Insights into CRAC channel gating and ion permeation

Abstract

The ORAI1 store-operated calcium channel, also known as the CRAC channel, has provided many surprises. The latest is that channel gating and ion selectivity are closely intertwined.The calcium release-activated calcium (CRAC) channel, originally defined in T lymphocytes and mast cells, is an oligomer composed of ORAI1 subunits. Its hallmarks are exquisite selectivity for Ca 2+ in physiological solutions, a narrow pore as defined by permeation of small organic cations, and a very small single-channel current. The channel is recruited to ER-plasma membrane junctions and activated by STIM proteins in response to ER Ca 2+ store depletion. The emerging picture of the STIM-ORAI pathway is reviewed in [1,2]. There has been relatively slow progress, however, in delineating the physical basis of channel gating and ion permeation. A new paper by McNally et al. [3] reports three important advances: It locates an external gate in the ORAI1 channel, implicates the gate region in ion selectivity, and in an aspect not discussed here, shows that STIM-ORAI stoichiometry can influence the ion selectivity of the channel.Previous work has established that the ORAI channel is not intrinsically restricted to conducting Ca 2+ . 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). 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].The new paper by McNally et al.[3] first probes entry of the organic cation 2-aminoethyl methanethiosulfonate (MTSEA + ) into the E106D channel. The pairing of channel and reagent is a calculated choice. The E106D channel has a larger pore diameter than the wild-type ORAI1 channel, but is gated normally by STIM1. The MTSEA + headgroup diameter, 3.8 Å, is comparable to the diameter of methylammonium, which carries current through E106D channels. Methanethiosulfonate reagents are typically used to modify cysteine residues introduced at specific sites in a channel...

Differential Regulation of Calcium Signalling Pathways by Components of Piper methysticum ('Awa)

“…In the current study, we tested the hypothesis that kava extracts would include compounds active at members of the TRP calcium permeant non‐selective cation channel family. RBL2H3 are used here as a convenient model system because they co‐express a range of calcium entry mechanisms including store operated calcium entry via I CRAC , and various TRPs (Stokes et al ., ; Stokes et al ., ; Turner et al ., ; Berna‐Erro et al ., ; Hogan, ; Srikanth and Gwack, ; Ru and Yao, ). Kava extracts from the Hawaiian Mahakea strain were applied to RBL2H3 in a single cell, whole cell patch clamp configuration.…”