Repeat polymorphisms underlie top genetic risk loci for glaucoma and colorectal cancer.

Many regions in the human genome vary in length among individuals due to variable numbers of tandem repeats (VNTRs). To assess the phenotypic impact of VNTRs genome-wide, we applied a statistical imputation approach to estimate the lengths of 9,561 autosomal VNTR loci in 418,136 unrelated UK Biobank participants and 838 GTEx participants. Association and statistical fine-mapping analyses identified 58 VNTRs that appeared to influence a complex trait in UK Biobank, 18 of which also appeared to modulate expression or splicing of a nearby gene. Non-coding VNTRs at TMCO1 and EIF3H appeared to generate the largest known contributions of common human genetic variation to risk of glaucoma and colorectal cancer, respectively. Each of these two VNTRs associated with a >2-fold range of risk across individuals. These results reveal a substantial and previously unappreciated role of non-coding VNTRs in human health and gene regulation.

Ion channels in innate and adaptive immunity.

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.

Structural Mechanism of EMRE-Dependent Gating of the Human Mitochondrial Calcium Uniporter.

Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.

Enjoy the Trip: Calcium in Mitochondria Back and Forth.

In the last 5 years, most of the molecules that control mitochondrial Ca(2+) homeostasis have been finally identified. Mitochondrial Ca(2+) uptake is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, a macromolecular structure that guarantees Ca(2+) accumulation inside mitochondrial matrix upon increases in cytosolic Ca(2+). Conversely, Ca(2+) release is under the control of the Na(+)/Ca(2+) exchanger, encoded by the NCLX gene, and of a H(+)/Ca(2+) antiporter, whose identity is still debated. The low affinity of the MCU complex, coupled to the activity of the efflux systems, protects cells from continuous futile cycles of Ca(2+) across the inner mitochondrial membrane and consequent massive energy dissipation. In this review, we discuss the basic principles that govern mitochondrial Ca(2+) homeostasis and the methods used to investigate the dynamics of Ca(2+) concentration within the organelles. We discuss the functional and structural role of the different molecules involved in mitochondrial Ca(2+) handling and their pathophysiological role.

TMCO1 Is an ER Ca(2+) Load-Activated Ca(2+) Channel.

Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading and disassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions.

Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.

The Ca(2+)-free form of calmodulin (apoCaM) often appears inert, modulating target molecules only upon conversion to its Ca(2+)-bound form. This schema has appeared to govern voltage-gated Ca(2+) channels, where apoCaM has been considered a dormant Ca(2+) sensor, associated with channels but awaiting the binding of Ca(2+) ions before inhibiting channel opening to provide vital feedback inhibition. Using single-molecule measurements of channels and chemical dimerization to elevate apoCaM, we find that apoCaM binding on its own markedly upregulates opening, rivaling the strongest forms of modulation. Upon Ca(2+) binding to this CaM, inhibition may simply reverse the initial upregulation. As RNA-edited and -spliced channel variants show different affinities for apoCaM, the apoCaM-dependent control mechanisms may underlie the functional diversity of these variants and explain an elongation of neuronal action potentials by apoCaM. More broadly, voltage-gated Na channels adopt this same modulatory principle. ApoCaM thus imparts potent and pervasive ion-channel regulation. PAPERCLIP:

Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility.

Spermatozoa must leave one organism, navigate long distances, and deliver their paternal DNA into a mature egg. For successful navigation and delivery, a sperm-specific calcium channel is activated in the mammalian flagellum. The genes encoding this channel (CatSpers) appear first in ancient uniflagellates, suggesting that sperm use adaptive strategies developed long ago for single-cell navigation. Here, using genetics, super-resolution fluorescence microscopy, and phosphoproteomics, we investigate the CatSper-dependent mechanisms underlying this flagellar switch. We find that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we find unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility.

Conservation of Ca2+/calmodulin regulation across Na and Ca2+ channels.

Voltage-gated Na and Ca2+ channels comprise distinct ion channel superfamilies, yet the carboxy tails of these channels exhibit high homology, hinting at a long-shared and purposeful module. For different Ca2+ channels, carboxyl-tail interactions with calmodulin do elaborate robust and similar forms of Ca2+ regulation. However, Na channels have only shown subtler Ca2+ modulation that differs among reports, challenging attempts at unified understanding. Here, by rapid Ca2+ photorelease onto Na channels, we reset this view of Na channel regulation. For cardiac-muscle channels (NaV1.5), reported effects from which most mechanistic proposals derive, we observe no Ca2+ modulation. Conversely, for skeletal-muscle channels (NaV1.4), we uncover fast Ca2+ regulation eerily similar to that of Ca2+ channels. Channelopathic myotonia mutations halve NaV1.4 Ca2+ regulation, and transplanting the NaV1.4 carboxy tail onto Ca2+ channels recapitulates Ca2+ regulation. Thus, we argue for the persistence and physiological relevance of an ancient Ca2+ regulatory module across Na and Ca2+ channels.

Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates.

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.

mTOR regulates lysosomal ATP-sensitive two-pore Na(+) channels to adapt to metabolic state.

Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na(+) channel (lysoNa(ATP)). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosome's membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNa(ATP) have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cell's metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.

A genetic program promotes C. elegans longevity at cold temperatures via a thermosensitive TRP channel.

Both poikilotherms and homeotherms live longer at lower body temperatures, highlighting a general role of temperature reduction in lifespan extension. However, the underlying mechanisms remain unclear. One prominent model is that cold temperatures reduce the rate of chemical reactions, thereby slowing the rate of aging. This view suggests that cold-dependent lifespan extension is simply a passive thermodynamic process. Here, we challenge this view in C. elegans by showing that genetic programs actively promote longevity at cold temperatures. We find that TRPA-1, a cold-sensitive TRP channel, detects temperature drop in the environment to extend lifespan. This effect requires cold-induced, TRPA-1-mediated calcium influx and a calcium-sensitive PKC that signals to the transcription factor DAF-16/FOXO. Human TRPA1 can functionally substitute for worm TRPA-1 in promoting longevity. Our results reveal a previously unrecognized function for TRP channels, link calcium signaling to longevity, and, importantly, demonstrate that genetic programs contribute to lifespan extension at cold temperatures.

Second cistron in CACNA1A gene encodes a transcription factor mediating cerebellar development and SCA6.

The CACNA1A gene, encoding the voltage-gated calcium channel subunit α1A, is involved in pre- and postsynaptic Ca(2+) signaling, gene expression, and several genetic neurological disorders. We found that CACNA1A coordinates gene expression using a bicistronic mRNA bearing a cryptic internal ribosomal entry site (IRES). The first cistron encodes the well-characterized α1A subunit. The second expresses a transcription factor, α1ACT, which coordinates expression of a program of genes involved in neural and Purkinje cell development. α1ACT also contains the polyglutamine (polyQ) tract that, when expanded, causes spinocerebellar ataxia type 6 (SCA6). When expressed as an independent polypeptide, α1ACT-bearing an expanded polyQ tract-lacks transcription factor function and neurite outgrowth properties, causes cell death in culture, and leads to ataxia and cerebellar atrophy in transgenic mice. Suppression of CACNA1A IRES function in SCA6 may be a potential therapeutic strategy.

STIM1 and ORAI1 form a novel cold transduction mechanism in sensory and sympathetic neurons.

Moderate coolness is sensed by TRPM8 ion channels in peripheral sensory nerves, but the mechanism by which noxious cold is detected remains elusive. Here, we show that somatosensory and sympathetic neurons express two distinct mechanisms to detect noxious cold. In the first, inhibition by cold of a background outward current causes membrane depolarization that activates an inward current through voltage-dependent calcium (CaV ) channels. A second cold-activated mechanism is independent of membrane voltage, is inhibited by blockers of ORAI ion channels and by downregulation of STIM1, and is recapitulated in HEK293 cells by co-expression of ORAI1 and STIM1. Using total internal reflection fluorescence microscopy we found that cold causes STIM1 to aggregate with and activate ORAI1 ion channels, in a mechanism similar to that underlying store-operated calcium entry (SOCE), but directly activated by cold and not by emptying of calcium stores. This novel mechanism may explain the phenomenon of cold-induced vasodilation (CIVD), in which extreme cold increases blood flow in order to preserve the integrity of peripheral tissues.

TPC proteins are phosphoinositide- activated sodium-selective ion channels in endosomes and lysosomes.

Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.

Drp1-Zip1 Interaction Regulates Mitochondrial Quality Surveillance System.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.

Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation-Contraction Complex.

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca2+-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.

MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy.

BACKGROUND: Calcium (Ca2+) uptake by mitochondria occurs via the mitochondrial Ca2+ uniporter. Mitochondrial Ca2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca2+]m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca2+-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS: MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.

Circular RNA circMYLK4 shifts energy metabolism from glycolysis to OXPHOS by binding to the calcium channel auxiliary subunit CACNA2D2.

Skeletal muscle is heterogeneous tissue, composed of fast-twitch fibers primarily relying on glycolysis and slow-twitch fibers primarily relying on oxidative phosphorylation. The relative expression and balance of glycolysis and oxidative phosphorylation in skeletal muscle are crucial for muscle growth and skeletal muscle metabolism. Here, we employed multi-omics approaches including transcriptomics, proteomics, phosphoproteomics, and metabolomics to unravel the role of circMYLK4, a differentially expressed circRNA in fast and slow-twitch muscle fibers, in muscle fiber metabolism. We discovered that circMYLK4 inhibits glycolysis and promotes mitochondrial oxidative phosphorylation. Mechanistically, circMYLK4 interacts with the voltage-gated calcium channel auxiliary subunit CACNA2D2, leading to the inhibition of Ca2+ release from the sarcoplasmic reticulum. The decrease in cytoplasmic Ca2+ concentration inhibits the expression of key enzymes, PHKB and PHKG1, involved in glycogen breakdown, thereby suppressing glycolysis. On the other hand, the increased fatty acid ß-oxidation enhances the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation. In general, circMYLK4 plays an indispensable role in maintaining the metabolic homeostasis of skeletal muscle.

Rac1 and Rac3 GTPases and TPC2 are required for axonal outgrowth and migration of cortical interneurons.

Rho GTPases, among them Rac1 and Rac3, are major transducers of extracellular signals and are involved in multiple cellular processes. In cortical interneurons, the neurons that control the balance between excitation and inhibition of cortical circuits, Rac1 and Rac3 are essential for their development. Ablation of both leads to a severe reduction in the numbers of mature interneurons found in the murine cortex, which is partially due to abnormal cell cycle progression of interneuron precursors and defective formation of growth cones in young neurons. Here, we present new evidence that upon Rac1 and Rac3 ablation, centrosome, Golgi complex and lysosome positioning is significantly perturbed, thus affecting both interneuron migration and axon growth. Moreover, for the first time, we provide evidence of altered expression and localization of the two-pore channel 2 (TPC2) voltage-gated ion channel that mediates Ca2+ release. Pharmacological inhibition of TPC2 negatively affected axonal growth and migration of interneurons. Our data, taken together, suggest that TPC2 contributes to the severe phenotype in axon growth initiation, extension and interneuron migration in the absence of Rac1 and Rac3.