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1.AbstractGenetically encoded reporters have greatly increased our understanding of biology, especially in neuroscience. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach we have engineered Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential. Unlike other luciferase-luciferin bioluminescent systems, AMBER encodes the genes to express both the luciferase and luciferin. AMBER is a voltage-gated luciferase coupling the functionalities of theCionavoltage-sensing domain (VSD) and bacterial luciferase, luxAB. When AMBER is co-expressed with the luciferin producing genes it reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods we show that AMBER modulates its enzymatic activity as a function of the membrane potential. AMBER shows several-fold increase in the luminescent (ΔL/L) signal upon switching from the off to on state when the cell is depolarized.In vivoexpression of AMBER inC. elegansallowed detecting pharyngeal pumping action and mechanosensory neural activity from multiple worms simultaneously. AMBER reports neural activity of multiple animals at the same time and can be used in social behavior assays to elucidate the role of membrane potential underlying behavior.2.Significance StatementThere have been many exciting advances in the development of genetically encoded voltage indicators to monitor intracelluar voltage changes. Most sensors employ fluorescence, which requires external light, potentially causing photobleaching or overheating. Consequently, there has been interest in developing luminescence reporters. However, they require addition of an exogenous substrate to produce light intracellularly. Here, we engineered a genetically encoded bioluminescent voltage indicator, AMBER, which unlike other bioluminescent activity indicators, does not require addition of an exogenous substrate. AMBER allows a large differential signal, a high signal-to-noise ratio, and causes minimal metabolic demand on cells. We used AMBER to record voltage activity in freely-moving C.elegans, demonstrating that AMBER is a important new tool for monitoring neuronal activity during social behavior.

Summary: The decision to consume or reject a food based on the degree of acidity is critical for animal survival. However, the gustatory receptors that detect sour compounds and influence feeding behavior have been elusive. Here, using the fly, Drosophila melanogaster, we reveal that a member of the ionotropic receptor family, IR7a, is essential for rejecting foods laced with high levels of acetic acid. IR7a is dispensable for repulsion of other acidic compounds, indicating that the gustatory sensation of acids occurs through a repertoire rather than a single receptor. The fly’s main taste organ, the labellum, is decorated with bristles that house dendrites of gustatory receptor neurons (GRNs). IR7a is expressed in a subset of bitter GRNs rather than GRNs dedicated to sour taste. Our findings indicate that flies taste acids through a repertoire of receptors, enabling them to discriminate foods on the basis of acid composition rather than just pH. : The receptors required for the gustatory responses to sour compounds have been elusive. Rimal et al. discover a Drosophila taste receptor, IR7a, which is required for rejecting a sour chemical. Surprisingly, IR7a is narrowly tuned to acetic acid, suggesting that carboxylic acids are detected by a repertoire of receptors. Keywords: sour, sourness, acid taste, proton, labeled line, feeding, Drosophila

Key points Receptor-operated activation of TRPC4 cation channels requires Gi/o proteins and phospholipase-Cδ1 (PLCδ1) activation by intracellular Ca2+ . Concurrent stimulation of the Gq/11 pathway accelerates Gi/o activation of TRPC4, which is not mimicked by increasing cytosolic Ca2+ . The kinetic effect of Gq/11 was diminished by alkaline intracellular pH (pHi ) and increased pHi buffer capacity. Acidic pHi (6.75-6.25) together with the cytosolic Ca2+ rise accelerated Gi/o -mediated TRPC4 activation. Protons exert their facilitation effect through Ca2+ -dependent activation of PLCδ1. The data suggest that the Gq/11 -PLCβ pathway facilitates Gi/o activation of TRPC4 through hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2 ) to produce the initial proton signal that triggers a self-propagating PLCδ1 activity supported by regenerative H+ and Ca2+ . The findings provide novel mechanistic insights into receptor-operated TRPC4 activation by coincident Gq/11 and Gi/o pathways and shed light on how aberrant activation of TRPC4 may occur under pathological conditions to cause cell damage. Abstract Transient Receptor Potential Canonical 4 (TRPC4) forms non-selective cation channels activated downstream from receptors that signal through G proteins. Our recent work suggests that TRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o proteins, with a co-dependence on phospholipase-Cδ1 (PLCδ1). The Gi/o -mediated TRPC4 activation is dually dependent on and bimodally regulated by phosphatidylinositol 4,5-bisphosphate (PIP2 ), the substrate hydrolysed by PLC, and intracellular Ca2+ . As a byproduct of PLC-mediated PIP2 hydrolysis, protons have been shown to play an important role in the activation of Drosophila TRP channels. However, how intracellular pH affects mammalian TRPC channels remains obscure. Here, using patch-clamp recordings of HEK293 cells heterologously co-expressing mouse TRPC4β and the Gi/o -coupled μ opioid receptor, we investigated the role of intracellular protons on Gi/o -mediated TRPC4 activation. We found that acidic cytosolic pH greatly accelerated the rate of TRPC4 activation without altering the maximal current density and this effect was dependent on intracellular Ca2+ elevation. However, protons did not accelerate channel activation by directly acting upon TRPC4. We additionally demonstrated that protons exert their effect through sensitization of PLCδ1 to Ca2+ , which in turn promotes PLCδ1 activity and further potentiates TRPC4 via a positive feedback mechanism. The mechanism elucidated here helps explain how Gi/o and Gq/11 co-stimulation induces a faster activation of TRPC4 than Gi/o activation alone and highlights again the critical role of PLCδ1 in TRPC4 gating.

Rhodopsin is a light receptor comprised of an opsin protein and a light-sensitive retinal chromophore. Despite more than a century of scrutiny, there is no evidence that opsins function in chemosensation. Here, we demonstrate that three Drosophila opsins, Rh1, Rh4, and Rh7, are needed in gustatory receptor neurons to sense a plant-derived bitter compound, aristolochic acid (ARI). The gustatory requirements for these opsins are light-independent and do not require retinal. The opsins enabled flies to detect lower concentrations of aristolochic acid by initiating an amplification cascade that includes a G-protein, phospholipase Cβ, and the TRP channel, TRPA1. In contrast, responses to higher levels of the bitter compound were mediated through direct activation of TRPA1. Our study reveals roles for opsins in chemosensation and raise questions concerning the original roles for these classical G-protein-coupled receptors.

Background and PurposeTransient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype-specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated.

Summary Many animals, ranging from vinegar flies to humans, discriminate a wide range of tastants, including sugars, bitter compounds, NaCl, and sour. However, the taste of Ca2+ is poorly understood, and it is unclear whether animals such as Drosophila melanogaster are endowed with this sense. Here, we examined Ca2+ taste in Drosophila and showed that high levels of Ca2+ are aversive. The repulsion was mediated by two mechanisms—activation of a specific class of gustatory receptor neurons (GRNs), which suppresses feeding and inhibition of sugar-activated GRNs, which normally stimulates feeding. The distaste for Ca2+, and Ca2+-activated action potentials required several members of the variant ionotropic receptor (IR) family (IR25a, IR62a, and IR76b). Consistent with the Ca2+ rejection, we found that high concentrations of Ca2+ decreased survival. We conclude that gustatory detection of Ca2+ represents an additional sense of taste in Drosophila and is required for avoiding toxic levels of this mineral.

Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications.

In the central nervous system, canonical transient receptor potential (TRPC) channels have been implicated in mediating neuronal excitation induced by stimulating metabotropic receptors, including group 1 metabotropic glutamate receptors (mGluRs). Lateral septal (LS) neurons express high levels of TRPC4 and group I mGluRs. However, to what extent native TRPC4-containing channels (TRPC4-cc) are activated as well as the impact of different levels of TRPC4-cc activation on neuronal excitability remain elusive. Here, we report that stimulating LS neurons with group I mGluR agonist, (S)-3,5-DHPG, causes either an immediate increase in firing rate or an initial burst followed by a pause of firing, which can be correlated with below-threshold-depolarization (BTD) or above-threshold-plateau-depolarization (ATPD), respectively, in whole-cell recordings. The early phase of BTD and the entire ATPD are completely absent in neurons from TRPC4−/− mice. Moreover, in the same LS neurons, BTD can be converted to ATPD at more depolarized potentials or with a brief current injection, suggesting that BTD and ATPD may represent partial and full activations of TRPC4-cc, respectively. We show that coincident mGluR stimulation and depolarization is required to evoke strong TRPC4-cc current, and Na+ and Ca2+ influx, together with dynamic changes of intracellular Ca(2+), are essential for ATPD induction. Our results suggest that TRPC4-cc integrates metabotropic receptor stimulation with intracellular Ca(2+) signals to generate two interconvertible depolarization responses to affect excitability of LS neurons in distinct fashions.

Individual classes of nanoparticles have made a tremendous impact on the biomedical sciences, with advances in imaging, single-molecule tracking, and cellular mechanotransduction. However, the future of nanotechnology will probably depend on the combination of attributes from several different nanomaterials. Here, one class of hybrid nanoparticles that possess both fluorescent and magnetic functionalities is described. These nanocomposites are created by combining fluorescent nanoparticles with magnetic iron oxide nanoparticles in an encapsulating micelle or solid polymer sphere. The resulting composites range from 10 to 500nm in size and display both fluorescent and magnetic properties of the constituent nano-particles. These particles are demonstrated as in vitro cellular labels, aprecursor to future in vivo studies; they will expand in vivo imaging options by providing the capability for both magnetic resonance (MR) and fluorescence imaging.

Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gβγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gβγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions.

Transient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype-specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated.Using fluorescence membrane potential and Ca(2+) assays and electrophysiological recordings, we characterized new 2-aminobenzimidazole-based small molecule inhibitors of TRPC4 and TRPC5 channels identified from cell-based fluorescence high-throughput screening.The original compound, M084, was a potent inhibitor of both TRPC4 and TRPC5, but was also a weak inhibitor of TRPC3. Structural modifications of the lead compound resulted in the identification of analogues with improved potency and selectivity for TRPC4 and TRPC5 channels. The aminobenzimidazole derivatives rapidly inhibited the TRPC4- and TRPC5-mediated currents when applied from the extracellular side and this inhibition was independent of the mode of activation of these channels. The compounds effectively blocked the plateau potential mediated by TRPC4-containing channels in mouse lateral septal neurons, but did not affect the activity of heterologously expressed TRPA1, TRPM8, TRPV1 or TRPV3 channels or that of the native voltage-gated Na(+) , K(+) and Ca(2) (+) channels in dissociated neurons.The TRPC4/C5-selective inhibitors developed here represent novel and useful pharmaceutical tools for investigation of physiological and pathophysiological functions of TRPC4/C5 channels.

Controlled transport of multiple individual nanostructures is crucial for nanoassembly and nanodelivery but is challenging because of small particle size. Although atomic force microscopy and optical and magnetic tweezers can control single particles, it is extremely difficult to scale these technologies for multiple structures. Here, we demonstrate a "nano-conveyer-belt" technology that permits simultaneous transport and tracking of multiple individual nanospecies in a selected direction. The technology consists of two components: nanocontainers, which encapsulate the nanomaterials transported, and nanoconveyer arrays, which use magnetic force to manipulate individual or aggregate nanocontainers. This technology is extremely versatile. For example, nanocontainers encapsulate quantum dots or rods and superparamagnetic iron oxide nanoparticles in

Heterogeneous nanostructures that possess multiple properties as a result of their differing constituent materials have attracted significant interest in the last few years. In particular, fluorescent-magnetic nanostructures have potential applications in imaging, separations, and single molecule tracking as a result of their fluorescent and magnetic properties. Here we report the synthesis of fluorescent-magnetic nanocomposites composed of fluorescent semiconductor quantum dots or graphitic carbon nanoparticles and magnetic iron oxide nanoparticles. We have developed synthetic strategies using either micellular or polymer encapsulation, yielding composites from ~10 - 100s of nms. Composites maintain the fluorescent and magnetic properties of their constituent materials. These composites can be used for in vitro and in vivo imaging using fluorescent or magnetic (e.g., MRI) modalities. Additionally, we describe the manipulation of these composites using magnetic instrumentation. In particular, we have designed a magnetic needle that can be used to manipulate nanocomposites. Particles as small as 30 nm can be manipulated while simultaneous observed through their fluorescent property. Single particle status can be confirmed through quantum dot blinking, demonstrating the potential of these composites for single molecule tracking.

Transient Receptor Potential Canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions, including smooth muscle contractility, synaptic transmission, and neurodegeneration. Unique among TRPC subfamily members, TRPC4 activation is dependent on Gi/o subgroup of heterotrimeric G proteins in addition to stimulation of phospholipase C pathway. Previously, the Gi/o, rather than Gbetgamma, subunits were suggested to mediate Gi/o-induced TRPC4 activation. Because the lifetime and availability of Galpha-GTP are regulated by Regulators of G protein signaling (RGS) and GoLoco domain-containing proteins via their GTPase-activating protein (GAP) and guanosine nucleotide dissociation inhibition (GDI) functions, respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch clamp recordings, we show that both RGS (RGS4, RGS6, RGS12, or RGS14) and GoLoco domain proteins (LGN, AGS3, RGS12, or RGS14) suppress receptor-mediated TRPC4 activation without eliciting any basal current. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco domains of AGS3. In addition, the Gi/o-specific RGS proteins, but not the GoLoco domain proteins, accelerate desensitization of receptor activation-evoked TRPC4 currents. We found that the inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS proteins that contain both the RGS and GoLoco domains, i.e. RGS12 and RGS14. Our data support the notion that the Galpha, but not Gbetagamma, arm of the Gi/o signaling is involved in TRPC4 activation and indicate that acting through Gi/o via GAP and GDI functions, RGS and GoLoco domain proteins can fine tune TRPC4 activity and thereby allow delicate regulation of cellular functions in nervous and other systems.

Transient Receptor Potential Canonical (TRPC) proteins form non-selective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show that coincident stimulation of Gi/o proteins and PLCδ1 (and not Gq/11-PLCβ) is necessary and sufficient for TRPC4 activation. In HEK293 cells co-expressing TRPC4 and Gi/o-coupled μ opioid receptor (μOR), μ agonist DAMGO elicited currents in a biphasic manner, with an initial slow phase preceding a second rapidly developing phase. While the currents were dependent on intracellular Ca2+ and PIP2, both Ca2+ and PIP2 also exhibited inhibitory effects on the channel. Depleting PIP2 abolished the biphasic kinetics and facilitated channel activation by weak Gi/o stimulation. Channel activation was inhibited by knocking down expression of PLCδ1 in both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A498 cells that endogenously express TRPC4. Channel activation was strongly suppressed by a dominant-negative PLCδ1 mutant or a constitutively active RhoA mutant. Our results demonstrate an integrative mechanism of TRPC4 for detection of coincident Gi/o, Ca2+, and PLC signaling, wherein TRPC4 and PLCδ1 are functionally coupled. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in the brain and other systems.

The effects of detergents on ion channel modulation have been studied before. Specifically, it has been noted that detergents used in the preparation of subunits of heterotrimeric G proteins can differentially modulate channel activity. A detailed analysis of the modulation, however, has not been performed. Here we show that detergents typically used in G protein subunit preparations, in sub-critical micelle concentrations, can affect the ion channel under consideration. TRPC4 and TRPC5 are closely homologous members of the Canonical Transient Receptor Potential family of non-selective cation channels. Activation of TRPC4 and TRPC5 elicits membrane depolarization and intracellular calcium signaling in neurons, vascular endothelium and smooth muscle cells. Both channels have been previously shown to be synergistically regulated by Gq-coupled receptor pathways and pertussis toxin sensitive Gi/o-coupled receptor pathways. The zwitterionic detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 14-50 μM) and nonionic surfactant lubrol (C12E10, 0.001-0.004%), which are typically used in G protein subunit preparations, were applied to the cytosolic side of inside-out membrane patches excised from HEK293 cells expressing TRPC4 or TRPC5. Although both CHAPS and lubrol caused ∼40-60% reduction in Popen under basal non-stimulated conditions, only CHAPS resulted in an increase in frequency of transitions between the open and closed states of the single channel activity. Therefore, the effects of detergents, especially at a single channel resolution, are significant and can affect results where proteins dissolved in these detergents are used. The mechanisms behind these phenomena have yet to be elucidated; however, it is likely that these detergents modify membrane elasticity in different ways, resulting in either partial solubilization of the ion channel or enhanced rigidity of the membrane surrounding the channel.

There has been great interest in the use of nanoparticles for imaging, particularly in multimodal applications (e.g., combination of MRI and fluorescence). Yet creating particles with multiple functionalities has been challenging. Here, we report the synthesis of pH sensitive, fluorescent-magnetic, nanocomposites created through a simple aqueous procedure. Separately synthesized superparamagnetic iron oxide nanoparticles and mercaptopropionic acid (MPA)-coated CdS quantum dots were crosslinked using 3-mercaptopropyl trimethoxysilane (MPS) as a bifunctional linker to yield CdS-iron oxide conjugates. Conjugates formed clusters of 0.1-1.0 microm diameter, with the smallest observed particle diameter approximately 50 nm. Particle solubility and photoluminescent (PL) intensity were sensitive to solution pH, with the highest PL intensity and stability obtained at pH values3.0 and MPS:Cd:Fe ratios of 1:10:1. pH sensitivity is believed to result from changes in nanoparticle solubility within the silica-based matrix. Given these unique properties, this material might find application in separation, pH sensitive detection (e.g., endosomal tracking) and biosensing.

TRPC4 is a member of the Canonical Transient Receptor Potential family of non-selective ion channels and is regulated by a set of intracellular and extracellular factors including G protein coupled receptor activation, calcium and membrane phospholipids. Sodium (Na+) and calcium (Ca2+) influx through the active TRPC4 channel elicits membrane depolarization and intracellular Ca2+ signaling in neurons, vascular endothelium and smooth muscle cells. Uniquely, TRPC4 is maximally activated only when two separate G protein pathways, Gq/11 and Gi/o, are co-stimulated, making it a coincidence detector of Gq/11- and Gi/o-coupled receptor co-activation. This function may be important for coordinating the actions of sympathetic (e.g. adrenergic) and parasympathetic (e.g. cholinergic) innervations. While a direct interaction between Gi/o-alpha subunits and the cytoplasmic C-terminal domain of TRPC4 has been suggested to underlie the activation mechanism by Gi/o proteins, the PLC-IP3-IP3R pathway is thought to be critical for the action of Gq/11. Using electrophysiological recordings of HEK293 cells heterologously expressing mouse TRPC4-beta, we examined the interdependence of Gi/o and Gq/11 pathways on TRPC4 activation. We show that whereas Gi/o (but not Gq/11) stimulation is necessary, it does not fully activate TRPC4 unless accompanied by a simultaneous rise in intracellular free Ca2+, which may or may not require activation of the Gq/11 pathway. Paradoxically, inhibiting calmodulin also facilitated TRPC4 activation by Gi/o signaling, but not to the full extent as that facilitated by increasing intracellular Ca2+ levels, indicating an inhibitory role played by calmodulin on TRPC4 activation and a calmodulin-independent positive regulation by Ca2+. Our findings indicate that TRPC4 is a coincidence sensor of (A) Gi/o stimulation and (B) intracellular [Ca2+] rise that can be triggered by receptor-PLC pathways, lysosomal Ca2+ release, ER Ca2+ store release and plasma membrane Ca2+ influx mechanisms.