Molecular biophysics of Orai store-operated Ca2+ channels

Abstract

Upon endoplasmic reticulum Ca(2+) store depletion, Orai channels in the plasma membrane are activated directly by endoplasmic reticulum-resident STIM proteins to generate the Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. After the molecular identification of Orai, a plethora of functional and biochemical studies sought to compare Orai homologs, determine their stoichiometry, identify structural domains responsible for the biophysical fingerprint of the CRAC current, identify the physiological functions, and investigate Orai homologs as potential therapeutic targets. Subsequently, the solved crystal structure of Drosophila Orai (dOrai) substantiated many findings from structure-function studies, but also revealed an unexpected hexameric structure. In this review, we explore Orai channels as elucidated by functional and biochemical studies, analyze the dOrai crystal structure and its implications for Orai channel function, and present newly available information from molecular dynamics simulations that shed light on Orai channel gating and permeation.

Figure 1

Orai1 structure-function mapping. (A) Annotated sequence of Orai1. (Circles) Residues; (bold) conservation in the three human Orai channels. Color-coded channel functions defined by mutational analysis (discussed in text) are highlighted from N- to C-terminus: N-terminal STIM1 and CaM binding; Ca²⁺-dependent inactivation (CDI); mutation that causes human SCID; constitutively active channel mutants; Ca²⁺ permeation; cation electrostatic attraction; second CDI site; TM3 residues that contribute to permeation and gating; and C-terminal STIM1 binding. (B) TM1 residues lining the Orai1 store-operated pore elucidated by functional analysis: selectivity filter E106, hydrophobic gate V102, gating hinge G98, L95, and basic gate R91. For clarity, only two TM1 domains, from two Orai1 monomers, are represented.

Figure 2

The dOrai crystal structure. (A) Side view showing hexameric assembly composed three α-subunits (green) with TM4 extended and three β-subunits (purple) with TM4 bent. (B) Pore view showing nine residues that line the conduction pathway, 55 Å in length. For clarity, only two of the six subunits are represented. (Red) Selectivity filter residue E178; (yellow) bound Ca²⁺; (gold) five hydrophobic residues; and (blue) basic residues, which bind two unidentified anions (orange). (C) View from the extracellular side showing concentric circles of α-helices with two rings of glutamates (red): the inner ring of TM1 E178 residues (corresponding to Orai1 E106) surrounding a Ca²⁺ ion at the center of the pore, and the outer ring formed by TM3 E262 residues (corresponding to Orai1 E190). (D) View from the intracellular side showing TM1 residues with positive charge (blue) at the center surrounding the anion (orange) in the pore, flanked by M4 extensions with three red stripes of negative charge at the C-terminal STIM-binding sites.

Figure 3

MD simulation of wild-type dOrai. (A) Configuration snapshot showing dOrai in secondary structure representation colored by subunit as in Fig. 2, with lipid tails (silver), lipid headgroups (yellow), and water (blue) shown in solid-sphere representation. (B) Extracellular view of dOrai with the water and lipids removed for clarity. In addition to the glutamate rings in TM1 (E178) and TM3 (E262) observed in the crystal structure, the full sequence shows another set of concentric acidic side chains formed by D182 and D184 in the TM1-TM2 connecting loop. (C) Cα RMSD from the initial configuration. Most simulation systems reached a steady state after ∼120 ns; WT+Gd³⁺ required only ∼50 ns. (D) Pore radius profiles of WT dOrai and the V174A mutant with Ca²⁺ or Gd³⁺ bound in the selectivity filter. The coordinate Z is defined as the distance (along the TM direction) from the center-of-mass for TM1 residues 144–180. The local minimum at Z ∼25 Å corresponds to the position of the E178 carboxyl groups, and the maximum at Z ∼20 Å corresponds to the position of the residue 174 side chain.