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Transient Receptor Potential Vanilloid subtype 3 (TRPV3) is a non-selective cation channel that is known to be activated by physiological temperature and endogenous ligands. Involvement of TRPV3 in different skin functions has been reported. In this work, we demonstrate that activation of TRPV3 by FPP, an endogenous ligand enhances skin wound healing and bacterial clearance there. We report for the first time that TRPV3 is endogenously expressed in macrophages and activation of TRPV3 results in efficient bacterial clearance. At the subcellular level, TRPV3 is present in the lysosome and also in the nucleolus. We demonstrate that pharmacological modulation of TRPV3 protects lysosomal functions at hyperthermic shock conditions. The localization of TRPV3 at the nucleolus is specific, more in case of LPS-treatment and dynamic with respect to the cell signalling. We demonstrate that at certain conditions, the nucleolar localization of TRPV3 is correlated with the presence of TRPV3 at the lysosome and with the cellular stress in general. We propose that TRPV3 act as a lysosomal regulator and sensor for cellular stress. These findings may have broad implications in understanding the cellular stress and TRPV3-induced channelopathies and may have clinical relevance to skin infection treatment.

The importance of TRPV4 in physiology and disease has been reported by several groups. Recently we have reported that TRPV4 localizes in the mitochondria in different cellular systems, regulates mitochondrial metabolism and electron transport chain functions. Here, we show that TRPV4 colocalizes with Cytochrome C (Cyt C), both in resting as well as in activated conditions. Amino acid region 592-630 of TRPV4 (termed as Fr592-630) that also covers TM4-Loop-TM5 region (which is also a hotspot of several pathogenic mutations) interacts with Cyt C, in a Ca

Microglia, the brain-resident macrophages, perform a myriad of functions directed towards development of neural circuits, and their maintenance. A plethora of ion channels aid in microglial activities that are critical for overall brain functioning. Notably, different functions of microglial cells are sensitive to minute temperature changes, as well as mechanical forces. Therefore, among all the players involved in the regulation of microglial functions, thermosensitive TRP ion channels are potentially important. In this study, we report the endogenous and functional presence of a heat-sensitive ion channel TRPV4 and a cold-sensitive ion channel TRPM8 in primary rat microglia and microglial cell line, N9. We demonstrate that pharmacological modulations of both these channels affect intracellular Ca

TRPV4 is a polymodal and non-selective cation channel that is activated by multiple physical and chemical stimuli.50 naturally occurring point-mutation of TRPV4 have been identified in human, most of which induce different diseases commonly termed as channelopathies. While, these mutations are either "gain-of-function" or "loss-of-function" in nature, the exact molecular and cellular mechanisms behind such diverse channelopathies are largely unknown. In this work, we analyze the evolutionary conservation of individual amino acids present in the lipid-water-interface (LWI) regions and the relationship of TRPV4 with membrane cholesterol. Our data suggests that the positive-negative charges and hydrophobic-hydrophilic amino acids form "specific patterns" in the LWI region which remain conserved throughout the vertebrate evolution and thus suggesting for the specific microenvironment where TRPV4 remain functional. Notably, Spondylometaphyseal Dysplasia, Kozlowski (SMDK) disease causing L596P mutation disrupts this pattern significantly at the LWI region. L596P mutant also sequesters Caveolin-1 differently, especially in partial cholesterol-depleted (~40 % reduction) conditions. L596P shows altered localization in membrane and enhanced Ca

Transient receptor potential (TRP) channels are a group of nonselective ion channels that are expressed in different cells constituting the gut tissue. These channels play important roles in the overall function and physiology of the gut tissue and are involved in different aspects of gastro, immune, neuronal, reproductive, and often behavioral functions. These functions can be substantially modulated either positively or negatively depending on the foods that we take in and the small and active compounds that are present in these foods. Different active compounds from plants, animals, microbes, mushrooms, as well as different toxins, peptides, small compounds, and certain metabolites from probiotic bacteria can either activate or inhibit these channels. The chemistry between TRPs and these small compounds have positive or negative aspects on the gut tissue depending on the molecular targets and/or quality, as well as the quantity of these compounds present in different foods. In this chapter, some of the specific aspects of TRP channels in the gut that can be specifically targeted by prebiotic, probiotic, postbiotic, and phyto-nutraceuticals factors for better health benefits are highlighted.

The understanding of molecules and their role in neurite initiation and/or extension is not only helpful to prevent different neurodegenerative diseases but also can be important in neuronal damage repair. In this work, we explored the role of transient receptor potential vanilloid 2 (TRPV2), a non-selective cation channel in the context of neurite functions. We confirm that functional TRPV2 is endogenously present in F11 cell line, a model system mimicking peripheral neuron. In F11 cells, TRPV2 localizes in specific subcellular regions enriched with filamentous actin, such as in growth cone, filopodia, lamellipodia and in neurites. TRPV2 regulates actin cytoskeleton and also interacts with soluble actin. Ectopic expression of TRPV2-GFP in F11 cell induces more primary and secondary neurites, confirming its role in neurite initiation, extension and branching events. TRPV2-mediated neuritogenesis is dependent on wildtype TRPV2 as cells expressing TRPV2 mutants reveal no neuritogenesis. These findings are relevant to understand the sprouting of new neurites, neuroregeneration and neuronal plasticity at the cellular, subcellular and molecular levels. Such understanding may have further implications in neurodegeneration and peripheral neuropathy.

With increased life expectancy, disorders in lifestyle and other clinical conditions, and the changes in the connective tissues such as in bone, impose diverse biomedical problems. Cells belong to osteogenic lineages are extremely specific for their surface requirements. Therefore, suitable surfaces are the critical bottle neck for successful bone tissue engineering. This study involves assessment of polysaccharide-based hydrogel which effectively allows growth, differentiation and mineralisation of osteogenic cells even in the absence of osteogenic inducing factors.Tamarind Kernel Polysaccharide was grafted with acrylic acid at different mole ratio. The critical parameter, surface morphology for bio application was assessed by SEM. MTT assay has been performed with hydrogels on Saos-2 cells. The biocompatibility and adhesion of different cell lines (F-11, Saos-2, Raw 264.7 and MSCs) on hydrogel surface was performed by Phalloidin and DAPI staining. Further the differentiation, mineralization and expression of different osteogenic markers, ALP assay, Alizarin Red staining and q-PCR was performed.The hydrogels show highly porous and interconnected pores. MTT assay demonstrates the hydrogel have no cytotoxicity towards Saos-2 cells and are suitable for proliferation of different lineage of cell lines. ALP, Alizarin red staining and q-PCR assay shows that the hydrogel surface enhances the differentiation, mineralization and expression of different osteogenic genes in Saos-2 cells in the absence of any osteogenic inducing factors. Conclusion Synthesized hydrogel surface triggers signalling events towards osteogenesis even in the absence of added growth factors. We proposed that this material can be used for effective bone tissue engineering in vitro at low cost.

TRPV3 is a non-selective cationic channel and is important for several physiological functions. It can be activated by physiological temperature and selective endogenous and exogenous compounds. TRPV3 is one of the key ion channel involved in Ca2+-signaling in keratinocyte and thus involved in skin-related functions. Recently, naturally occurring mutations in TRPV3, namely G573A, G573S, G573C and W692G have been detected which are linked with the development of pathophysiological conditions such as Olmsted Syndrome (OS) and other skin disorders. Our qualitative and quantitative data suggests that these naturally occurring TRPV3 mutants are mainly restricted in the ER. Expression of OS-mutants cause impaired vesicular trafficking resulting reduced surface localization of these mutants and other membrane proteins too. OS-mutants also cause reduced cell adhesion, altered distribution and less number of lysosomes. Our data confirms that TRPV3 is a lysosomal protein suggesting that Olmsted Syndrome is a lysosomal disorder. These findings may have a broad implication in the context of keratinocyte functions, skin-degeneration and in skin-cancer.

Transient receptor potential Vanilloid ion channel sub type 4 (TRPV4) is involved in complex Ca2+-signaling. At least one copy of TRPV4 is present in all vertebrates and is involved in several physiological processes including sensory process and point mutations in TRPV4 leads to development of different pathophysiological disorders in human. R616Q mutant of TRPV4 has been referred as “gain-of-function” mutant causing abnormality in bone cells and develop pathophysiological condition known as “Brachyolmia”. In this work, we demonstrated that R616Q mutation is located in a very critical position of TRPV4 containing a cholesterol-binding motif sequence which is highly conserved in all vertebrates. Accordingly, TRPV4-Wt but not the TRPV4-R616Q localizes preferably in cholesterol-enriched lipid rafts in osteogenic cell line Saos2 and in DRG-neuron derived F11 cell line. Further, FRAP experiment suggest TRPV4-Wt but not the TRPV4-R616Q mutant is more mobile especially in cholesterol-reduced lipid membrane. GST-tagged TM4-Loop-TM5 fragment containing TRPV4-Wt but not R616Q sequence interacts with cholesterol, forms high-molecular weight complex and also show band shift in SDS-PAGE. TRPV4 is expressed in Mesenchymal stem cells and the localization of TRPV4 in lipid raft is dependent on temperature and cholesterol. Our data suggests that TRPV4-R616Q mutant behaves as a “loss-of-interaction” with cholesterol.

Transient receptor potential channel subfamily A member 1 (TRPA1) is a non-selective cationic channel, identified initially as a cold sensory receptor. TRPA1 responds to diverse exogenous and endogenous stimuli associated with pain and inflammation. However, the information on the role of TRPA1 toward T-cell responses remains scanty. In silico data suggest that TRPA1 can play an important role in the T-cell activation process. In this work, we explored the endogenous expression of TRPA1 and its function in T cells. By reverse transcription polymerase chain reaction (RT-PCR), confocal microscopy and flow cytometry, we demonstrated that TRPA1 is endogenously expressed in primary murine splenic T cells as well as in primary human T cells. TRPA1 is primarily located at the cell surface. TRPA1-specific activator namely allyl isothiocyanate (AITC) increases intracellular calcium ion (Ca2+) levels while two different inhibitors namely A-967079 as well as HC-030031 reduce intracellular Ca2+ levels in T cells; TRPA1 inhibition also reduces TCR-mediated calcium influx. TRPA1 expression was found to be increased during αCD3/αCD28 (TCR) or Concanavalin A (ConA)-driven stimulation in T cells. TRPA1-specific inhibitor treatment prevented induction of cluster of differentiation 25 (CD25), cluster of differentiation 69 (CD69) in ConA/TCR stimulated T cells and secretion of cytokines like tumor necrosis factor (TNF), interferon γ (IFN-γ), and interleukin 2 (IL-2) suggesting that endogenous activity of TRPA1 may be involved in T-cell activation. Collectively these results may have implication in T cell-mediated responses and indicate possible role of TRPA1 in immunological disorders.

TRPA1 is a non-selective cation channel originated in invertebrates. The genomic locus containing TRPA1 gene remains highly conserved and retained in all vertebrates. TRPA1 gene is evolutionarily selected, yet maintained as a highly diverged protein. Throughout the vertebrate evolution, the extracellular loops of TRPA1 become most diverged indicating that TRPA1 may be involved in detecting large spectrum and uncertain stimulus which is critical for adaptive benefit. We tested the expression of TRPA1 in mature sperm from different vertebrates. This is the first report demonstrating that TRPA1 is expressed endogenously in mature spermatozoa of multiple species representing entire vertebrate phyla. However, its specific localization within sperm remains species-specific. Accordingly, we report that in rodents TRPA1 expression correlates with different stages of spermatogenesis. We propose that presence of endogenous TRPA1 in testes and in mature sperm provides reproductive benefit.

Patho-physiologies related to skin are diverse in nature such as burns, skin ulcers, atopic dermatitis, psoriasis etc. which impose severe bio-medical problems and thus enforce requirement of new and healthy skin prepared through tissues engineering methodologies. However, fully functional and biodegradable matrix for attachment, growth, proliferation and differentiation of the relevant cells is not available. In the present study, we introduce a set of hydrogels synthesized by incorporation of a synthetic monomer (Hydroxyethlmethacryate) with a semi-synthetic polymer backbone (carboxy methyl tamarind, CMT) in different mole ratios. We termed these materials as CMT:HEMA based hydrogels and these were characterized by different physico-chemical techniques, namely by X-Ray Diffraction, SEM and Dynamic Light Scattering. Biocompatibility studies with HaCaT, NIH-3T3 and mouse dermal fibroblasts confirm that this material is biocompatible. MTT assay further confirmed that this material does not have any cytotoxic effects. Assays for mitochondrial functionality such as ATP assay and mitochondrial reactive oxygen (ROS) generation also suggest that this material is safe and does not have any cytotoxicity. Hemolytic assay with red blood cells and acute skin irritation test on SD Rats confirmed that this material is suitable for ex-vivo application in future. We suggest that this hydrogel is suitable for in-vivo applications and may have clinical and commercial importance against skin disorders.

TRPV1 is a thermo-sensitive ion channel involved in neurosensory and other physiological functions. The trans-membrane helices of TRPV1 undergo quick and complex conformational changes governed by thermodynamic parameters and membrane components leading to channel opening. However, the molecular mechanisms underlying such events are poorly understood. Here we analysed the molecular evolution of TRPV1 at the lipid-water-interface region (LWI), typically defined as a layer of 6 Å thickness on each side of the membrane with less availability of free water. Amino acids demarcating the end of the trans-membrane helices are highly conserved. Residues present in the inner leaflet are more conserved and have been preferentially selected over others. Amino acids with snorkeling properties (Arginine and Tyrosine) undergo specific selection during the vertebrate evolution in a cholesterol-dependent and/or body temperature manner. Results suggest that H-bond formation between the OH- group of cholesterol and side chain of Arg557 or Arg575 at the inner leaflet is a critical parameter that can regulate channel functions. Different LWI mutants of TRPV1 have altered membrane localization and deficient colocalization with lipid raft markers. These findings may help to understand the lipid-protein interactions, and molecular basis of different neuronal functions. Such findings may have broad importance in the context of differential sensory responses, pathophysiologies, and application of pharmacological drugs such as anaesthetics acting on TRPVs.

TRPV4 is involved in several physiological and sensory functions as well as with several diseases and genetic disorders, though the molecular mechanisms for these are unclear. In this work we have analyzed molecular evolution and structure-function relationship of TRPV4 using sequences from different species. TRPV4 has evolved during early vertebrate origin (450million years). Synteny analysis confirms that TRPV4 has coevolved with two enzymes involved in sterol biosynthesis, namely MVK and GLTP. Cholesterol-recognizing motifs are present within highly conserved TM4-Loop4-TM5 region of TRPV4. TRPV4 is present in lipid raft where it co-localizes with Caveolin1 and Filipin. TM4-Loop4-TM5 region as well as Loop4 alone can physically interact with cholesterol, its precursor mevalonate and derivatives such as stigmasterol and aldosterone. Mobility of TRPV4-GFP depends on membrane cholesterol level. Molecular evolution of TRPV4 shared striking parallelism with the cholesterol bio-synthesis pathways at the genetic, molecular and metabolic levels. We conclude that interaction with sterols and cholesterol-dependent membrane dynamics have influence on TRPV4 function. These results may have importance on TRPV4-medaited cellular functions and pathophysiology.

Helicobacter pylori induces the antiapoptotic protein myeloid cell leukemia 1 (Mcl1) in human gastric epithelial cells (GECs). Apoptosis of oncogenic protein Mcl1-expressing cells is mainly regulated by Noxa-mediated degradation of Mcl1. We wanted to elucidate the status of Noxa in H. pylori–infected GECs. For this, various GECs such as AGS, MKN45, and KATO III were either infected with H. pylori or left uninfected. The effect of infection was examined by immunoblotting, immunoprecipitation, chromatin immunoprecipitation assay, in vitro binding assay, flow cytometry, and confocal microscopy. Infected GECs, surgical samples collected from patients with gastric adenocarcinoma as well as biopsy samples from patients infected with H. pylori showed significant up-regulation of both Mcl1 and Noxa compared with noninfected samples. Coexistence of Mcl1 and Noxa was indicative of an impaired Mcl-Noxa interaction. We proved that Noxa was phosphorylated at Ser13 residue by JNK in infected GECs, which caused cytoplasmic retention of Noxa. JNK inhibition enhanced Mcl1-Noxa interaction in the mitochondrial fraction of infected cells, whereas overexpression of nonphosphorylatable Noxa resulted in enhanced mitochondria-mediated apoptosis in the infected epithelium. Because phosphorylation-dephosphorylation can regulate the apoptotic function of Noxa, this could be a potential target molecule for future treatment approaches for H. pylori–induced gastric cancer.—Rath, S., Das, L., Kokate, S. B., Pratheek, B. M., Chattopadhyay, S., Goswami, C., Chattopadhyay, R., Crowe, S. E., Bhattacharyya, A. Regulation of Noxa-mediated apoptosis in Helicobacter pylori–infected gastric epithelial cells.

C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.

Sperm cells exhibit extremely high sensitivity in response to slight changes in temperature, osmotic pressure and/or presence of various chemical stimuli. In most cases throughout the evolution, these physico-chemical stimuli trigger Ca (2+)-signaling and subsequently alter structure, cellular function, motility and survival of the sperm cells. Few reports have recently demonstrated the presence of Transient Receptor Potential (TRP) channels in the sperm cells from higher eukaryotes, mainly from higher mammals. In this work, we have explored if the sperm cells from lower vertebrates can also have thermo-sensitive TRP channels. In this paper, we demonstrate the endogenous presence of one specific thermo-sensitive ion channel, namely Transient Receptor Potential Vanilloid family member sub type 1 (TRPV1) in the sperm cells collected from fresh water teleost fish, Labeo rohita. By using western blot analysis, fluorescence assisted cell sorting (FACS) and confocal microscopy; we confirm the presence of this non-selective cation channel. Activation of TRPV1 by an endogenous activator NADA significantly increases the quality as well as the duration of fish sperm movement. The sperm cell specific expression of TRPV1 matches well with our in silico sequence analysis. The results demonstrate that TRPV1 gene is conserved in various fishes, ranging from 1-3 in copy number, and it originated by fish-specific duplication events within the last 320 million years (MY). To the best of our knowledge, this is the first report demonstrating the presence of any thermo-sensitive TRP channels in the sperm cells of early vertebrates as well as of aquatic animals, which undergo external fertilization in fresh water. This observation may have implications in the aquaculture, breeding of several fresh water and marine fish species and cryopreservation of fish sperms.

Antithrombin III (ATIII) performs a critical anticoagulant function by precluding the activation of blood clotting proteinases, aided by its two cofactors, heparin and heparan sulfate. Though several studies have been carried out on physiological, biochemical and structural perspectives on ATIII, so far there are lim- ited studies on the molecular evolution of ATIII. Herein, we carried out molecular phylogenetic analyses of ATIII genes, combining gene structures, synteny and sequence-structural features for ATIII spanning 50 vertebrate genomes. ATIII is maintained over 450 MY on same genomic loci in vertebrates with few changes in ray-finned fishes and lost one intron 262c in tetrapods and coelacanth. In ray-finned fishes, ATIII gene has additional intron at the position 262c, which shared by group V1 members, corroborating that it is lost in other vertebrates and also in lobed-finned fish coelacanth (Latimeria chalumnae). We found that heparin binding basic residues, hD helix, three pairs of Cys-Cys salt bridges, N-glycosylation sites, serpin motifs and inhibitory reactive center loop (RCL) of ATIII protein are highly conserved. Using 1092 human genomes available from 1000G project, we also compiled 1997 ATIII variants, which reveals 76.2% single nucleotide polymorphisms (SNPs), 11.8% deletions and 8.1% insertions as three major classes of gene variations. These understandings may have medical importance as well.

Every individual varies in character and so do their sensory functions and perceptions. The molecular mechanism and the molecular candidates involved in these processes are assumed to be similar if not same. So far several molecular factors have been identified which are fairly conserved across the phylogenetic tree and are involved in these complex sensory functions. Among all, members belonging to Transient Receptor Potential (TRP) channels have been widely characterized for their involvement in thermo-sensation. These include TRPV1 to TRPV4 channels which reveal complex thermo-gating behavior in response to changes in temperature. The molecular evolution of these channels is highly correlative with the thermal response of different species. However, recent 2504 human genome data suggest that these thermo-sensitive TRPV channels are highly variable and carry possible deleterious mutations in human population. These unexpected findings may explain the individual differences in terms of complex sensory functions.

Transient Receptor Potential Vanilloid sub-type 4 (TRPV4) is a non-selective cationic channel involved in regulation of temperature, osmolality and different ligand-dependent Ca(2+)-influx. Recently, we have demonstrated that TRPV4 is conserved in all vertebrates. Now we demonstrate that TRPV4 is endogenously expressed in all vertebrate sperm cells ranging from fish to mammals. In human sperm, TRPV4 is present as N-glycosylated protein and its activation induces Ca(2+)-influx. Its expression and localization differs in swim-up and swim-down cells suggesting that TRPV4 is an important determining factor for sperm motility. We demonstrate that pharmacological activation or inhibition of TRPV4 regulates Ca(2+)-wave propagation from head to tail. Such findings may have wide application in male fertility-infertility, contraception and conservation of endangered species as well.

TRP channels are localized at specialized sub-cellular compartments like filopodial tips, ciliary structures, growth cones and spines that have importance in the context of several sensory functions. Several motor proteins largely regulate these localizations. Recent studies indicate that both physical and genetic interactions exist between TRP channels with actin and microtubule-based motor proteins. These two groups of proteins share specialized and fine regulation underlying physiological functions. Indeed, mutations causing loss of these interactions and regulations result in development of pathophysiological disorders and syndromes. In this review we analyze the recent progress made in cell-biological, biochemical, electrophysiological and genetic studies and summarize the multi-dimensional crosstalk between TRP channels with different motor proteins.

Recently, we described estrogen and agonists of the G-protein coupled estrogen receptor GPR30 to induce protein kinase C (PKC)e-dependent pain sensitization. PKCe phosphorylates the ion channel transient receptor potential, vanilloid subclass I (TRPV1) close to a novel microtubule-TRPV1 binding site. We now modeled the binding of tubulin to the TRPV1 C-terminus. The model suggests PKCe phosphorylation of TRPV1-S800 to abolish the tubulin-TRPV1 interaction. Indeed, in vitro PKCe phosphorylation of TRPV1 hindered tubulin-binding to TRPV1. In vivo, treatment of sensory neurons and F-11 cells with estrogen and the GPR30 agonist, G-1, resulted in microtubule destabilization and retraction of microtubules from filopodial structures. We found estrogen and G-1 to regulate the stability of the microtubular network via PKC phosphorylation of the PKCe-phosphorylation site TRPV1-S800. Microtubule disassembly was not, however, dependent on TRPV1 ion conductivity. TRPV1 knock-down in rats inverted the effect of the microtubule-modulating drugs, Taxol and Nocodazole, on estrogen-induced and PKCe-dependent mechanical pain sensitization. Thus, we suggest the C-terminus of TRPV1 to be a signaling intermediate downstream of estrogen and PKCe, regulating microtubule-stability and microtubule-dependent pain sensitization.

More than 150 participants from 25 countries gathered in Stockholm during 25(th) to 27(th) Sept 2009 to attend the meeting "TRP channels: from sensory signaling to human disease" and enjoyed an international, intensive and vibrant meeting. This meeting shed lights on the recent advances made in this field of research in different sectors of biology, and identified directions for future research and the areas where TRP channels could be used as potential targets for prevention and treatment of human diseases. The participants of this meeting shared their recent largely unpublished data, state-of-the-art techniques and their critical views which would push research in this field forward in the new decade. Another major outcome of this meeting was the realization that extensive work remains to be done to develop the necessary tools and enhance the quality of research in this area so that the prevailing controversies can be resolved. In this report we summarize the latest scientific excitements, some critical issues, as well as some future directions for research that were addressed and discussed in this meeting.

Much work has focused on the electrophysiological properties of transient receptor potential channels. Recently, a novel aspect of importance emerged: the interplay of transient receptor potential channels with the cytoskeleton. Recent data suggest a direct interaction and functional repercussion for both binding partners. The bi-directionality of physical and functional interaction renders therefore, the cytoskeleton a potent integration point of complex biological signalling events, from both the cytoplasm and the extracellular space. In this minireview, we focus mostly on the interaction of the cytoskeleton with transient receptor potential vanilloid channels. Thereby, we point out the functional importance of cytoskeleton components both as modulator and as modulated downstream effector. The resulting implications for patho-biological situations are discussed.

While the importance of Ca2+ channel activity in axonal path finding is established, the underlying mechanisms are not clear. Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones. These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N-arachidonoyl-dopamine (NADA), and affect the integrity of microtubule cytoskeleton. Using TRPV1-transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retraction, and collapse and formation of varicosities along neurites. These changes were due to TRPV1-activation-mediated disassembly of microtubules and are partly Ca2+-independent. Prolonged activation with very low doses (1 nm) of NADA results in shortening of neurites in the majority of isolectin B4-positive dorsal root ganglia neurones. We postulate that TRPV1 activation plays an inhibitory role in sensory neuronal extension and motility by regulating the disassembly of microtubules. This might have a role in the chronification of pain.

The vanilloid receptor TRPV1 plays a well-established functional role in the detection of a range of chemical and thermal noxious stimuli, such as those associated with tissue inflammation and the resulting pain. TRPV1 activation results in membrane depolarization, but may also trigger intracellular Ca2+-signalling events. In a proteomic screen for proteins associated with the C-terminal sequence of TRPV1, we identified β-tubulin as a specific TRPV1-interacting protein. We demonstrate that the TRPV1 C-terminal tail is capable of binding tubulin dimers, as well as of binding polymerized microtubules. The interaction is Ca2+-sensitive, and affects microtubule properties, such as microtubule sensitivity towards low temperatures and nocodazole. Our data thus provide compelling evidence for the interaction of TRPV1 with the cytoskeleton. The Ca2+-sensitivity of this interaction suggests that the microtubule cytoskeleton at the cell membrane may be a downstream effector of TRPV1 activation.

The three cerebral cavernous malformations (CCMs) genes namely CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10 have been identified for which mutations cause cerebral cavernous malformations. However, the protein products of these genes involved in forming CCM signaling, are still poorly understood imposing an urgent need to understand these genes and their signaling processes in details. So far involvement of CCM3/PDCD10 in the cavernous angioma has been characterized from biochemical and biophysical analyses. However, there is no comprehensive study illustrating the phylogenetic history and comprehensive genetic variants of CCM3/PDCD10. Herein, we explored the phylogenetic history and genetic variants of CCM3/PDCD10 gene. Synteny analyses revealed that CCM3/PDCD10 gene shared same genomic loci from Drosophila to human and the gene structure of CCM3/PDCD10 is conserved from human to Branchiostoma floridae for about 500 MYs with some changes in sea urchin and in insects. The conserved CCM3/PDCD10 is characterized by presence of indels in the N-terminal dimerization domain. We identified 951 CCM3/PDCD10 variants by analysis of 1092 human genomes with top three variation classes belongs to 84% SNPs, 6.9% insertions and 6.2% deletions. We identified 22 missense mutations in the human CCM3/PDCD10 protein and out of which three mutations are deleterious. We also identified four stop-codon gaining mutations at the positions E34*, E68*, E97* and E140*, respectively. This study is the first comprehensive analysis of the CCM3/PDCD10 gene based on phylogenetic origin and genetic variants. This study corroborates that the evolution of CCM proteins with tubular organization evolvements by endothelial cells.

The importance of Ca(2+) signalling and temperature in the context of T cell activation is well known. However, the molecular identities of key players involved in such critical regulations are still unknown. In this work we explored the endogenous expression of transient receptor potential vanilloid (TRPV) channels, a group of thermosensitive and non-selective cation channels, in T cells. Using flow cytometry and confocal microscopy, we demonstrate that members belonging to the TRPV subfamily are expressed endogenously in the human T cell line Jurkat, in primary human T cells and in primary murine splenic T cells. We also demonstrate that TRPV1- and TRPV4-specific agonists, namely resiniferatoxin and 4α-phorbol-12,13-didecanoate, can cause Ca(2+) influx in T cells. Moreover, our results show that expression of these channels can be upregulated in T cells during concanavalin A-driven mitogenic and anti-CD3/CD28 stimulated TCR activation of T cells. By specific blocking of TRPV1 and TRPV4 channels, we found that these TRPV inhibitors may regulate mitogenic and T cell receptor mediated T cell activation and effector cytokine(s) production by suppressing tumour necrosis factor, interleukin-2 and interferon-γ release. These results may have broad implications in the context of cell-mediated immunity, especially T cell responses and their regulations, neuro-immune interactions and molecular understanding of channelopathies.

Remodeling of bone by tissue engineering is a realistic option for treating several bone-related pathophysiological ailments such as osteoporosis, bone tumor, bone cancer or abnormal bone development. But, these possibilities are hindered due to lack of proper natural and biodegradable surface on which bone precursor cells can adhere efficiently and grow further. Here we describe the synthesis and characterization of a new hydrogel as an effective surface which can acts as a material for bone tissue engineering. This hydrogel has been prepared by chemically grafting a semi-synthetic polymer with a synthetic monomer, namely hydroxyethyl methacrylate (HEMA). Carboxy methyl tamarind (CMT) was selected as the semi-synthetic polymer. The hydrogel was prepared at different mole ratios and at the ratio of 1:10 (CMT:HEMA) yielded the best hydrogel as characterized by several physico-chemical analysis such as UV spectroscopy, FT-IR spectroscopy and swelling properties. We further demonstrate that this material is suitable for effective adhesion, growth and further clustering of bone precursor cells (RAW 264.7). This material is also compatible for growing other sensitive cells such as neuronal cells (Neuro2a) and human umbilical vein endothelial cells (HUVEC) demonstrating that this surface does not possess any cytotoxicity and is compatible for primary human cells too. We conclude that the hydrogel made of CMT:HEMA at a ratio of 1:10 can be suitable for bone tissue engineering and thus may have clinical as well as commercial application in future.

Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus, L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of -6.066 kcal mol(-1) Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management.

Existence of microtubule cytoskeleton at the membrane and submembranous regions, referred as 'membrane tubulin' has remained controversial for a long time. Since we reported physical and functional interaction of Transient Receptor Potential Vanilloid Sub Type 1 (TRPV1) with microtubules and linked the importance of TRPV1-tubulin complex in the context of chemotherapy-induced peripheral neuropathy, a few more reports have characterized this interaction in in vitro and in in vivo condition. However, the cross-talk between TRPs with microtubule cytoskeleton, and the complex feedback regulations are not well understood. Sequence analysis suggests that other than TRPV1, few TRPs can potentially interact with microtubules. The microtubule interaction with TRPs has evolutionary origin and has a functional significance. Biochemical evidence, Fluorescence Resonance Energy Transfer analysis along with correlation spectroscopy and fluorescence anisotropy measurements have confirmed that TRPV1 interacts with microtubules in live cell and this interaction has regulatory roles. Apart from the transport of TRPs and maintaining the cellular structure, microtubules regulate signaling and functionality of TRPs at the single channel level. Thus, TRPV1-tubulin interaction sets a stage where concept and parameters of 'membrane tubulin' can be tested in more details. In this review, I critically analyze the advancements made in biochemical, pharmacological, behavioral as well as cell-biological observations and summarize the limitations that need to be overcome in the future.

Transient receptor potential vanilloid sub type 4 (TRPV4) is a member of non-selective cation channel that is important for sensation of several physical and chemical stimuli and also involved in multiple physiological functions. Recently it gained immense medical and clinical interest as several independent studies have demonstrated that mutations in the TRPV4 gene can results in genetic disorders like Brachyolmia, Charcot-Marie-Tooth disease type 2C, Spinal Muscular Atrophy and Hereditary Motor and Sensory Neuropathy type 2. Close analysis of the data obtained from these naturally occurring as well as other TRPV4 mutants suggest that it is not the altered channel activity of these mutants per se, but the involvement and interaction of other factors that seem to modulate oligomerization, trafficking and degradation of TRPV4 channels. Also, these factors can either enhance or reduce the activity of TRPV4. In addition, there are some potential signaling events that can also be involved in these genetic disorders. In this review, we analyzed how and what extent certain cellular and molecular functions like oligomerization, surface expression, ubiquitination and functional interactions might be affected by these mutations.

BACKGROUND: TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCepsilon and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca2+-influx via TRPV4. CONCLUSIONS AND SIGNIFICANCE: TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients.

Background Transient Receptor Potential Vanilloid sub type 1 (TRPV1), commonly known as capsaicin receptor can detect multiple stimuli ranging from noxious compounds, low pH, temperature as well as electromagnetic wave at different ranges. In addition, this receptor is involved in multiple physiological and sensory processes. Therefore, functions of TRPV1 have direct influences on adaptation and further evolution also. Availability of various eukaryotic genomic sequences in public domain facilitates us in studying the molecular evolution of TRPV1 protein and the respective conservation of certain domains, motifs and interacting regions that are functionally important. Methodology and Principal Findings Using statistical and bioinformatics tools, our analysis reveals that TRPV1 has evolved about ∼420 million years ago (MYA). Our analysis reveals that specific regions, domains and motifs of TRPV1 has gone through different selection pressure and thus have different levels of conservation. We found that among all, TRP box is the most conserved and thus have functional significance. Our results also indicate that the tubulin binding sequences (TBS) have evolutionary significance as these stretch sequences are more conserved than many other essential regions of TRPV1. The overall distribution of positively charged residues within the TBS motifs is conserved throughout evolution. In silico analysis reveals that the TBS-1 and TBS-2 of TRPV1 can form helical structures and may play important role in TRPV1 function. Conclusions and Significance Our analysis identifies the regions of TRPV1, which are important for structure – function relationship. This analysis indicates that tubulin binding sequence-1 (TBS-1) near the TRP-box forms a potential helix and the tubulin interactions with TRPV1 via TBS-1 have evolutionary significance. This interaction may be required for the proper channel function and regulation and may also have significance in the context of Taxol®-induced neuropathy.

Around 35 years ago, several laboratories reported the presence of cytoskeletal proteins in biochemically enriched membrane fractions. It was quickly confirmed that actin and interacting proteins localize to submembranous regions, spurring an interest in their role and organization there. By contrast, evidence for the presence of microtubules at the membrane was contradictory and often criticized as a preparational artefact. Retrospectively, this can mostly be attributed to the use of an inappropriate model system, namely the erythrocyte membrane, which is mostly void of tubulin. However, direct and specific adsorption of tubulin on artificially prepared liposomes and the in vitro interaction of the tubulin–colchicine complex with brain microsomal membrane were shown. Reconstitution of ciliary membrane with attached tubulin then became possible and membranes were found to act as microtubule nucleation centres. Further biochemical studies indicated that a-tubulin could act as an integral membrane protein. Nonetheless, doubts about the interaction of tubulin with membrane were maintained for a long time. Recent advances in imaging techniques and increasing cell biological data have changed this notion fundamentally. The presence of tubulin and several microtubule-associated proteins in membranous structures has been demonstrated. This led to the prediction of novel functions for the microtubule cytoskeleton in submembranous domains and to the concept of ‘membrane-tubulin’. Microtubules interact directly with numerous proteins at the membrane and form scaffolds. These include proteins as diverse as ion channels, receptors, ion pumps and others. Such interactions lead, among other effects, to the sequestration of protein complexes at the plasma membrane to form, for example, signalplex. Membrane-tubulins play a critical role in cell division, in the positioning of complexes within the lipid bilayer and the proper positioning of intracellular organelles. A large number of questions remain to be addressed. How dynamic is membrane-bound tubulin? What is the true biochemical nature of tubulin at the membrane? Does tubulin reside at the membrane as monomeric, dimeric or short oligomeric units? What determines the specific submembranous localization of tubulin? And last, but not least, what is the influence of microtubule on the functionality of the nearby (trans)-membrane proteins? This minireview series summarizes evidence that, like actin, the microtubule cytoskeleton is part of several functional complexes at the membrane. Roychowdhury and Rasenick give an overview of membrane-bound heterotrimeric G proteins and their interaction with the microtubule cytoskeleton. They also describe the ramifications of such cross-talk for cellular function. Arce et al. describe the importance of microtubules in the context of molecular ion pumps such as the Na,KATPase. In particular, they emphasize how these pumps are regulated by a subset of specifically modified tubulin, namely acetylated tubulin. Chen et al. focus on how interactions between polycystin 2 and the cytoskeleton are linked with disease. And lastly, Goswami and Hucho summarize the physical and functional interplay of TRP channels with the cytoskeleton. This minireview series aims thereby to shed light on a rapidly growing and exciting novel subcellular domain, the membranous microtubule cytoskeleton.

The Vanilloid Receptor 1 is a heat-sensitive, nonselective cation channel expressed by primary sensory neurons involved in nociception. To find and study potential interacting proteins of VR1 we cloned the full N- and C-termini without transmembrane sequences of VR1 into the bacterial expression vector pMALc2x. We here report the expression and purification of the VR1 fragments fused to the maltose binding protein (MBP). In addition, we assessed the subcellular targeting of N- and C-terminal VR1 fragments expressed in the cell line F-11. This cell line has recently been shown to be particularly suited for the expression of VR1 (1). Preliminary data suggest that the VR1 N-terminal fragment is targeted to membranes despite the lack of predicted transmembrane regions.