Structural determinants of ion permeation in CRAC channels

Abstract

CRAC channels generate Ca 2؉ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca 2؉ selectivity, low Cs ؉ permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiolreactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca 2؉ selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd 2؉ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.Orai1 ͉ STIM1 ͉ store-operated channels C a 2ϩ release-activated Ca 2ϩ (CRAC) channels are the principal route of Ca 2ϩ entry in immune cells and orchestrate functions such as gene expression, motility, and the release of inflammatory mediators (1). Mutations in CRAC channels give rise to devastating immunodeficiencies and abnormalities in muscle, skin, and teeth, highlighting their importance for various organ systems (1). The recent discoveries of STIM1 (the ER Ca 2ϩ sensor), and Orai1 (the CRAC channel pore subunit) have provided major breakthroughs to illuminate the molecular basis of CRAC channel function (2). However, while the identification of these proteins has produced rapid progress in our understanding of the cellular events underlying channel activation, the molecular mechanisms of ion selectivity and permeation remain unclear.CRAC channels are distinguished by an extraordinarily high selectivity for Ca 2ϩ over monovalent ions (P Ca /P Na Ͼ 1,000), a very low unitary conductance (Ͻ1 pS), and low permeability to Cs ϩ and larger monovalent cations (3). The structural underpinnings of these characteristics have been the focus of much debate but are largely unknown. As with most ion channels, the pore properties of CRAC channels are likely shaped by the arrangement and chemistry of pore-lining residues. Thus, to understand the basis of the unique permeation properties of CRAC channels, the residues lining the ion transport pathway need to be elucidated.Orai1 bears little sequence homology to other ion channel proteins, and consequently, there are few clues regarding the contribution of the different parts of the molecule for pore formation. Electrophysiological studies indicate that the exquisite Ca 2ϩ selectivity of CRAC...

Orai1 pore residues control CRAC channel inactivation independently of calmodulin

“…Also, the inner pore structures of human Orai1 and dOrai could differ, although this seems unlikely given the high degree of sequence conservation between the two species (94% identity in the M1/pore region, corresponding to residues 76-107 of human Orai1). Further, cysteine scanning of side chain accessibility in the middle to upper pore region (R91-E106 of human Orai1; McNally et al, 2009;Zhou et al, 2010) gave results consistent with the dOrai crystal structure. The dOrai structure presumably reflects a closed channel state (Hou et al, 2012), and in theory the open or inactivated states of human Orai1 could have a more open inner pore than that seen in the dOrai structure, possibly allowing CaM binding.…”

Insights into CRAC channel gating and ion permeation

“…Their point of departure is the reversible block of CRAC current through R91C channels upon oxidative crosslinking of neighboring R91C residues. This recalls the partial block of the open R91C channel by Cd 2+ [6], and both findings suggest that pinning TM1 helices together at their cytoplasmic ends occludes the pore. That the channel can be occluded or pinned closed in these artificial cases does not imply that the gating movement of the wild-type channel involves widening the inner mouth of the pore.…”

“…Na + and other monovalent ions can permeate the channel when no divalent ions are present, but the channel is selective for Ca 2+ in physiological solutions, because binding of Ca 2+ at a site in the pore prevents Na + permeation. E106 in transmembrane helix 1 (TM1) of the human ORAI1 channel was implicated in Ca 2+ binding by electrophysiology [4,5], and the physical proximity of E106 sidechains from separate ORAI1 subunits was confirmed by disulfide crosslinking of E106C monomers [6,7]. An unusual feature of the ORAI1 channel is that its constituent TM1 helices are in proximity and line the pore along its entire length (Figure 1).…”

“…An unusual feature of the ORAI1 channel is that its constituent TM1 helices are in proximity and line the pore along its entire length (Figure 1). This was apparent from the block of currents by Cd 2+ traversing the channel and from the disulfide crosslinking of ORAI1 monomers, when cysteine residues were introduced at positions 88, 91, 95, 98, or 102 in TM1 [6,7]. Cd 2+ block at V102C is of particularly high affinity, indicating that the V102C sidechains are close together in the permeation pathway [6].…”

“…This was apparent from the block of currents by Cd 2+ traversing the channel and from the disulfide crosslinking of ORAI1 monomers, when cysteine residues were introduced at positions 88, 91, 95, 98, or 102 in TM1 [6,7]. Cd 2+ block at V102C is of particularly high affinity, indicating that the V102C sidechains are close together in the permeation pathway [6]. The protein crosslinking experiments further point to limited flexibility of the helical backbone from F99-M104 in comparison to other parts of TM1, placing V102 in a narrow and relatively rigid part of the pore that could serve as an energy barrier to ion passage [7].…”