Trafficking of through mitochondria. The inner mitochondrial membrane generates the electrochemical potential for accumulation via a low-affinity channel (MiCa). A channel (voltage-dependent anion channel, VDAC) may, at least in part, control the passage of from the cytosol across the outer mitochondrial membrane to MiCa. gradients around open channels within either the PM or ER (e.g., ) allow near closely apposed mitochondria to reach levels sufficient to allow mitochondrial uptake. A exchanger allows efflux from the mitochondria to the cytosol, from where it may again be accumulated into the ER by SERCA. This interplay allows mitochondria to modulate the feedback effects of on channels, it allows to modulate mitochondrial behavior, and it provides a route for silently moving into the ER from the extracellular space.
Key structural features of . Two views of the three-dimensional structure of the are shown viewed from the side (left, with the transmembrane region uppermost) or from the cytosol. The scale bar applies to both structures. Modified from Ref. 96 . The sequence of a single subunit is shown highlighting the SD, IBC, six TMDs, and the pore lying between TMDs 5 and 6. Structures of the SD [PDB code 1XZZ (Ref. 30 )] and IBC [PDB code 1N4K (Ref. 29 )] are also shown.
Targeting of to the ER and their retention within ER are mediated by TMD. [(A) and (B)] COS-7 cells were transiently transfected with the indicated constructs derived from and tagged with enhanced yellow fluorescent protein (EYFP) (shown in the left panels). The central panels show the distribution of calreticulin, an ER luminal protein identified by antibody staining and visualized with Alexa633. The right panels show the overlaid images with EYFP shown in yellow and calreticulin in red. (A) demonstrates that whereas the N terminal of (NT) is cytosolic, TMD1-2 allow both ER targeting and retention. (B) shows that each of the indicated pairs of TMD retain ICAM-1 (a PM protein) in the ER. Scale . (B) reproduced from Ref. 52 . Copyright © 2004. Reprinted with permission from the American Society for Biochemistry and Molecular Biology.
Expression of functional in the PM. (A) Intracellular signals evoked by anti-IgM (to activate the BCR, added at the arrow head) are shown for populations of DT40 cells stimulated in the absence of extracellular or in the presence of extracellular alone or with 300 nM to fully inhibit store-operated entry. The results demonstrate that the sustained entry evoked by the BCR is only partially mediated by store-operated entry. (B) Whole-cell patch-clamp recording from a DT40 cell at −100 mV, with as the charge-carrier, and with present (b) or absent (a) from the pipette solution; C denotes the closed state. (C) Current-voltage relationships for -activated currents in whole-cell recordings (PM) or excised nuclear patches (nucleus) from cells expressing mutant . R1 denotes wild-type rat , has the native Glu-2547 changed to Ala, and has the native Val-2548 changed to Ile. The important point is that although the single channel conductances (from the slopes of the curves) differ for in the PM and nuclear envelope, the effects of the mutations are similar in each membrane. This provides compelling evidence that the -evoked currents across the PM are directly mediated by . Panels (A)–(C) are reproduced from Ref. 68 . Copyright © 2006. Reprinted with permission from AAAS.
-evoked clustering of . (A) Typical recordings from patches excised from the nuclear envelope of DT40 cells expressing . The patch pipette contained and 200 nM free . The patches shown include one (top), two, or three (bottom) . The closed (C) and open (O1–O3) states are shown. (B) Typical single -mediated current recorded with and free in the pipette solution. The lower trace shows a patch with two on an enlarged timescale. Red arrows highlight the near-synchronous opening and closing of the two channels. (C) At resting , low concentrations of rapidly drive into cluster of approximately four , wherein each is independently gated but to only 50% of the observed for a lone . Within a cluster, an is more likely to experience the increase in arising from an active neighbor, but it is also primed to respond most to it because increased reverses the inhibition caused by clustering and the gating of becomes coupled.
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