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Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.

The plasma membrane protein Orai forms the pore of the calcium release—activated calcium (CRAC) channel and generates sustained cytosolic calcium signals when triggered by depletion of calcium from the endoplasmic reticulum. The crystal structure of Orai from Drosophila melanogaster, determined at 3.35 angstrom resolution, reveals that the calcium channel is composed of a hexameric assembly of Orai subunits arranged around a central ion pore. The pore traverses the membrane and extends into the cytosol. A ring of glutamate residues on its extracellular side forms the selectivity filter. A basic region near the intracellular side can bind anions that may stabilize the closed state. The architecture of the channel differs markedly from other ion channels and gives insight into the principles of selective calcium permeation and gating.

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

The mechanism of ion channel voltage gating—how channels open and close in response to voltage changes—has been debated since Hodgkin and Huxley's seminal discovery that the crux of nerve conduction is ion flow across cellular membranes. Using all-atom molecular dynamics simulations, we show how a voltage-gated potassium channel (KV) switches between activated and deactivated states. On deactivation, pore hydrophobic collapse rapidly halts ion flow. Subsequent voltage-sensing domain (VSD) relaxation, including inward, 15-angstrom S4-helix motion, completes the transition. On activation, outward S4 motion tightens the VSD-pore linker, perturbing linker—S6-helix packing. Fluctuations allow water, then potassium ions, to reenter the pore; linker-S6 repacking stabilizes the open pore. We propose a mechanistic model for the sodium/potassium/caldum voltage-gated ion channel superfamily that reconciles apparently conflicting experimental data.

Store-operated Ca²⁺ entry is mediated by Ca²⁺ release-activated Ca²⁺ (CRAC) channels following Ca²⁺ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca²⁺ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.

Calcium in the endoplasmic reticulum STIM1 is the messenger that signals endoplasmic reticulum Ca 2+ store depletion and subsequently activates the store-operated channel ORAI. However, its precise mechanism of action is unclear. Here, Luik et al . show that STIM oligomerzation, not the concentration of Ca 2+ in the endoplasmic reticulum, is the key event that triggers ORAI activation. Ca 2+ -release-activated Ca 2+ (CRAC) channels generate sustained Ca 2+ signals that are essential for a range of cell functions, including antigen-stimulated T lymphocyte activation and proliferation 1 , 2 . Recent studies 3 have revealed that the depletion of Ca 2+ from the endoplasmic reticulum (ER) triggers the oligomerization of stromal interaction molecule 1 (STIM1), the ER Ca 2+ sensor, and its redistribution to ER–plasma membrane (ER–PM) junctions 4 , 5 , 6 , 7 , 8 where the CRAC channel subunit ORAI1 accumulates in the plasma membrane and CRAC channels open 9 , 10 , 11 , 12 . However, how the loss of ER Ca 2+ sets into motion these coordinated molecular rearrangements remains unclear. Here we define the relationships among [Ca 2+ ] ER , STIM1 redistribution and CRAC channel activation and identify STIM1 oligomerization as the critical [Ca 2+ ] ER -dependent event that drives store-operated Ca 2+ entry. In human Jurkat leukaemic T cells expressing an ER-targeted Ca 2+ indicator, CRAC channel activation and STIM1 redistribution follow the same function of [Ca 2+ ] ER , reaching half-maximum at ∼200 µM with a Hill coefficient of ∼4. Because STIM1 binds only a single Ca 2+ ion 5 , the high apparent cooperativity suggests that STIM1 must first oligomerize to enable its accumulation at ER–PM junctions. To assess directly the causal role of STIM1 oligomerization in store-operated Ca 2+ entry, we replaced the luminal Ca 2+ -sensing domain of STIM1 with the 12-kDa FK506- and rapamycin-binding protein (FKBP12, also known as FKBP1A) or the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR, also known as FRAP1). A rapamycin analogue oligomerizes the fusion proteins and causes them to accumulate at ER–PM junctions and activate CRAC channels without depleting Ca 2+ from the ER. Thus, STIM1 oligomerization is the critical transduction event through which Ca 2+ store depletion controls store-operated Ca 2+ entry, acting as a switch that triggers the self-organization and activation of STIM1–ORAI1 clusters at ER–PM junctions.

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction. Large transmembrane proteins of the Piezo family assemble as tetramers to form a new class of ion channel that can be activated by mechanical force. Piezo ion channel feels the force Many tissues are able to detect and respond to mechanical forces, and this mechanical sensitivity has been implicated in many biological processes and diseases, including touch, pain, deafness and hypertension. The conversion of mechanical force into biological signals, or 'mechanotransduction', is thought to involve specialized cation channels. In a pair of papers, Ardem Patapoutian and colleagues establish that the large transmembrane proteins of the 'Piezo' family — conserved from animals to plants and protozoa — are among the long-sought-after mechanically activated ion channels. Coste et al . show that the Drosophila melanogaster Piezo protein induces mechanically activated cationic currents in human embryonic kidney cells, establishing functional conservation. Comparison of the mechanically activated currents induced by mouse and fly Piezos reveals ion-channel activities with unique pore properties, suggesting that Piezos are bona fide ion channels. Kim et al . show that D. melanogaster Piezo is essential for sensing mechanical pain in fruitflies, giving the first demonstration that Piezos are physiologically relevant mechanosensors in vivo .

Calcium pump Two groups report the molecular identification of the long-sought CRAC channel as a plasma membrane protein known variously as olf186-F, Orai and CRACM1. The CRAC channel is of fundamental importance to Ca 2+ signalling mechanisms in cell biology. Experimental results suggest an alternative mode for stimulation of Atm by double-strand breaks, in which Atm autophosphorylation at Ser 1987 (like trans-phosphorylation of downstream substrates) is a consequence rather than a cause of Atm activation. Stimulation of immune cells causes depletion of Ca 2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca 2+ entry through store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation 1 , 2 . Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca 2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane 3 , 4 , 5 . We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca 2+ entry downstream of STIM 6 , 7 , 8 . Combined overexpression of Orai and Stim in Drosophila cells 8 , or Orai1 and STIM1 in mammalian cells 9 , 10 , 11 , leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca 2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs + . These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.

P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X 4 receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit–subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in β-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the ‘gate’ is defined by an ∼8 Å slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening. P2X 4 and ASIC1 at the gate P2X receptors are ATP-gated non-selective cation channels involved in nociception and inflammatory responses, whose structures were unknown. Kawate et al . now present the crystal structure of the zebrafish P2X 4 receptor in a closed state. The trimeric structure reveals some of the molecular underpinnings of ligand-binding, cation entry and channel gating. A related paper presents the structure of chicken acid-sensing ion channel 1 (ASIC1) in a desensitized state. Like P2X receptors, ASICs are trimeric, but they belong to an entirely different family of ion channels. The structure determination of ASIC1 shows how ion permeation and desensitization may occur, and comparison of ASIC and P2X structures suggests that these functionally distinct channels employ similar mechanistic principles. P2X receptors are ATP-gated cation channels that are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. The crystal structure of the zebrafish P2X 4 channel is now solved in its closed state, revealing some of the molecular underpinnings of ligand-binding, cation entry and channel gating.

STIM1-mediated gating of CRAC channels occurs through a mechanism in which ion selectivity and gating are closely coupled, and the residue V102 is identified as a candidate for the channel gate. Ion selectivity in CRAC channels Store-operated CRAC channels (Ca 2+ release-activated Ca 2+ channels) are implicated in the physiology of numerous cell types, underlie several disease processes, and have emerged as major targets for drug development. The molecular components of CRAC channels are well known, but the mechanisms of channel permeation and gating remain obscure. Here it is shown that the ion selectivity of the prototypic store-operated CRAC channel, Orai1, is not an intrinsic property of the channel as had been assumed. Rather, it is a tunable feature bestowed on the otherwise non-selective channel by the endoplasmic reticulum Ca 2+ sensor and CRAC channel activator, STIM1. 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 1 . 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 2 . 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.