number of S domains to the V102C monomer causes a progressive rightward shift in V_rev of I_CRAC. d, A plot of V102C Orai1 V_rev (in the 20 mM Ca²⁺ Ringer's solution) against current density of WT Orai1 channels tagged to zero, one, or two S domains.