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...

The ability to detect hot temperatures is critical to maintaining body temperature and avoiding injury in diverse animals from insects to mammals. Zebrafish embryos, when given a choice, actively avoid hot temperatures and display an increase in locomotion similar to that seen when they are exposed to noxious compounds such as mustard oil. Phylogenetic analysis suggests that the single zebrafish ortholog of TRPV1/2 may have arisen from an evolutionary precursor of the mammalian TRPV1 and TRPV2. As opposed to TRPV2, mammalian TRPV1 is essential for environmentally relevant heat sensation. In the present study, we provide evidence that the zebrafish TRPV1 ion channel is also required for the sensation of heat. Contrary to development in mammals, zebrafish TRPV1 ϩ neurons arise during the first wave of somatosensory neuron development, suggesting a vital importance of thermal sensation in early larval survival. In vitro analysis showed that zebrafish TRPV1 acts as a molecular sensor of environmental heat (Ն25°C) that is distinctly lower than the sensitivity of the mammalian form (Ն42°C) but consistent with thresholds measured in behavioral assays. Using in vivo calcium imaging with the genetically encoded calcium sensor GCaMP3, we show that TRPV1-expressing trigeminal neurons are activated by heat at behaviorally relevant temperatures. Using knock-down studies, we also show that TRPV1 is required for normal heat-induced locomotion. Our results demonstrate for the first time an ancient role for TRPV1 in the direct sensation of environmental heat and show that heat sensation is adapted to reflect species-dependent requirements in response to environmental stimuli.

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...

We investigated the cellular and molecular mechanisms underlying arrhythmias in heart failure. A genetically engineered mouse lacking the expression of the muscle LIM protein (MLP ؊/؊ ) was used in this study as a model of heart failure. We used electrocardiography and patch clamp techniques to examine the electrophysiological properties of MLP ؊/؊ hearts. We found that MLP ؊/؊ myocytes had smaller Na ؉ currents with altered voltage dependencies of activation and inactivation and slower rates of inactivation than control myocytes. These changes in Na ؉ currents contributed to longer action potentials and to a higher probability of early afterdepolarizations in MLP ؊/؊ than in control myocytes. Western blot analysis suggested that the smaller Na ؉ current in MLP ؊/؊ myocytes resulted from a reduction in Na ؉ channel protein. Interestingly, the blots also revealed that the ␣-subunit of the Na ؉ channel from the MLP ؊/؊ heart had a lower average molecular weight than in the control heart. Treating control myocytes with the sialidase neuraminidase mimicked the changes in voltage dependence and rate of inactivation of Na ؉ currents observed in MLP ؊/؊ myocytes. Neuraminidase had no effect on MLP ؊/؊ cells thus suggesting that Na ؉ channels in these cells were sialic acid-deficient. We conclude that deficient glycosylation of Na ؉ channel contributes to Na ؉ current-dependent arrhythmogenesis in heart failure.