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The exceptionally high temperature sensitivity of certain transient receptor potential (TRP) family ion channels is the molecular basis of hot and cold sensation in sensory neurons. The laws of thermodynamics dictate that opening of these specialized TRP channels must involve an unusually large conformational standard-state enthalpy, ΔHo: positive ΔHo for heat-activated and negative ΔHo for cold-activated TRPs. However, the molecular source of such high-enthalpy changes has eluded neurobiologists and biophysicists. Here we offer a general, unifying mechanism for both hot and cold activation that recalls long-appreciated principles of protein folding. We suggest that TRP channel gating is accompanied by large changes in molar heat capacity, ΔCP. This postulate, along with the laws of thermodynamics and independent of mechanistic detail, leads to the conclusion that hot- and cold-sensing TRPs operate by identical conformational changes.

Mitochondria are integral components of cellular calcium (Ca²⁺) signaling. Calcium stimulates mitochondrial adenosine 5'-triphosphate production, but can also initiate apoptosis. In turn, cytoplasmic Ca²⁺ concentrations are regulated by mitochondria. Although several transporter and ion-channel mechanisms have been measured in mitochondria, the molecules that govern Ca²⁺ movement across the inner mitochondrial membrane are unknown. We searched for genes that regulate mitochondrial Ca²⁺ and H⁺ concentrations using a genome-wide Drosophila RNA interference (RNAi) screen. The mammalian homolog of one Drosophila gene identified in the screen, Letm1, was found to specifically mediate coupled Ca²⁺/H⁺ exchange. RNAi knockdown, overexpression, and liposome reconstitution of the purified Letm1 protein demonstrate that Letm1 is a mitochondrial Ca²⁺/H⁺ antiporter.

Primary cilia are known as specialized calcium signalling compartments on the cell surface, but the ionic permeability and other physiological properties of these protrusions are unknown—this is one of two studies identifying the ion channels that densely populate primary cilia, with direct measurements revealing cilia as a unique, functionally independent calcium signalling compartment that modulates hedgehog signalling pathways. Calcium handling in primary cilia Primary cilia are organelles that project from most cell surfaces, where they are thought to be involved in controlling cell growth and cell division and also to act as specialized calcium signalling compartments. The ionic permeability and other physiological properties of these protrusions are unknown. In two studies, David Clapham and colleagues identify the ion channels that densely populate primary cilia and conduct direct measurements to reveal cilia as a unique, functionally independent calcium signalling compartment that modulates hedgehog signalling pathways. A primary cilium is a solitary, slender, non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane 1 . Housing components of the cell division apparatus between cell divisions, primary cilia also serve as specialized compartments for calcium signalling 2 and hedgehog signalling pathways 3 . Specialized sensory cilia such as retinal photoreceptors and olfactory cilia use diverse ion channels 4 , 5 , 6 , 7 . An ion current has been measured from primary cilia of kidney cells 8 , but the responsible genes have not been identified. The polycystin proteins (PC and PKD), identified in linkage studies of polycystic kidney disease 9 , are candidate channels divided into two structural classes: 11-transmembrane proteins (PKD1, PKD1L1 and PKD1L2) remarkable for a large extracellular amino terminus of putative cell adhesion domains and a G-protein-coupled receptor proteolytic site, and the 6-transmembrane channel proteins (PKD2, PKD2L1 and PKD2L2; TRPPs). Evidence indicates that the PKD1 proteins associate with the PKD2 proteins via coiled-coil domains 10 , 11 , 12 . Here we use a transgenic mouse in which only cilia express a fluorophore and use it to record directly from primary cilia, and demonstrate that PKD1L1 and PKD2L1 form ion channels at high densities in several cell types. In conjunction with an accompanying manuscript 2 , we show that the PKD1L1–PKD2L1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established hedgehog pathways.

Magnesium (Mg²⁺) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg²⁺ transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg²⁺ influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg²⁺. Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg²⁺ concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg²⁺ or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg²⁺ transport system.

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na⁺-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4–S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π–π bonds and cation–π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

The transient receptor potential ion channel subfamily M, member 7 (TRPM7), is a ubiquitously expressed protein that is required for mouse embryonic development. TRPM7 contains both an ion channel and an α-kinase. The channel domain comprises a nonselective cation channel with notable permeability to Mg2+ and Zn2+. Here, we report the closed state structures of the mouse TRPM7 channel domain in three different ionic conditions to overall resolutions of 3.3, 3.7, and 4.1 Å. The structures reveal key residues for an ion binding site in the selectivity filter, with proposed partially hydrated Mg2+ ions occupying the center of the conduction pore. In high [Mg2+], a prominent external disulfide bond is found in the pore helix, which is essential for ion channel function. Our results provide a structural framework for understanding the TRPM1/3/6/7 subfamily and extend the knowledge base upon which to study the diversity and evolution of TRP channels.

The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its molecular components include the EF-hand-containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex). Quantitative mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and essential MCU regulator (EMRE), a previously uncharacterized protein. EMRE is a 10-ki loda Ito n, m etazoa ç-specific protein with a single transmembrane domain. In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and additionally bridges the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU.

Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels. Members of the transient receptor potential (TRP) ion channels conduct cations into cells upon activation by a variety of signals. Here authors present the cryo-EM structure of TRPC4 in its unliganded (apo) state, which provides molecular insights into TRPC4's ion selectivity and TPR channel evolution.

The crystal structure of Na v Rh, a NaChBac orthologue from the marine Rickettsiales sp. HIMB114 , defines an ion binding site within the selectivity filter, and reveals several conformational rearrangements that may underlie the electromechanical coupling mechanism. High-resolution sodium channel structures There are many published structures for potassium channels, but structural information on voltage-gated sodium (Na v ) channels is much more scare, despite their importance in the initiation and propagation of action potentials in nerve cells, muscle cells and in the heart. Bacterial Na v channels provide a good model system for structure–function analyses, and here two groups report the X-ray crystal structure of bacterial Na v channels apparently in 'inactivated' conformations. Nieng Yan and colleagues determined the structure of Na v Rh from the marine bacterium known as alpha proteobacterium HIMB114 at 3.05-ångström resolution. William Catterall and colleagues report crystallographic snapshots of the Na v Ab channel from Arcobacter butzleri in two potentially inactivated states at 3.2-ångström resolution. Comparisons of these newly obtained structures with previously published data on Na v Ab in a 'pre-open' state reveal conformational rearrangements that may underlie the electromechanical coupling mechanism of these channels. This work is relevant to channelopathies and more widely to the design of neuroactive drugs. Voltage-gated sodium (Na v ) channels are essential for the rapid depolarization of nerve and muscle 1 , and are important drug targets 2 . Determination of the structures of Na v channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na v channels, exemplified by the Na + -selective channel of bacteria (NaChBac) 3 , provides a useful model system for structure–function analysis. Here we report the crystal structure of Na v Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 ( Rickettsiales sp. HIMB114 ; denoted Rh), at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr 178 and Leu 179 constitute an inner site within the selectivity filter where a hydrated Ca 2+ resides in the crystal structure. The outer mouth of the Na + selectivity filter, defined by Ser 181 and Glu 183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na v Rh is in an ‘inactivated’ conformation. Comparison of Na v Rh with Na v Ab 4 reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

During the mitochondrial permeability transition, a large channel in the inner mitochondrial membrane opens, leading to the loss of multiple mitochondrial solutes and cell death. Key triggers include excessive reactive oxygen species and mitochondrial calcium overload, factors implicated in neuronal and cardiac pathophysiology. Examining the differential behavior of mitochondrial Ca2+ overload in Drosophila versus human cells allowed us to identify a gene, MCUR1, which, when expressed in Drosophila cells, conferred permeability transition sensitive to electrophoretic Ca2+ uptake. Conversely, inhibiting MCUR1 in mammalian cells increased the Ca2+ threshold for inducing permeability transition. The effect was specific to the permeability transition induced by Ca2+, and such resistance to overload translated into improved cell survival. Thus, MCUR1 expression regulates the Ca2+ threshold required for permeability transition.