Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85β subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85β coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

Although a large number of ion channels are now believed to be regulated by phosphoinositides, particularly phosphoinositide 4,5-bisphosphate (PIP2), the mechanisms involved in phosphoinositide regulation are unclear. For the TRP superfamily of ion channels, the role and mechanism of PIP2 modulation has been especially difficult to resolve. Outstanding questions include: is PIP2 the endogenous regulatory lipid; does PIP2 potentiate all TRPs or are some TRPs inhibited by PIP2; where does PIP2 interact with TRP channels; and is the mechanism of modulation conserved among disparate subfamilies? We first addressed whether the PIP2 sensor resides within the primary sequence of the channel itself, or, as recently proposed, within an accessory integral membrane protein called Pirt. Here we show that Pirt does not alter the phosphoinositide sensitivity of TRPV1 in HEK-293 cells, that there is no FRET between TRPV1 and Pirt, and that dissociated dorsal root ganglion neurons from Pirt knock-out mice have an apparent affinity for PIP2 indistinguishable from that of their wild-type littermates. We followed by focusing on the role of the C terminus of TRPV1 in sensing PIP2. Here, we show that the distal C-terminal region is not required for PIP2 regulation, as PIP2 activation remains intact in channels in which the distal C-terminal has been truncated. Furthermore, we used a novel in vitro binding assay to demonstrate that the proximal C-terminal region of TRPV1 is sufficient for PIP2 binding. Together, our data suggest that the proximal C-terminal region of TRPV1 can interact directly with PIP2 and may play a key role in PIP2 regulation of the channel.TRPV1 ion channels are capsaicin-, heat-, and acid-activated non-selective cation channels expressed in nociceptors of the peripheral nervous system as well as in the neurons of the hippocampus and cortex (1). TRPV1 is an essential component of inflammatory hyperalgesia, as TRPV1 knock-out mice show essentially no hyperalgesia in response to thermal and chemical stimuli during inflammation (2). The role of TRPV1 in nociception has made it an attractive target for pain therapies. In addition, the large physical size of its pore has been shown to allow cationic local anesthetics to enter nociceptors (3), raising the possibility of identifying more specific analgesics that do not interfere with non-painful sensation.Like many ion channels, TRPV1 is regulated by G-proteincoupled receptors (GPCR) 3 and receptor-tyrosine kinases (RTK), both of which generally sensitize nociceptors (4 -5). Bradykinin, a GPCR ligand, and nerve growth factor, an RTK ligand, are released in response to tissue injury and sensitize TRPV1 to subsequent activation by noxious stimuli (6). Because both GPCRs and RTKs can activate phospholipase C (PLC), degradation of PI(4,5)P 2 (PIP2) by PLC was originally proposed to constitute the common mechanism for bradykinin-and nerve growth factor-mediated sensitization of TRPV1 (6). In this PLC model of hyperalgesia, PIP2 is tonically bound to TRPV1 and inhibits chann...

Cell migration is a fundamental process in animal development, including development of the nervous system. In C. elegans, the bilateral QR and QL neuroblasts undergo initial anterior and posterior polarizations and migrations before they divide to produce neurons. A subsequent Wnt signal from the posterior instructs QL descendants to continue their posterior migration. Nck-interacting kinases (NIK kinases) have been implicated in cell and nuclear migration as well as lamellipodia formation. Studies here show that the C. elegans MIG-15 NIK kinase controls multiple aspects of initial Q cell polarization, including the ability of the cells to polarize, to maintain polarity, and to migrate. These data suggest that MIG-15 acts independently of the Wnt signal that controls QL descendant posterior migration. Furthermore, MIG-15 affects the later migrations of neurons generated from Q cell division. Finally, a mosaic analysis indicates that MIG-15 acts cell autonomously in Q descendant migration.

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Bamboo charcoal could be an effective amendment for nitrogen conservation and heavy metal stabilization in sludge composts. Further research into the effect of BC-amended sludge compost material on soil properties, bioavailability of heavy metals, and nutrient turnover in soil needs to be carried out prior to the application of BC-sludge compost in agriculture.

Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P 2 ) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P 3 ) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P 2 -specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P 3 -specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P 2 inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P 2 , and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.Although they make up only 1-5% of the total anionic lipids in a cell membrane (1-3), phosphoinositides have a prominent role in lipid signaling (4). Both phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P 2 ) 3 and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P 3 ) have been implicated in protein trafficking, cell motility, Ca 2ϩ signaling, cell growth, endo-and exocytosis, and ion transporter and channel regulation (4, 5). Changes in the plasma membrane PI(4,5)P 2 concentration that result from activation of cell surface receptors are believed to regulate the function of many ion channels and transporters, including K ϩ channels, voltage-gated Ca 2ϩ channels, inositol trisphosphate receptors, and the family of transient receptor potential (TRP) channels (4).Several different phosphoinositides have been implicated in modulation of TRP channels. The original TRP channel from Drosophila is thought to be gated by either diacyl glycerol or a polyunsaturated fatty acid (6). This is in contrast to original studies that implied an inhibitory role for PI(4,5)P 2 on both TRP and TRPL. A recent study demonstrated phosphoinositide binding by TRPC6, a TRP channel involved in vasodilation, but did not determine whether PI(4,5)P 2 or PI(3,4,5)P 3 was the endogenous ligand (7). Finally, it has been shown that PI(4,5)P 2 can alter the function of TRPM4, TRPM5, TRPM8, TRPM7, and TRPV5 (8 -14), but whether PI(4,5)P 2 is indeed the physiological regulator has not been addressed.For the transient receptor potential vanilloid-type 1 (TRPV1) channel responsible for transduction of painful thermal...

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Adenoviral vectors have shown great promise as vaccine carriers and in gene transfer to correct underlying genetic diseases. Traditionally, construction of adenoviral vectors is complex and time consuming. In this paper, we provide an improved method for efficient generation of novel adenoviral vectors by using direct cloning. We introduce a feasible and detailed protocol for the development of chimpanzee adenoviruses (Ads) as molecular clones, as well as for the generation of recombinant virus from the molecular clones. Recombinant viruses are genetically stable and induce potent immune responses in animals. Generation of new Ad molecular clones or new recombinant Ad can be achieved in 2 months or 2 weeks, respectively.