Bidirectional Allosteric Coupling between PIP2 Binding and the Pore of the Oncochannel TRPV6.

The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.

Nanojunctions: Specificity of Ca2+ signaling requires nano-scale architecture of intracellular membrane contact sites.

Spatio-temporal definition of Ca2+ signals involves the assembly of signaling complexes within the nano-architecture of contact sites between the sarco/endoplasmic reticulum (SR/ER) and the plasma membrane (PM). While the requirement of precise spatial assembly and positioning of the junctional signaling elements is well documented, the role of the nano-scale membrane architecture itself, as an ion-reflecting confinement of the signalling unit, remains as yet elusive. Utilizing the Na+/Ca2+ Exchanger-1 / SR/ER Ca2+ ATPase-2-mediated ER Ca2+ refilling process as a junctional signalling paradigm, we provide here the first evidence for an indispensable cellular function of the junctional membrane architecture. Our stochastic modeling approach demonstrates that junctional ER Ca2+ refilling operates exclusively at nano-scale membrane spacing, with a strong inverse relationship between junctional width and signaling efficiency. Our model predicts a breakdown of junctional Ca2+ signaling with loss of reflecting membrane confinement. In addition we consider interactions between Ca2+ and the phospholipid membrane surface, which may support interfacial Ca2+ transport and promote receptor targeting. Alterations in the molecular and nano-scale membrane organization at organelle-PM contacts are suggested as a new concept in pathophysiology.

Calcium transport and sensing in TRPC channels - New insights into a complex feedback regulation.

Canonical TRP (TRPC) channels are a still enigmatic family of signaling molecules with multimodal sensing features. These channels enable Ca2+ influx through the plasma membrane to control a diverse range of cellular functions. Based on both regulatory- and recently uncovered structural features, TRPC channels are considered to coordinate Ca2+ and other divalent cations not only within the permeation path but also at additional sensory sites. Analysis of TRPC structures by cryo-EM identified multiple regulatory ion binding pockets. With this review, we aim at an overview and a critical discussion of the current concepts of divalent sensing by TRPC channels.

Arriving at the next level of complexity in IP3R and SOCE signaling.

The endoplasmic reticulum (ER) has long been recognized as the master regulator of cellular Ca2+ signaling. In this context, IP3R channels may be envisioned as this conductor's baton, which enables virtuous orchestration of cellular Ca2+ signaling tunes. IP3Rs serve the generation of spatiotemporally defined Ca2+ changes and are key for the ER´s function as an autonomous Ca2+ signaling unit, which is able to govern its own refilling from the extracellular Ca2+ pool. As yet, IP3R signaling has been primarily attributed to its precisely-tunable Ca2+ channel function and IP3-mediated control over Ca2+ levels within signaling domains. A recent report from the Hasan laboratory [1] provides evidence for an as yet overlooked function of IP3R1 in terms of supporting STIM/Orai-mediated SOCE in neurons. IP3R1 is demonstrated to remarkably facilitate productive STIM-Orai interactions and SOCE by a process that is triggered by IP3 but independent of the receptors' function as an ER Ca2+ channel.

TRPC3 Regulates Islet Beta-Cell Insulin Secretion.

Insulin release is tightly controlled by glucose-stimulated calcium (GSCa) through hitherto equivocal pathways. This study investigates TRPC3, a non-selective cation channel, as a critical regulator of insulin secretion and glucose control. TRPC3's involvement in glucose-stimulated insulin secretion (GSIS) is studied in human and animal islets. TRPC3-dependent in vivo insulin secretion is investigated using pharmacological tools and Trpc3-/- mice. TRPC3's involvement in islet glucose uptake and GSCa is explored using fluorescent glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose and calcium imaging. TRPC3 modulation by a small-molecule activator, GSK1702934A, is evaluated in type 2 diabetic mice. TRPC3 is functionally expressed in human and mouse islet beta cells. TRPC3-controlled insulin secretion is KATP -independent and primarily mediated by diacylglycerol channel regulation of the cytosolic calcium oscillations following glucose stimulation. Conversely, glucose uptake in islets is independent of TRPC3. TRPC3 pharmacologic inhibition and knockout in mice lead to defective insulin secretion and glucose intolerance. Subsequently, TRPC3 activation through targeted small-molecule enhances insulin secretion and alleviates diabetes hallmarks in animals. This study imputes a function for TRPC3 at the onset of GSIS. These insights strengthen one's knowledge of insulin secretion physiology and set forth the TRPC3 channel as an appealing candidate for drug development in the treatment of diabetes.

TRPC3 governs the spatiotemporal organization of cellular Ca2+ signatures by functional coupling to IP3 receptors.

Communication between TRPC channels and IP3 receptors (IP3R) is considered pivotal in the generation of spatiotemporal Ca2+signaling patterns. Here we revisited the role of TRPC3-IP3R coupling for local Ca2+ signaling within TRPC3-harbouring micro/nanodomains using R-GECO as a reporter, fused to the channel´s C-terminus. Cytoplasmic Ca2+ changes at TRPC3 originated from both the entry of Ca2+ through the TRPC channel and Ca2+ mobilization via IP3R. Local Ca2+ changes at TRPC3 channels expressed in HEK293 cells were predominantly biphasic with IP3R-dependent initial Ca2+ transients, while exclusively monophasic signals were recorded when all three IP3R isoforms were lacking. Abrogation of Ca2+ entry through TRPC3 by point mutations, which impair Ca2+ permeability (E630Q), cation permeation (E630K), or DAG sensitivity (G652A), promoted microdomain Ca2+ oscillations. Ca2+ signals at E630Q, E630K, and G652A channels featured initial Ca2+ transients along with oscillatory activity. Similarly, when extracellular Ca2+ was omitted, IP3R-mediated Ca2+ transients and Ca2+ oscillations were promoted at the cytoplasmic face of wild-type TRPC3 channels. By contrast, oscillations, as well as initial Ca2+ transients, were virtually lacking, when the TRPC3 channels were sensitized by preexposure to low-level PLC activity. TIRF imaging provided evidence for dynamic colocalization of TRPC3 and IP3R. We suggest that TRPC3-mediated Ca2+ entry controls IP3R activity at ER-PM junctions to determine Ca2+ signaling signatures and enable specificity of downstream signaling.

Differences in circulating obesity-related microRNAs in Austrian and Japanese men: A two-country cohort analysis.

Background: The prevalence of obesity is higher in Western countries than in East Asian countries. It remains unknown whether microRNAs (miRNAs) are involved in the pathogenesis of the ethnic difference in obesity. The purpose of this study was to determine whether expression levels of circulating obesity-associated miRNAs are different in Europeans and Asians. Methods: The subjects were middle-aged healthy male Austrians (n = 20, mean age of 49.9 years) and Japanese (n = 20, mean age of 48.7 years). Total miRNAs in serum from each subject were analyzed using the 3D-Gene miRNA Oligo chip. miRNAs that showed significant differences between the Austrian and Japanese groups were uploaded into Ingenuity Pathway Analysis (IPA). Results: Among 16 miRNAs that were revealed to be associated with obesity in previous studies and showed expression levels that were high enough for a reasonable comparison, serum levels of 3 miRNAs displayed significant differences between the Austrian and Japanese groups: miR-125b-1-3p was significantly lower with a fold change of -2.94 and miR-20a-5p and miR-486-5p were significantly higher with fold changes of 1.73 and 2.38, respectively, in Austrians than in Japanese. In IPA including all 392 miRNAs that showed significant differences between Austrians and Japanese, three canonical pathways including leptin signaling in obesity, adipogenesis pathway and white adipose tissue browning pathway were identified as enriched pathways. Conclusions: miRNAs are thought to be involved in the ethnic difference in the prevalence of obesity, which may in part be caused by different expression levels of miR-125b-1-3p, miR-20a-5p and miR-486-5p.

Exploring TRPC3 Interaction with Cholesterol through Coarse-Grained Molecular Dynamics Simulations.

Transient receptor potential canonical 3 (TRPC3) channel belongs to the superfamily of transient receptor potential (TRP) channels which mediate Ca2+ influx into the cell. These channels constitute essential elements of cellular signalling and have been implicated in a wide range of diseases. TRPC3 is primarily gated by lipids and its surface expression has been shown to be dependent on cholesterol, yet a comprehensive exploration of its interaction with this lipid has thus far not emerged. Here, through 80 µs of coarse-grained molecular dynamics simulations, we show that cholesterol interacts with multiple elements of the transmembrane machinery of TRPC3. Through our approach, we identify an annular binding site for cholesterol on the pre-S1 helix and a non-annular site at the interface between the voltage-sensor-like domain and pore domains. Here, cholesterol interacts with exposed polar residues and possibly acts to stabilise the domain interface.

Characterization of DAG Binding to TRPC Channels by Target-Dependent cis-trans Isomerization of OptoDArG.

Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel-lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable cis conformation to the trans conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand-protein interactions. Cis photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal cis-trans isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by cis PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), cis OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of cis OptoDArG-induced currents in the dark was sensitive to mutations in the L2 lipid coordination site of TRPC channels. We conclude that the binding of cis OptoDArG to TRPC channels promotes transition of cis OptoDArG to the trans conformation. This process is suggested to provide valuable information on DAG-ion channel interactions and may enable highly selective photopharmacological interventions.

Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state.

Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca2+ signaling function. Single particle cryo-EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure-guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG-probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C-DAG pathway.

Two-Dimensional Interfacial Exchange Diffusion Has the Potential to Augment Spatiotemporal Precision of Ca2+ Signaling.

Nano-junctions between the endoplasmic reticulum and cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca2+ signals. Herein, we propose that 2D Ca2+ exchange diffusion on the negatively charged phospholipid surface lining nano-junctions participates in guiding Ca2+ from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca2+ flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca2+ trajectories within nano-junctions are discussed in order to substantiate its possible requirements. Finally, we analyze recent literature on Ca2+ lipid interactions, which suggests that 2D interfacial Ca2+ diffusion may represent an important mechanism of signal transduction in biological systems characterized by high phospholipid surface to aqueous volume ratios.

TRPC3, an underestimated, universal pacemaker channel?

Transient receptor potential channel canonical 3 (TRPC3) is a cation channel with poor Ca2+ selectivity and significant constitutive activity. One of the channels' features is its striking ability to couple in a surprisingly versatile manner to different down-stream signaling pathways, thereby serving cellular functions in a tissue specific manner. Expression of this protein is prominent in excitable cells, and its activity has repeatedly been implicated in electrical pacemaking. Previous studies demonstrated a linkage between constitutive activity of TRPC3 and neuronal firing in hippocampus and cerebellum. A most recent report from the Park laboratory corroborates the concept of TRPC3 functioning as a critical element in the neuronal pacemaking machinery for dopaminergic neurons of substantia nigra. Conclusively, mechanistic coupling between TRPC3 activity and firing frequency appears evident for different types of neurons, highlighting the potential of TRPC3 as a universal as well as multifunctional pacemaker channel.

Ethnic Differences in Serum Levels of microRNAs Potentially Regulating Alcohol Dehydrogenase 1B and Aldehyde Dehydrogenase 2.

Ethnic difference is known in genetic polymorphisms of aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B), which cause Asian flushing by blood vessel dilation due to accumulation of acetaldehyde. We investigated ethnic differences in microRNAs (miRNAs) related to ALDH2 and ADH1B. miRNA levels in serum were totally analyzed by using miRNA oligo chip arrays and compared in Austrian and Japanese middle-aged men. There were no ALDH2- and ADH1B-related miRNAs that had previously been reported in humans and that showed significantly different serum levels between Austrian and Japanese men. With the use of miRNA prediction tools, we identified four and five miRNAs that were predicted to target ALDH2 and ADH1B, respectively, and they had expression levels high enough for comparison. Among the ADH1B-related miRNAs, miR-150-3p, -3127-5p and -4314 were significantly higher and miR-3151-5p was significantly lower in Austrian compared with Japanese men, while no significant difference was found for miR-449b-3p. miR-150-3p and miR-4314 showed relatively high fold changes (1.5 or higher). The levels of ALDH2-related miRNAs (miR-30d-5p, -6127, -6130 and -6133) were not significantly different between the countries. miR-150-3p and miR-4314 are candidates of miRNAs that may be involved in the ethnic difference in sensitivity to alcohol through modifying the expression of ADH1B.

Erratum: Potassium ions promote hexokinase-II dependent glycolysis.

[This corrects the article DOI: 10.1016/j.isci.2021.102346.].

Potassium ions promote hexokinase-II dependent glycolysis.

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of KV1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism.

Mechanisms and significance of Ca2+ entry through TRPC channels.

The transient receptor potential (TRP) superfamily of plasma membrane cation channels has been recognized as a signaling hub in highly diverse settings of human physiopathology. In the past three decades of TRP research, attention was focused mainly on the channels Ca2+ signaling function, albeit additional cellular functions, aside of providing a Ca2+ entry pathway, have been identified. Our understanding of Ca2+ signaling by TRP proteins has recently been advanced by a gain in high-resolution structure information on these pore complexes, and by the development of novel tools to investigate their role in spatiotemporal Ca2+ handling. This review summarizes recent discoveries as well as remaining, unresolved aspects of the canonical subfamily of transient receptor potential channels (TRPC) research. We aim at a concise overview on current mechanistic concepts of Ca2+ entry through- and Ca2+ signaling by TRPC channels.

Blood levels of microRNAs associated with ischemic heart disease differ between Austrians and Japanese: a pilot study.

Mortality from ischemic heart disease (IHD) is significantly lower in Japan than in Western countries. The purpose of this study was to investigate differences in circulating microRNA (miRNA) levels related to IHD in Austrians and Japanese. Participants were middle-aged healthy male Austrians (n = 20) and Japanese (n = 20). Total miRNAs in serum from each participant were analyzed using the 3D-Gene miRNA Oligo chip. Twenty-one miRNAs, previously reported as associated with IHD, were compared between Austrians and Japanese. The expression levels of miR-106a-5p, miR-135a-3p, miR-150-3p, miR-16-5p, miR-17-5p. miR-191-5p, miR-320b, miR-451a, miR-486-5p, miR-663b, and miR-92a-3p were significantly higher, while the miR-2861 expression level was significantly lower in Austrians as compared to Japanese. Both in Austrians and Japanese, there were significant positive correlations between serum expression levels of each pair of the above miRNAs except for miR-2861. The expression level of miR-2861 showed significant positive correlations with the expression levels of miR-106a-5p, miR-150-3p, miR-17-5p, miR-486-5p, miR-663b and miR-92a-3p in Austrians but not in Japanese. In pathway analysis, proinflammatory cytokine production in foam cells and collagen synthesis in vascular smooth muscle cells were associated with differentially expressed miRNAs. Difference in miRNA levels may contribute to lower cardiovascular risk in Japan than in Western countries.

TRIC-A shapes oscillatory Ca2+ signals by interaction with STIM1/Orai1 complexes.

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A-mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A-dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release-activated Ca2+ channel 1 (Orai1) within ER-plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.

Light-Mediated Control over TRPC3-Mediated NFAT Signaling.

Canonical transient receptor potential (TRPC) channels were identified as key players in maladaptive remodeling, with nuclear factor of activated T-cells (NFAT) transcription factors serving as downstream targets of TRPC-triggered Ca2+ entry in these pathological processes. Strikingly, the reconstitution of TRPC-NFAT signaling by heterologous expression yielded controversial results. Specifically, nuclear translocation of NFAT1 was found barely responsive to recombinant TRPC3, presumably based on the requirement of certain spatiotemporal signaling features. Here, we report efficient control of NFAT1 nuclear translocation in human embryonic kidney 293 (HEK293) cells by light, using a new photochromic TRPC benzimidazole activator (OptoBI-1) and a TRPC3 mutant with modified activator sensitivity. NFAT1 nuclear translocation was measured along with an all-optical protocol to record local and global Ca2+ pattern generated during light-mediated activation/deactivation cycling of TRPC3. Our results unveil the ability of wild-type TRPC3 to produce constitutive NFAT nuclear translocation. Moreover, we demonstrate that TRPC3 mutant that lacks basal activity enables spatiotemporally precise control over NFAT1 activity by photopharmacology. Our results suggest tight linkage between TRPC3 activity and NFAT1 nuclear translocation based on global cellular Ca2+ signals.

Pharmaco-Optogenetic Targeting of TRPC Activity Allows for Precise Control Over Mast Cell NFAT Signaling.

Canonical transient receptor potential (TRPC) channels are considered as elements of the immune cell Ca2+ handling machinery. We therefore hypothesized that TRPC photopharmacology may enable uniquely specific modulation of immune responses. Utilizing a recently established TRPC3/6/7 selective, photochromic benzimidazole agonist OptoBI-1, we set out to test this concept for mast cell NFAT signaling. RBL-2H3 mast cells were found to express TRPC3 and TRPC7 mRNA but lacked appreciable Ca2+/NFAT signaling in response to OptoBI-1 photocycling. Genetic modification of the cells by introduction of single recombinant TRPC isoforms revealed that exclusively TRPC6 expression generated OptoBI-1 sensitivity suitable for opto-chemical control of NFAT1 activity. Expression of any of three benzimidazole-sensitive TRPC isoforms (TRPC3/6/7) reconstituted plasma membrane TRPC conductances in RBL cells, and expression of TRPC6 or TRPC7 enabled light-mediated generation of temporally defined Ca2+ signaling patterns. Nonetheless, only cells overexpressing TRPC6 retained essentially low basal levels of NFAT activity and displayed rapid and efficient NFAT nuclear translocation upon OptoBI-1 photocycling. Hence, genetic modification of the mast cells' TRPC expression pattern by the introduction of TRPC6 enables highly specific opto-chemical control over Ca2+ transcription coupling in these immune cells.