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Mammalian cyclic nucleotide-gated (CNG) channels play crucial roles in visual and olfactory signal transduction. In olfactory sensory neurons, the native CNG channel functions as a heterotetramer consisting of CNGA2, CNGA4, and CNGB1b subunits and is activated by cAMP. Calmodulin (CaM) modulates the activity of the olfactory CNG channel, enabling rapid adaptation to odorants. Here we present cryo-EM structures of the native human olfactory CNGA2/A4/B1b channel in both CaM-bound closed and cAMP-bound open states, elucidating the molecular basis of the 2:1:1 subunit stoichiometry in channel assembly and the asymmetrical channel gating upon cAMP activation. Combining structural and functional analyses with AlphaFold prediction, we define two distinct CaM binding sites (CaM1 and CaM2) on the N- and C-terminal regions of CNGB1b, respectively, shedding light on the molecular mechanism of Ca 2+ /CaM-mediated rapid inhibition of the native olfactory CNG channel. CNG channels are essential for vision and smell. Xue et al. reveal structures of the native human olfactory CNG channel in CaM-bound closed and cAMP-bound open states, elucidating mechanisms of assembly, gating, and calmodulin-mediated inhibition.

Organellar two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region, yet exhibit drastically different ion selectivity. Plant TPC1 functions as a nonselective cation channel on the vacuole membrane, whereas mammalian TPC channels have been shown to be endo/lysosomal Na⁺-selective or Ca2+-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 from Arabidopsis thaliana (AtTPC1) and compared its selectivity with the selectivity of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is selective for Ca2+ over Na⁺, but nonselective among monovalent cations (Li⁺, Na⁺, and K⁺). Our results also confirm that HsTPC2 is a Na⁺-selective channel activated by phosphatidylinositol 3,5-bisphosphate. Guided by our recent structure of AtTPC1, we converted AtTPC1 to a Na⁺-selective channel by mimicking the selectivity filter of HsTPC2 and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Na⁺-selective AtTPC1 mutant elucidates the structural basis for Na+ selectivity in mammalian TPCs.

Na + /Ca 2+ exchangers (NCX) transport Ca 2+ in or out of cells in exchange for Na + . They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca 2+ homeostasis. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na + and Ca 2+ . Here we present the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states, elucidating key structural elements important for NCX ion exchange function and its modulation by cytosolic Ca 2+ and Na + . We demonstrate that the interactions between the ion-transporting transmembrane (TM) domain and the cytosolic regulatory domain define the activity of NCX. In the inward-facing state with low cytosolic [Ca 2+ ], a TM-associated four-stranded β-hub mediates a tight packing between the TM and cytosolic domains, resulting in the formation of a stable inactivation assembly that blocks the TM movement required for ion exchange function. Ca 2+ binding to the cytosolic second Ca 2+ -binding domain (CBD2) disrupts this inactivation assembly which releases its constraint on the TM domain, yielding an active exchanger. Thus, the current NCX1 structures provide an essential framework for the mechanistic understanding of the ion transport and cellular regulation of NCX family proteins. Here authors present the cryo-EM structures of human cardiac NCX1, elucidating key structural elements for ion exchange and modulation by cytosolic Ca2 + and Na + .

ZnT8 is a Zn 2+ /H + antiporter that belongs to SLC30 family and plays an essential role in regulating Zn 2+ accumulation in the insulin secretory granules of pancreatic β cells. However, the Zn 2+ /H + exchange mechanism of ZnT8 remains unclear due to the lack of high-resolution structures. Here, we report the cryo-EM structures of human ZnT8 (HsZnT8) in both outward- and inward-facing conformations. HsZnT8 forms a dimeric structure with four Zn 2+ binding sites within each subunit: a highly conserved primary site in transmembrane domain (TMD) housing the Zn 2+ substrate; an interfacial site between TMD and C-terminal domain (CTD) that modulates the Zn 2+ transport activity of HsZnT8; and two adjacent sites buried in the cytosolic domain and chelated by conserved residues from CTD and the His-Cys-His (HCH) motif from the N-terminal segment of the neighboring subunit. A comparison of the outward- and inward-facing structures reveals that the TMD of each HsZnT8 subunit undergoes a large structural rearrangement, allowing for alternating access to the primary Zn 2+ site during the transport cycle. Collectively, our studies provide the structural insights into the Zn 2+ /H + exchange mechanism of HsZnT8.

TRPM4 is a calcium-activated, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) -modulated, non-selective cation channel that belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the electron cryo-microscopy structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide-binding domain and the C-terminal coiled-coil participate in the tetrameric assembly of the channel; ATP binds at the nucleotide-binding domain and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. The S1–S4 domain and the post-S6 TRP domain form the central gating apparatus that probably houses the Ca 2+ - and PtdIns(4,5)P 2 -binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and reveal the molecular architecture of the TRPM family. Electron cryo-microscopy structures of mouse TRPM4, a calcium-activated, non-selective cation channel, in the apo and ATP-bound states. Scoping out TRPM channels Melastatin-related transient receptor potential (TRPM) ion channels are the largest group of the TRP superfamily and, as such, are widespread throughout the body with diverse physiological roles including heat and taste sensation and regulating ion homeostasis. For example, TRPM4 is a Ca 2+ -activated non-selective channel expressed in many of the central organs including the brain and heart, and is involved in the cardiac rhythm, breath pacemaking and insulin secretion. In this issue of Nature , two groups report the structure of TRPM4 by electron cryo-microscopy. Wei Lü and colleagues solved the structure of human TRPM4, which shows an umbrella-like structure, bound to Ca(ɪɪ) and decavanadate. Youxing Jiang and colleagues report the structure of mouse TRPM4 with and without ATP, which inhibits channel activity. These studies provide the first structural insights into the TRPM family.

Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel – independently of the membrane potential – by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.

Two-pore channels (TPCs) contain two copies of a Shaker -like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here we present the crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana , AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca 2+ . Ca 2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TM domains. Luminal Ca 2+ or Ba 2+ can modulate voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. Our Ba 2+ -bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels. The X-ray crystal structure of a two-pore channel from Arabidopsis thaliana reveals the structure and the mechanism of voltage gating of this class of ubiquitous cation-selective ion channels. Characterization of the two-pore channel AtTPC1 The X-ray crystal structure of the two-pore channel AtTPC1 from Arabidopsis thaliana reveals the structure and mechanism of voltage gating of a type of cation-selective ion channel ubiquitously expressed in the organelles of animal and plant cells. AtTPC1 is activated by both voltage and cytosolic Ca 2+ , and voltage activation can be inhibited by luminal Ca 2+ . Youxing Jiang and colleagues determined the crystal structure of AtTPC1 to 3.3 Å resolution and find that, as predicted, two AtTPC1 subunits make up the functional channel. Alexander Kintzer and Robert Stroud report the AtTPC1 crystal structure at 2.87 Å resolution, revealing the mechanisms of ion permeation, channel activation, and location of regulatory sites and voltage-sensing domains.

The structure of mouse transient receptor potential mucolipin 1 (TRPML1), a cation channel located within endosomal and lysosomal membranes, is resolved using single-particle electron cryo-microscopy. Closing in on ion channels Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca( II )-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation. Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells 1 , 2 , its loss-of-function mutations are the direct cause of type IV mucolipidosis, an autosomal recessive lysosomal storage disease 3 , 4 , 5 , 6 . Here we present the single-particle electron cryo-microscopy structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis analysis, the TRPML1 structure reveals that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P 2 ) binds to the N terminus of the channel—distal from the pore—and the helix–turn–helix extension between segments S2 and S3 probably couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion-binding sites, and the conserved acidic residues form the luminal Ca 2+ -blocking site that confers luminal pH and Ca 2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing several luminal ion passages to the pore and creating a negative electrostatic trap, with a preference for divalent cations, at the luminal entrance. The structure also reveals two equally distributed S4–S5 linker conformations in the closed channel, suggesting an S4–S5 linker-mediated PtdInsP 2 gating mechanism among TRPML channels 7 , 8 .

Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel whose loss-of-function mutations directly cause the lysosomal storage disorder mucolipidosis type IV (MLIV). TRPML1 can be allosterically regulated by various ligands including natural lipids and small synthetic molecules and the channel undergoes a global movement propagated from ligand-induced local conformational changes upon activation. In this study, we identified a functionally critical residue, Tyr404, at the C-terminus of the S4 helix, whose mutations to tryptophan and alanine yield gain- and loss-of-function channels, respectively. These allosteric mutations mimic the ligand activation or inhibition of the TRPML1 channel without interfering with ligand binding and both mutant channels are susceptible to agonist or antagonist modulation, making them better targets for screening potent TRPML1 activators and inhibitors. We also determined the high-resolution structure of TRPML1 in complex with the PI(4,5)P 2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P 2 -bound TRPML1 structure at the same hotspot for agonists and antagonists, providing a plausible structural explanation for the inhibitory effect of sphingomyelin on agonist activation.

The otopetrin (OTOP) proteins were recently characterized as extracellular proton-activated proton channels. Several recent OTOP channel structures demonstrated that the channels form a dimer with each subunit adopting a double-barrel architecture. However, the structural mechanisms underlying some basic functional properties of the OTOP channels remain unresolved, including extracellular pH activation, proton conducting pathway, and rapid desensitization. In this study, we performed structural and functional characterization of the Caenorhabditis elegans OTOP8 (CeOTOP8) and mouse OTOP2 (mOTOP2) and illuminated a set of conformational changes related to the proton-conducting process in OTOP. The structures of CeOTOP8 reveal the conformational change at the N-terminal part of TM12 that renders the channel in a transiently proton-transferring state, elucidating an inter-barrel, Glu/His-bridged proton passage within each subunit. The structures of mOTOP2 reveal the conformational change at the N-terminal part of TM6 that exposes the central glutamate to the extracellular solution for protonation. In addition, the structural comparison between CeOTOP8 and mOTOP2, along with the structure-based mutagenesis, demonstrates that an inter-subunit movement at the OTOP channel dimer interface plays a central role in regulating channel activity. Combining the structural information from both channels, we propose a working model describing the multi-step conformational changes during the proton conducting process. The otopetrin (OTOP) proteins function as proton-activated proton channels. Here, the authors perform a structural characterization of C. elegans OTOP8 and mouse OTOP2 channels, providing mechanistic insights into the proton-conducting process in OTOP.