EF-hand Domain Research Articles

Genomic collation revealed the significance of Piriformospora indica (Serendipita indica) PiEF-hand protein in vesicle trafficking and fungal hyphal growth

EF-hand domain proteins are Ca2+ binding proteins with helix-loop-helix structural pattern and classified as CaMs, CMLs, CBLs, CDPKs, Rbohs, Myosin light chains (Mlcs) and others. Mlcs are important proteins that function through binding with IQ motifs of Myosins and respond to changes in Ca2+ ion concentration. Present study reports in silico analysis of Piriformospora indica EF-hand (PiEF-hand) protein annotated as probable myosin regulatory light chain Cdc4. The genomic analysis revealed sequence & structure attributes, orthologs identification, evolutionary perspective, protein-protein interactions and functionality. PiEF-hand protein is 141 aa long acidic protein lacking cysteine. Structural analysis highlighted four EF-hand motifs and conservation of Myosin heavy chain IQ motif interacting sites. Further, evolutionary relationships across 79 eukaryotic organisms were drawn classifying orthologs in two groups- CaMs and Mlcs. The PiEF-hand protein is grouped along with Mlcs underlining its possible role as myosin light chain. Separate sequence logos of each EF-hand motifs for CaMs and Mlcs revealed poor sequence conservation among Mlcs, whereas, CaMs EF-hand motifs were well conserved. The protein-protein interaction network analysis glimpsed involvement of PiEF-hand protein in vesicular trafficking and polarized fungal hyphal growth.

Identification and Characterization of Calcium-Dependent Protein Kinase (CDPK) Gene Families Across the Whole Genome in Cyclocarya paliurus

Cyclocarya paliurus, as a medicinal plant, grows widely in Karst ecosystem due to its great tolerance to high calcium levels. Previous studies suggested that Calcium-dependent protein kinases (CDPKs) are crucial for transmitting Ca2+ signal, and thus affecting the plant calcium tolerance. This mechanism has not yet been sufficiently examined for Cyclocarya paliurus (C. paliurus). In this study, 25 CpCDPKs genes were computationally examined and categorized as four groups (CDPK I, CDPK II, CDPK III, and CDPK IV) according to their gene structures, motif and phylogenetic relationships. Both EFhand and protein kinase domain were found in CpCDPK proteins. This work lays the groundwork for further investigation into the roles of CpCDPK in Cyclocarya paliurus.

Genome-Wide Identification and Characterization of Calmodulin and Calmodulin-like Genes Family in Tea Plant and Their Roles under Abiotic Stress

As an important Ca2+ sensor, calmodulin (CaM) and calmodulin-like protein (CML) play core roles in plant growth, development, and response to environmental stimuli. The CaM/CML gene family has been well characterized in various plant species, such as Arabidopsis thaliana, rice, and tomato; however, in the tea plant, the CaM/CML gene family has not been systematically and comprehensively characterized. In the present study, a total of 5 CsCaM and 60 CsCML proteins were identified from the tea plant genome, which were unevenly distributed on the 14 chromosomes of the tea plant. All the proteins contained two to four EF-hand domains. Meanwhile, an integrated analysis of physicochemical properties, sequence structure, motif identification, phylogeny, gene duplication, promoter cis-elements, and RNA-seq expression profiles in the CsCaM/CML gene family was performed. Transcriptome analysis revealed that CsCaM/CMLs were differentially expressed in different tissues of the tea plant, suggesting their potential roles in plant growth and development. The expression profiles associated with various stress treatments revealed that CsCaM/CML genes were involved in a wide range of abiotic factors, including cold and drought stress. Quantitative real-time PCR (qRT-PCR) was also used to validate the differences in expression under abiotic stress. Overall, these findings enhanced our understanding of CsCaM/CML genes and provided useful information for further research into their molecular functions in abiotic stress response, and in multiple physiological processes in the tea plant.

Large Rab GTPase Rab44 regulates microtubule-dependent retrograde melanosome transport in melanocytes

Melanosomes are melanin-containing organelles in melanocytes, and they are responsible for skin and hair pigmentation in mammals. The intracellular distribution of melanosomes is mainly determined by the balance between their anterograde transport on actin filaments and retrograde transport on microtubules. Although we have shown previously that melanoregulin and Rab36 serve as cargo receptors on melanosomes for retrograde transport, their knockdown does not completely inhibit retrograde melanosome transport, suggesting the existence of an additional cargo receptor(s) in melanocytes. In this study, we investigated the possible involvement of an atypical large Rab, Rab44, which also contains EF-hand domains and a coiled-coil domain, in retrograde melanosome transport in mouse melanocytes (Rab27A-deficient melan-ash cells). Our results showed that Rab44 localizes on mature melanosomes through lipidation of its C-terminal Rab-like GTPase domain, and that its knockdown results in suppression of retrograde melanosome transport. In addition, our biochemical analysis indicated that Rab44 interacts with the dynein–dynactin motor complex via its coiled-coil domain–containing middle region. Since simultaneous depletion of Rab44, melanoregulin, and Rab36 resulted in almost complete inhibition of retrograde melanosome transport, we propose that Rab44 is the third cargo receptor. We also showed that the N-terminal region of Rab44, which contains EF-hand domains, is required for both retrograde melanosome transport and its Ca2+-modulated activities. Our findings indicated that Rab44 is a third melanosomal cargo receptor, and that, unlike other cargo receptors previously described, its transport function is regulated by Ca2+.

Strongyloides venezuelensis-derived venestatin ameliorates asthma pathogenesis by suppressing receptor for advanced glycation end-products-mediated signaling

IntroductionEF-hand Ca2+-binding proteins such as S100 protein family members are recognized by the receptor for advanced glycation end-products (RAGE) and are involved in the pathogenesis of asthma/allergic airway inflammation (AAI). Venestatin, an EF-hand Ca2+-binding protein, which is secreted by the parasitic helminth Strongyloides venezuelensis, binds with RAGE and suppresses RAGE-mediated inflammatory responses after parasite invasion. In this study, we evaluated the effect of venestatin on pathogenesis in a house dust mite (HDM) murine model of asthma/AAI. MethodsMice were intranasally treated with HDM, HDM with recombinant venestatin, or HDM with synthetic peptides, which were designed based on the EF-hand Ca2+-binding domain of venestatin. Pro-inflammatory responses in the lungs of mice were assessed. ResultsHDM treatment induced inflammatory cell infiltration, phosphorylation of the mitogen-activated protein kinase and inhibitor κB, and production of the cytokines tumor necrosis factor-α and interleukin-5 in the lungs. Co-administration of recombinant venestatin with HDM suppressed these pro-inflammatory responses. Treatment with synthetic peptides reduced inflammatory cell infiltration in a RAGE-dependent manner. ConclusionThe EF-hand domain of venestatin may have potential therapeutic benefits in asthma.

EF-hand domain-containing proteins in Triticum aestivum: Insight into their roles in stress response and signalling

In plants, Ca2+ is an integral element in several stresses related signallings. During environmental stresses, the cytosolic Ca2+ concentration tends to rise rapidly, which is further co-ordinate with EF-hand domain-containing proteins. In our study, a total of 586 EF-hand domain-containing proteins were identified in Triticum aestivum as calmodulin (CaM)/Calmodulin-like (CML), calcineurin B-like (CBL), protein kinase (CDPK, CRK, CCaMK), respiratory burst oxidase homologs (RBOHs), miscellaneous proteins and un-categorized EF-hand proteins (UEPs). These proteins either carried the EF-hand domain singly or along with other domains. Phylogenetically, the proteins were clustered into eight groups, each with diverse types of proteins. The genes were found on all the chromosomes and sub-genomes of T. aestivum. The gene structure analysis revealed their conserved architecture. The diverse expression patterns of genes indicated their involvement in plant growth and development. The modulated expression of various genes to heat, drought, combined heat drought, salt and two fungal stresses unveiled their functioning in defence responses. Moreover, the altered expression under CaCl2 treatment suggested their role in stress signalling. Furthermore, the miRNA interaction analysis revealed their interaction with various plant development and stress resistance associated miRNAs, which also showed their participation in these phenomenon. Additionally, protein-protein and protein-chemical analyses revealed the interaction of EF-hand proteins with various Ca2+ binding/sensing proteins which regulate Ca2+- mediated signallings.This study will be useful in the functional characterization of potential candidate genes in future studies.

SPERM FACTORS AND EGG ACTIVATION: The structure and function relationship of sperm PLCZ1.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca2+) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency. PLCZ1 is the smallest PLC in size, comprising four N-terminal EF-hand domains, followed by X and Y catalytic domains, which are separated by the XY-linker, and ending with a C-terminal C2 domain. The EF hands are responsible for the high Ca2+ sensitivity of PLCZ1. The X and Y catalytic domains are responsible for the catalysis of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] substrate to produce the Ca2+-mobilising messenger, inositol 1,4,5-trisphosphate (IP3), while the XY-linker plays multiple roles in the unique mode of PLCZ1 action. Finally, the C2 domain has been proposed to facilitate the anchoring of PLCZ1 to intracellular vesicles through its direct interactions with specific phosphoinositides. This review discusses recent advances in the structure and function relationship of PLCZ1 and the potential binding partners of this important sperm-specific protein in the sperm and oocyte. The unravelling of all the remaining hidden secrets of sperm PLCZ1 should help us to understand the precise mechanism of fertilization, as well as enabling the diagnosis and treatment of currently unknown forms of PLCZ1 -linked human infertility.

Genome-wide alternative splicing profile in the posterior kidney of brown trout (Salmo trutta) during proliferative kidney disease

BackgroundThe cnidarian myxozoan parasite Tetracapsuloides bryosalmonae causes chronic proliferative kidney disease (PKD) in salmonids. This parasite is a serious threat to wild and cultured salmonids. T. bryosalmonae undergoes intra-luminal sporogonic development in the kidney of brown trout (Salmo trutta) and the viable spores are released via urine. We investigated the alternative splicing pattern in the posterior kidney of brown trout during PKD.ResultsRNA-seq data were generated from the posterior kidney of brown trout collected at 12 weeks post-exposure to T. bryosalmonae. Subsequently, this data was mapped to the brown trout genome. About 153 significant differently expressed alternatively spliced (DEAS) genes, (delta PSI = 5%, FDR P-value < 0.05) were identified from 19,722 alternatively spliced events. Among the DEAS genes, the least and most abundant alternative splicing types were alternative 5′ splice site (5.23%) and exon skipping (70.59%), respectively. The DEAS genes were significantly enriched for sodium-potassium transporter activity and ion homeostasis (ahcyl1, atp1a3a, atp1a1a.1, and atp1a1a.5). The protein-protein interaction network analysis enriched two local network clusters namely cation transporting ATPase C-terminus and Sodium/potassium ATPase beta chain cluster, and mixed inclusion of Ion homeostasis and EF-hand domain cluster. Furthermore, the human disease-related salmonella infection pathway was significantly enriched in the protein-protein interaction network.ConclusionThis study provides the first baseline information about alternative splicing in brown trout during PKD. The generated data lay a foundation for further functional molecular studies in PKD - brown trout infection model. The information generated from the present study can help to develop therapeutic strategies for PKD in the future.

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure.

Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.

Structural and Functional Characterization of PLCβ3

Phospholipase C β (PLCβ) plays an important role in cardiovascular diseases and opioid analgesia. PLCβ catalyzes the hydrolysis of the inner membrane lipid phosphatidylinositol‐4,5‐bisphosphate (PIP2) to inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control responses to extracellular signals. PLCβ is a downstream effector of G‐protein coupled receptors (GPCRs) and is activated by both the Gαq and Gβγ subunits. Activation by Gα q requires a unique 400 amino acid C‐terminal region of the lipase, which is subdivided into the proximal and distal C‐terminal domains (CTDs). These domains are required for allosteric activation by Gα q and for membrane binding. Our lab and others have reported that PLCβ is more flexible in solution, as compared to crystal structures of the protein. In small angle X‐ray scattering (SAXS) experiments, the solution structure of PLCβ had additional density that cannot be accounted by the crystal structure. Additionally, crosslinking the PH and EF hand domains decreased this density and the maximum particle diameter. We propose that this additional density corresponds to an open conformation, wherein the PH and EF hand domains are extended from the core of the lipase, and that this conformation of PLCβ represents an autoinhibited state. To test this hypothesis, we mutated residues in PLCβ3 at the interface between the PH and EF hand domains to disrupt their interaction, and then measured changes in PLCβ basal activity and Gβγ‐stimulated activity. These mutations had no significant impact on basal activity, however in many of these mutants, there was a decrease in apparent Gβγ activation. Since these mutations should stabilize an open conformation of PLCβ, this suggests the open conformation has less ability to bind to or be activated by Gβγ. We are currently working to solve the structure of PLCβ3 in solution using single particle cryo‐electron microscopy (cryo‐EM), which will allow us to determine a solution structure of PLCβ to a higher resolution than SAXS studies. We hypothesize that PLCβ3 will exist in the open, autoinhibited conformation, and will also shed light on possible allosteric interactions between the distal CTD and the core.

What Is Parvalbumin for?

Parvalbumin (PA) is a small, acidic, mostly cytosolic Ca2+-binding protein of the EF-hand superfamily. Structural and physical properties of PA are well studied but recently two highly conserved structural motifs consisting of three amino acids each (clusters I and II), which contribute to the hydrophobic core of the EF-hand domains, have been revealed. Despite several decades of studies, physiological functions of PA are still poorly known. Since no target proteins have been revealed for PA so far, it is believed that PA acts as a slow calcium buffer. Numerous experiments on various muscle systems have shown that PA accelerates the relaxation of fast skeletal muscles. It has been found that oxidation of PA by reactive oxygen species (ROS) is conformation-dependent and one more physiological function of PA in fast muscles could be a protection of these cells from ROS. PA is thought to regulate calcium-dependent metabolic and electric processes within the population of gamma-aminobutyric acid (GABA) neurons. Genetic elimination of PA results in changes in GABAergic synaptic transmission. Mammalian oncomodulin (OM), the β isoform of PA, is expressed mostly in cochlear outer hair cells and in vestibular hair cells. OM knockout mice lose their hearing after 3–4 months. It was suggested that, in sensory cells, OM maintains auditory function, most likely affecting outer hair cells’ motility mechanisms.

Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM.

Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryogenic electron microscopy of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca2+ conditions revealed an open and a closed-inactivated conformation. Ca2+ binding to the high-affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca2+-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly knit subunit interface contributed by a fully occupied Ca2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca2+ activation prerequisite for Ca2+ inactivation and provides for a seamless transition from inactivated to closed conformations.

Disrupted regulation of voltage-gated calcium channels by pathogenic calmodulin variants

Calmodulin (CaM) is an essential sensor of intracellular calcium in multiple cell types, including cardiac myocytes and neurons. In these cells, calcium ions enter the cell through voltage-gated calcium channels (VGCCs) and bind to CaM in one of its four EF-hand domains. These binding events trigger an array of important downstream cellular processes, including calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF) of multiple VGCC subtypes. In this way, CaM tunes VGCC activity and facilitates calcium signaling cascades.

Distinct mechanism of interactions between NCS1 and Tb3+/Eu3+

Neuronal calcium sensor 1 (NCS1) is a highly conserved protein which is expressed in neurons mainly. NCS1, including with all NCS family members, contains four EF-hand domains. Ca2+ ion binds to three of the EF-hands with varying affinities except EF-hand 1. NCS1 undergoes binding with many intracellular proteins including AFR1, CaV2.1, D2R, InsP3Retc. It is involved in wide number of cellular functions exocytosis, neurite outgrowth, neuroprotection etc. NCS1 is found in association with several neuronal diseases for some vertebrates.

1H, 13C and 15N chemical shift backbone resonance NMR assignment of tobacco calmodulin 2.

Calcium is a ubiquitous second messenger regulating numbers of cellular processes in living organisms. It encodes and transmits information perceived by cells to downstream sensors, including calmodulin (CaM), that initiate cellular responses. In plants, CaM has been involved in the regulation of plant responses to biotic and abiotic environmental cues. Plant CaMs possess a cysteine residue in their first calcium-binding motif EF-hand, which is not conserved in other eucaryotic organisms. In this work, we report the near-complete backbone chemical shift assignment of tobacco CaM2 with calcium. These results will be useful to study the impact of this particular EF-hand domain regarding CaM interaction with partners involved in stress responses.

Genome Wide Identification of Respiratory Burst Oxidase Homolog (Rboh) Genes in Citrus sinensis and Functional Analysis of CsRbohD in Cold Tolerance.

Respiratory burst oxidase homologs (Rbohs) are critical enzymes involved in the generation of reactive oxygen species (ROS) that play an important role in plant growth and development as well as various biotic and abiotic stresses in plants. Thus far, there have been few reports on the characterization of the Rboh gene family in Citrus. In this study, seven Rboh genes (CsRbohA~CsRbohG) were identified in the Citrus sinensis genome. The CsRboh proteins were predicted to localize to the cell membrane. Most CsRbohs contained four conserved domains, an EF-hand domain, and a transmembrane region. Phylogenetic analysis demonstrated that the CsRbohs were divided into five groups, suggesting potential distinct functions and evolution. The expression profiles revealed that these seven CsRboh genes displayed tissue-specific expression patterns, and five CsRboh genes were responsive to cold stress. Fourteen putative cis-acting elements related to stress response, hormone response, and development regulation were present within the promoters of CsRboh genes. The in-silico microRNA target transcript analyses indicated that CsRbohE might be targeted by csi-miR164. Further functional and physiological analyses showed that the knockdown of CsRbohD in trifoliate orange impaired resistance to cold stress. As a whole, our results provide valuable information for further functional studies of the CsRboh genes in response to cold stress.

Electron cryo-tomography structure of axonemal doublet microtubule from Tetrahymena thermophila.

Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, collectively named the microtubule inner proteins (MIPs). We used cryoET to study axoneme DMT isolated from Tetrahymena We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP RIB72A/B binds to the luminal wall of DMT by multiple DM10 domains. We found FAP115, an MIP-containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of A-tubule. It contacts both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. We observed substantial structure heterogeneity in DMT in an FAP115 knockout strain, showing extensive structural defects beyond the FAP115-binding site. The defects propagate along the axoneme. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

Functional characterization of myosin III light chain b of sea perch (Lateolabrax japonicus) in response to fish virus infection

Myosins are key motor proteins of vertebrates that are important in cell-cell adhesion and migration; they are associated with viral infections. Herein, a myosin III light chain b gene from sea perch (Lateolabrax japonicus), designated Ljmyo3b, was characterized and its positive role in fish virus pathogenesis was revealed. The cDNA of Ljmyo3b encoded a 150-amino acid protein with two classical EF-hand domains; it shared the closest genetic relationship with Larimichthys crocea myo3b. Cellular distribution analysis showed that Ljmyo3b was localized in the cytoplasm and nucleus of LJB cells, and also on the cell surface, which was further confirmed by the protease protection assay. Spatial expression analysis revealed that Ljmyo3b was more abundant in the immune tissues of sea perch post red spotted grouper nervous necrosis virus (RGNNV) or viral hemorrhagic septicemia virus (VHSV) infection. In RGNNV- and VHSV-treated LJB cells, Ljmyo3b expression was significantly upregulated at 2, 4, 24, and 48 hpi. Furthermore, the ectopic expression of Ljmyo3b facilitated RGNNV and VHSV infection in LJB cells at 2, 4, and 24 hpi. Taken together, these results suggest that Ljmyo3b is involved in mediating RGNNV and VHSV infection in fish.

Astragalus polysaccharide improve the proliferation and insulin secretion of mouse pancreatic β cells induced by high glucose and palmitic acid partially through promoting miR-136-5p and miR-149-5p expression

ABSTRACT The current drugs for the treatment of type 2 diabetes mellitus (T2DM) can cause side effects after long-time use. Hence, the novel drugs were urgent need to developed for T2DM patients. In this study, the effect of astragalus polysaccharide on dysfunctional insulin cells was investigated to clarify whether astragalus polysaccharide could be a novel drug for T2DM treatment. MIN6 cells (mouse pancreatic β-cell line) were treated with high glucose (HG)+ palmitic acid (PA) and then treated with astragalus polysaccharide. The proliferation, apoptosis, and insulin secretion were measured using CCK8, flow cytometry, and ELISA, respectively. Pancreatic and duodenal homeobox 1 (PDX1), miR-136-5p, and miR-149-5p expression levels were measured by RT-qPCR. The combination of EF-hand domain family member 2 (EFHD2) and miR-136-5p or miR-149-5p was analyzed by luciferase reporter assay. EFHD2 protein level was measured by western blot. We found that HG+PA treatment reduced MIN6 cell viability, insulin secretion, and PDX1 expression and promoted MIN6 cell apoptosis. Astragalus polysaccharide treatment reversed the effect of HG+PA on MIN6 cells. Additionally, astragalus polysaccharide treatment promoted miR-136-5p and miR-149-5p expression. Silencing of miR-136-5p and miR-149-5p expression partially reversed the therapeutic effects of astragalus polysaccharide. Furthermore, EFHD2 was the target of miR-136-5p and miR-149-5p. Meanwhile, astragalus polysaccharide treatment inhibited EFHD2 protein level in HG+PA treated MIN6 cell. Finally, EFHD2 overexpression partially reversed the therapeutic effects of astragalus polysaccharide. In conclusion, astragalus polysaccharide treatment improved proliferation and insulin secretion in HG+PA-treated MIN6 cells partially by promoting miR-136-5p and miR-149-5p expression to inhibit EFHD2 expression.

Voltage-gating and cytosolic Ca2+ activation mechanisms of Arabidopsis two-pore channel AtTPC1

Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca2+-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca2+ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca2+ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca2+-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca2+ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.