Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze–fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)–plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze–fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic “particles”; TG treatment led to aggregation of these particles into sharply delimited “puncta” positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.Specialized junctions linking the endoplasmic reticulum (ER) to the plasma membrane (PM) were first described by Porter and Palade (1) in skeletal and cardiac muscle. In skeletal muscle, excitation–contraction coupling is mediated by direct physical contact between voltage-gated Ca²⁺ channels (dihydropyridine receptors) in invaginated transverse tubules of the PM and Ca²⁺-release channels (ryanodine receptors) in ER membrane (2). A second type of ER-PM junction mediates inside-out signaling by linking depletion of Ca²⁺ in the ER lumen to Ca²⁺ influx across the PM in a process termed store-operated Ca²⁺ entry (SOCE). In addition to being a mechanism of ionic homeostasis, SOCE supports long-lasting Ca²⁺ signals in many cell types. ER stromal interacting molecule (STIM) and PM Orai proteins were identified by RNAi screening as required for SOCE (3–7). Overexpression of both proteins is required to amplify Ca²⁺ influx through Orai channels (7–10). In Drosophila, STIM and Orai are the sole members of a gene family, which in mammals, includes two STIM and three Orai proteins. STIM1 and Orai1 are predominant in the immune system; human mutations in either gene can cause lethal severe combined immune deficiencies (SCID) (11). ER STIM proteins trigger SOCE by sensing ER Ca²⁺ store depletion, translocating as oligomers to the PM, and binding to PM Orai proteins to promote clustering and channel opening (3, 12–16). These events have been extensively documented by microscopy of cells expressing fluorescently tagged proteins. Numerous studies have defined domains and amino acid residues of STIM1 and Orai1 that are vital for channel function (17, 18).ER-PM junctions underlying SOCE have been visualized by electron microscopy (EM), using either HRP-tagged STIM1 (13, 19) or immunogold labeling of STIM1 (20). However, little is known about the nanometer-scale subcellular organization of STIM and Orai proteins, although they define a basic unit of Ca²⁺ signaling. Here, through a close examination of transmission and freeze–fracture electron micrographs of transfected cells expressing STIM1 and Orai1, we further define the microanatomy of the ER-PM, as well as of ER-ER junctions in store-depleted and untreated cells. These images provide direct candidate signatures for STIM1 molecules bridging the ER-PM and ER-ER gaps and for individual Orai1 channels in puncta. Taken together, our observations provide visual confirmation of STIM1 and Orai1 function, constrain models of STIM1 and Orai1 assembly and interaction, and suggest new aspects of molecular interactions between STIM1 and Orai1.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.     

POST, partner of stromal interaction molecule 1 (STIM1), targets STIM1 to multiple transporters

Specialized proteins in the plasma membrane, endoplasmic reticulum (ER), and mitochondria tightly regulate intracellular calcium. A unique mechanism called store-operated calcium entry is activated when ER calcium is depleted, serving to restore intra-ER calcium levels. An ER calcium sensor, stromal interaction molecule 1 (STIM1), translocates within the ER membrane upon store depletion to the juxtaplasma membrane domain, where it interacts with intracellular domains of a highly calcium-selective plasma membrane ion channel, Orai1. STIM1 gates Orai1, allowing calcium to enter the cytoplasm, where it repletes the ER store via calcium-ATPases pumps. Here, we performed affinity purification of Orai1 from Jurkat cells to identify partner of STIM1 (POST), a 10-transmembrane-spanning segment protein of unknown function. The protein is located in the plasma membrane and ER. POST-Orai1 binding is store depletion-independent. On store depletion, the protein binds STIM1 and moves within the ER to localize near the cell membrane. This protein, TMEM20 (POST), does not affect store-operated calcium entry but does reduce plasma membrane Ca(2+) pump activity. Store depletion promotes STIM1-POST complex binding to smooth ER and plasma membrane Ca(2+) ATPases (SERCAs and PMCAs, respectively), Na/K-ATPase, as well as to the nuclear transporters, importins-β and exportins.     

Stromal Interaction Molecule 1 Promotes the Replication of vvIBDV by Mobilizing Ca2+ in the ER

Infectious bursal disease virus (IBDV) is one of the main threats to the poultry industry worldwide. Very virulent IBDV (vvIBDV) is a fatal virus strain that causes heavy mortality in young chicken flocks. Ca²⁺ is one of the most universal and versatile signalling molecules and is involved in almost every aspect of cellular processes. Clinical examination showed that one of the characteristics of vvIBDV-infected chickens was severe metabolic disorders, and the chemical examination showed that their serum Ca²⁺ level decreased significantly. However, there are limited studies on how vvIBDV infection modulates the cellular Ca²⁺ level and the effect of Ca²⁺ level changes on vvIBDV replication. In our study, we found Ca²⁺ levels in the endoplasmic reticulum (ER) of vvIBDV-infected B cells were higher than that of mock-infected cells, and the expression level of stromal interaction molecule 1 (STIM1), an ER Ca²⁺ sensor, was significantly upregulated due to vvIBDV infection. The knock-down expression of STIM1 led to decreased Ca²⁺ level in the ER and suppressed vvIBDV replication, while the over-expressed STIM1 led to ER Ca²⁺ upregulation and promoted vvIBDV replication. We also showed that the inhibition of Ca²⁺-release-activated-Ca²⁺ (CRAC) channels could reduce vvIBDV infection by blocking Ca²⁺ from entering the ER. This study suggests a new mechanism that STIM1 promotes the replication of vvIBDV by mobilizing Ca²⁺ in the ER.     

STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels

Ca²⁺-dependent inactivation (CDI) is a key regulator and hallmark of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, a prototypic store-operated Ca²⁺ channel. Although the roles of the endoplasmic reticulum Ca²⁺ sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342–448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470–491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68–91) that binds CaM in a Ca²⁺-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.     

Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1(-/-) mice

Agonist-induced Ca(2+) entry via store-operated Ca(2+) (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(-/-) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(-/-) mice. Deletion of TRPC1 also eliminated sustained Ca(2+)-dependent potassium channel activity, which depends on Ca(2+) entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca(2+) signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(-/-) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.     

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na⁺/Ca²⁺ exchanger (NCX) occurs via its cytoplasmic Ca²⁺-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca²⁺-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca²⁺-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca²⁺. Striking differences in the electrostatic potential between the Ca²⁺-bound and -free forms strongly suggest that Ca²⁺-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C₂-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca²⁺-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca²⁺ is influenced by the closely located cassette exons. We further propose that Ca²⁺-binding to CBD2 induces a second electrostatic switch, required to alleviate Na⁺-dependent inactivation of Na⁺/Ca²⁺ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.     

Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1)

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.     

Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal endocytosis

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca²⁺ plays critical roles in exocytosis–endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca²⁺ in endocytosis have been reported; however, Ca²⁺ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca²⁺ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis–endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca²⁺-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca²⁺-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca²⁺ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.

Endocytosis and subsequent vesicle recycling are spatiotemporally coupled to exocytosis, which is critical for neurons and endocrinal cells to maintain the integrity of plasma membrane architecture, intracellular homeostasis, and sustained neurotransmission (1–3). In addition to triggering vesicular exocytosis, neural activity/Ca²⁺ also play an executive role in the coupling of endocytosis to exocytosis (1, 2, 4–6). Following a pioneering study 40 y ago (7), extensive studies have been conducted and showed that Ca²⁺ triggers and facilitates vesicle endocytosis in neurons and nonneuronal secretory cells (1, 8–11). Accumulating evidence also shows that intracellular Ca²⁺ may inhibit endocytosis (12–15), which has been challenged greatly due to the apparently lower occurrences in few preparations and the missing underlining mechanisms, making the endocytic role of Ca²⁺ a four-decades–long dispute (1, 2, 4, 6).Machineries and regulators involved in exocytosis–endocytosis coupling have been extensively studied for over 30 y. The soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) and synaptophysin play critical dual roles in exocytosis and endocytosis during neurotransmission (2, 3, 16, 17). Calmodulin and synaptotagmin-1 (Syt1) are currently known primary Ca²⁺ sensors facilitating endocytosis (1, 9, 16, 18, 19). Ca²⁺/calmodulin activate calcineurin, which dephosphorylates endocytic proteins (e.g., dynamin, synaptojanin, and amphiphysin) to facilitate clathrin-mediated endocytosis (CME) and clathrin-independent fast endocytosis (1, 2). Syt1 is a dual Ca²⁺ sensor for both exocytosis and endocytosis (5, 16, 18–20). It promotes CME through binding with the endocytic adaptors adaptor protein-2 (AP-2) and stonin-2 (21–24). In contrast to the well-established Ca²⁺ sensors that promote endocytosis, the mechanism of Ca²⁺-dependent inhibition in endocytosis remains unknown.CME is the classical but slow endocytosis pathway for vesicle retrieval under resting conditions or in response to mild stimulation, while the accumulated Ca²⁺ also triggers calmodulin/calcineurin-dependent bulk endocytosis, which takes up a large area of plasma membrane to fulfill the urgent requirement for high-speed vesicle exocytosis (1–3). They cooperate with kiss-and-run and ultrafast endocytosis to ensure both sufficient and precise membrane retrieval following exocytosis (3, 25–27). These endocytic pathways are all initiated from membrane invagination and are critically controlled by neural activity. However, how the switch between different endocytic modes is precisely determined remains largely unknown.Here, by combining electrophysiological recordings, confocal live imaging, superresolution stimulated emission depletion (STED) imaging, in vitro liposome manipulation, and electron microscope imaging of individual endocytic vesicles, we define Syt1 as a primary and bidirectional Ca²⁺ sensor for endocytosis, which promotes CME but inhibits bulk endocytosis, probably by mediating membrane remodeling. The balance between the facilitatory and inhibitory effects of Syt1 on endocytosis offers a fine-tuning mechanism to ensure both efficient and precise coupling of endocytosis to exocytosis. By including a non-Ca²⁺–binding Syt as the constitutive brake, this work also explains the four-decades–long puzzle about the positive and negative Ca²⁺ effects on endocytosis.     

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.

Voltage-gated ion channels (VGICs), such as voltage-gated potassium channel (Kv), sodium channel (Nav), and calcium channel (Cav), are activated by depolarization of membrane potential and play essential roles in electrical signal transduction (1–3). VGICs sense the membrane potential by voltage-sensing domains (VSDs), which consist of four transmembrane helices S1 to S4. In most VGICs, VSDs are stabilized in the resting state by hyperpolarizing (negative) membrane potential, and the channel gate stays closed. At depolarizing (relatively positive) membrane potential, VSDs are activated, and their depolarization-induced conformational changes are coupled to the S5–S6 pore domain, resulting in the opening of the channel gate.While dozens of VGIC structures have been determined over the past two decades, only very few VSDs in these structures were captured in the resting state. That is because VGICs for structural studies in vitro are solubilized in detergent micelle or lipid nanodisc, making it difficult to recapitulate the resting state under hyperpolarizing (negative) membrane potential. The structure of plant two-pore channel (TPC) from Arabidopsis thaliana (AtTPC1) was among the first to capture a resting-state VSD (4, 5). In addition, mutagenesis combined with cross-linking, ion bridge, or toxin binding have been used to trap the structures of VGICs in resting state in several recent studies, including structures of the bacterial sodium channel NavAb (6), the eukaryotic sodium channel chimera Nav1.7-NavPaS (7), and the hyperpolarization-activated cyclic nucleotide-gated ion channel HCN (8). To fully understand the similarities and differences of the voltage-gating mechanism among different VGICs, it will be essential to visualize the structures of various VGICs in both activated and resting state. To this end, we are using AtTPC1 as a model to elucidate the structural mechanism of voltage gating.TPCs belong to the VGIC superfamily and are ubiquitously expressed in animals and plants (9, 10). While animal TPCs (TPC1 and TPC2) are endolysosomal sodium channels, the plant TPC (TPC1) is a nonselective cation channel responsible for generating the slow vacuole (SV) current (11, 12). TPCs function as homodimer with each subunit comprising two homologous Shaker-like 6-transmembrane segment domains (6-TM I and 6-TM II) (9, 10, 13), thereby equivalent to a classical VGIC with four VSDs and one pore domain.Plant TPC is involved in many important physiological processes, such as germination and stomatal opening (12), jasmonate biosynthesis (14, 15), modulation of Ca²⁺ waves induced by salinity stress (16), and plant–pathogen interaction (17). AtTPC1, the most well-studied plant TPC from A. thaliana, is activated by the membrane depolarization and cytosolic Ca²⁺ but inhibited by vacuolar Ca²⁺ (18, 19). Previously, we determined the crystal structure of AtTPC1 in closed state (Protein Data Bank [PDB]: 5E1J, AtTPC1_5E1J) (4). We demonstrated that between the two VSDs within each AtTPC1 subunit, only the second one (VSDII) senses the membrane potential and adopts a resting state in the structure whereas the first one (VSDI) lacks several key features essential for voltage sensing and therefore does not contribute to the voltage-dependent gating. Ca²⁺ activation occurs at the EF-hand domain containing two EF-hand motifs. However, Ca²⁺ binding at EF hand 1 appears to play a structural role and does not contribute to Ca²⁺ activation; Ca²⁺ binding at EF hand 2 is central for Ca²⁺ activation and it adopts an unbound state in the structure (4, 19). We also identified the luminal divalent inhibition site in AtTPC1 where Ca²⁺ or Ba²⁺ binding can stabilize the voltage-sensing VSDII in a resting state. Based on our structural and electrophysiological analysis, we proposed that the conformational changes triggered by the binding of Ca²⁺ to cytosolic EF-hand domain are coupled with the pair pore-lining inner helices from the 6-TM I (IS6), whereas the conformational changes of VSDII activated by membrane potential are coupled with the pair of inner helices from the 6-TM II (IIS6) (4). In order to understand the structural basis underlying multistimuli gating of AtTPC1, here we determined AtTPC1 structures in both closed and partially open conformation under different Ca²⁺ conditions, revealing the structural mechanism of voltage gating and Ca²⁺ modulation of AtTPC1.     

From the Cover: Salt stress-induced Ca2+ waves are associated with rapid,long-distance root-to-shoot signaling in plants

Their sessile lifestyle means that plants have to be exquisitely sensitive to their environment, integrating many signals to appropriate developmental and physiological responses. Stimuli ranging from wounding and pathogen attack to the distribution of water and nutrients in the soil are frequently presented in a localized manner but responses are often elicited throughout the plant. Such systemic signaling is thought to operate through the redistribution of a host of chemical regulators including peptides, RNAs, ions, metabolites, and hormones. However, there are hints of a much more rapid communication network that has been proposed to involve signals ranging from action and system potentials to reactive oxygen species. We now show that plants also possess a rapid stress signaling system based on Ca²⁺ waves that propagate through the plant at rates of up to ∼400 µm/s. In the case of local salt stress to the Arabidopsis thaliana root, Ca²⁺ wave propagation is channeled through the cortex and endodermal cell layers and this movement is dependent on the vacuolar ion channel TPC1. We also provide evidence that the Ca²⁺ wave/TPC1 system likely elicits systemic molecular responses in target organs and may contribute to whole-plant stress tolerance. These results suggest that, although plants do not have a nervous system, they do possess a sensory network that uses ion fluxes moving through defined cell types to rapidly transmit information between distant sites within the organism.Plants are constantly tailoring their responses to current environmental conditions via a complex array of chemical regulators that integrate developmental and physiological programs across the plant body. Environmental stimuli are often highly localized in nature, but the subsequent plant response is often elicited throughout the entire organism. For example, soil is a highly heterogeneous environment and the root encounters stimuli that are presented in a patchy manner. Thus, factors including dry or waterlogged regions of the soil, variations in the osmotic environment, and stresses such as elevated levels of salt are all likely to be encountered locally by individual root tips, but the information may have to be acted on by the plant as a whole.In animals, long-range signaling to integrate activities across the organism occurs through rapid ionic/membrane potential-driven signaling through the nervous system in addition to operating via long-distance chemical signaling. Plants have also been proposed to possess a rapid, systemic communication network, potentially mediated through signals ranging from changes in membrane potential/ion fluxes (1–3) and levels of reactive oxygen species (ROS) (4, 5) to altered hydraulics in the vasculature (6). Even so, the molecular mechanisms behind rapid, systemic signaling in plants and whether such signals indeed carry regulatory information remains largely unknown. Suggestions that Ca²⁺ channels play a role in signals that occlude sieve tube elements (7), or that mediate systemic electrical signaling (2) in response to remote wounding, highlight Ca²⁺-dependent signaling events as a strong candidate for mediating some of these long-range responses. Similarly, cooling of roots elicits Ca²⁺ increases in the shoot within minutes (8), suggesting systemic signals can elicit Ca²⁺-dependent responses at distal sites within the plant. However, despite extensive characterization of Ca²⁺ signals (reviewed in ref. 9), their roles in a possible plant-wide communication network remain poorly understood. Therefore, to visualize how Ca²⁺ might act in local and systemic signaling, we generated Arabidopsis plants expressing the highly sensitive, GFP-based, cytoplasmic Ca²⁺ sensor YCNano-65 (10). We observed that a range of abiotic stresses including H₂O₂, touch, NaCl, and cold shock triggered Ca²⁺ increases at the point of application. However, NaCl also elicited a Ca²⁺ increase that moved away from the point of stress application. Propagation of this Ca²⁺ increase was associated with subsequent systemic changes in gene expression. We also report that this salt stress-induced long-distance Ca²⁺ wave is dependent on the activity of the ion channel protein Two Pore Channel 1 (TPC1), which also appears to contribute to whole-plant stress tolerance.     

Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions,regulates Ca2+ dynamics in T cells

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca²⁺ entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca²⁺ sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER–PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER–PM junctions to regulate Ca²⁺ signaling. Silencing or genetic manipulation of JP4 decreased ER Ca²⁺ content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca²⁺-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate–JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca²⁺ homeostasis and mediate SOCE in T cells.The endoplasmic reticulum (ER)–plasma membrane (PM) junctions are ubiquitous structures essential for intermembrane communications (1–3). These junctions play an important role in lipid transfer and regulation of Ca²⁺ dynamics, including ER Ca²⁺ homeostasis and Ca²⁺ entry after receptor stimulation (1, 4). Four major categories of components of the ER–PM junctions have been identified so far: (i) dyad/triad junctional proteins in the heart and skeletal muscle (e.g., junctophilins and junctin), (ii) ER-resident vesicle-associated membrane protein-associated proteins (VAPs) that form the lipid transfer machinery by interacting with phospholipid-binding proteins, (iii) extended synaptogamin-like proteins (E-Syts) that tether membranes, and (iv) the Orai1–stromal interaction molecule 1 (STIM1) complex that forms the primary Ca²⁺ channel in T cells, the Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Among these proteins, the dyad/triad junctional proteins and the Orai1–STIM1 complex are known to play a crucial role in Ca²⁺ dynamics, including excitation–contraction coupling in muscle and store-operated Ca²⁺ entry (SOCE) in immune cells, respectively (2, 5).Stimulation of T-cell receptors (TCRs) triggers activation of SOCE primarily mediated by the PM-resident Orai1 channels and ER-resident STIM1 protein that senses ER Ca²⁺ concentration (6–11). Upon store depletion, STIM1 translocates and interacts with Orai1 at the preformed ER–PM junctions (12, 13). STIM1 uses two major mechanisms to translocate into the ER–PM junctions: by interactions with phosphatidylinositol-4,5-bisphosphate (PIP₂) in the PM via its C-terminal polybasic residues and by interaction with Orai1 or the ER-resident junctate proteins (14, 15). Recently, septin filaments were shown to play a role in PIP₂ enrichment at the ER–PM junctions before STIM1 recruitment (16). Subsequently, membrane-tethering VAP and E-Syt proteins were shown to be important for PIP₂ replenishment after store depletion (17). The importance of protein interaction in STIM1 recruitment was demonstrated by a STIM1ΔK mutant truncated in its C-terminal polybasic domain. Interaction with Orai1 or junctate facilitated recruitment of this PIP₂ binding-deficient mutant into the junctions (15, 18, 19). It was thought that the roles of dyad/triad junctional proteins are limited to muscle cells. However, identification of junctate as a STIM1-interacting partner implied that some components (or homologs) of ER–PM junctions in excitable cells may be shared in immune cells.The junctophilin family consists of four genes (JP1, JP2, JP3, and JP4) that are expressed in a tissue-specific manner and are known to form ER–PM junctions in excitable cells (20, 21). Junctophilins contain eight repeats of the membrane occupation and recognition nexus (MORN) motifs that bind to phospholipids in the N terminus and a C-terminal ER membrane-spanning transmembrane segment (20, 22). In this study, we observed expression of JP4 in both human and mouse T cells, which was further enhanced by TCR stimulation. Depletion or deficiency of JP4 reduced ER Ca²⁺ content, SOCE, and activation of the nuclear factor of activated T cells (NFAT) and ERK mitogen-activated protein kinase (MAPK) pathways. Mechanistically, JP4 depletion reduced accumulation of STIM1 at the junctions without affecting the number and length of the ER–PM junctions. We observed a direct interaction between the cytoplasmic regions of JP4 and STIM1, and, correspondingly, overexpression of the STIM1-interacting JP4 fragment had a dominant negative effect on SOCE. Finally, we identified a protein complex consisting of JP4 and junctate at the ER–PM junctions, which may have a synergistic effect in recruiting STIM1 to the junctions. Therefore, our studies identify a PIP₂-independent, but protein interaction-mediated, mechanism by which the junctate–JP4 complex recruits STIM1 into the ER–PM junctions to maintain ER Ca²⁺ homeostasis and activate SOCE in T cells.     

Diabetes-induced activation of protein kinase C inhibits store-operated Ca<Superscript>2+</Superscript> uptake in rat retinal microvascular smooth muscle

Aims/Hypothesis To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca²⁺ influx pathways in retinal microvascular smooth muscle cells.Methods Cytosolic Ca²⁺ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca²⁺ influx was tested by measuring rises in [Ca²⁺]_i with KCl (100 mmol/l) and store-operated Ca²⁺ influx was assessed by depleting [Ca²⁺]_i stores with Ca²⁺ free medium containing 5 µmol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca²⁺ on adding 2 mmol/l or 10 mmol/l Ca²⁺solution.Results Ca²⁺ entry through voltage-dependent L-type Ca²⁺ channels was unaffected by diabetes. In contrast, store-operated Ca²⁺ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca²⁺ influx. Diabetic rats injected daily with insulin had store-operated Ca²⁺ influx rates similar to non-diabetic control rats. The reduced Ca²⁺ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKC antagonist LY379196 (100 nmol/l) also reversed the poor Ca²⁺ influx although its action was less efficacious than staurosporine.Conclusion/interpretation These results show that store-operated Ca²⁺ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKC seems to play a role in mediating this effect.Abbreviations DAG Diacylglycerol - PKC protein kinase C - [Ca²⁺]_i intracellular calcium concentration - STZ streptozotocin - SPP staurosporine - SR sarcoplasmic reticulum - MVSM microvascular smooth muscle - CPA cyclopiazonic acid - PMA phorbol myristate acetate - VDCC voltage-dependent Ca²⁺ channels     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.