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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 p85beta subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85beta 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.

We investigated the contribution of sialic acid residues to the [K.sup.+] currents involved in the repolarization of mouse ventricular myocytes. Ventricular [K.sup.+] currents had a rapidly inactivating component followed by slowly decaying and sustained components. This current was produced by the summation of three distinct currents: [I.sub.to], which contributed to the transient component; [I.sub.ss], which contributed to the sustained component; and [I.sub.K,slow], which contributed to both components. Incubation of ventricular myocytes with the sialidase neuraminidase reduced the amplitude of [I.sub.to] without altering [I.sub.K,slow] and [I.sub.ss]. We found that the reduction in [I.sub.to] amplitude resulted from a depolarizing shift in the voltage of activation and a reduction in the conductance of [I.sub.to]. Expression of Kv4.3 channels, a major contributor to [I.sub.to] in the ventricle, in a sialylation-deficient Chinese hamster ovary cell line (lec2) mimicked the effects of neuraminidase on the ventricular [I.sub.to]. Furthermore, we showed that sialylated glycolipids have little effect on the voltage dependence of [I.sub.to]. Finally, consistent with its actions on [I.sub.to], neuraminidase produced an increase in the duration of the action potential of ventricular myocytes and the frequency of early afterdepolarizations. We conclude that sialylation of the proteins forming Kv4 channels is important in determining the voltage dependence and conductance of [I.sub.to] and that incomplete glycosylation of these channels could lead to arrhythmias. glycosylation; Kv4.3; arrhythmias; mouse ventricular myocytes; transient outward currents

Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EOF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5' position of the phosphoinositide. [ABSTRACT FROM AUTHOR]

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 TRPV 1 and TRPV2. As opposed to TRPV2, mammalian TRPV 1 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 TRPV 1+ 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 TRPV 1 acts as a molecular sensor of environmental heat ( >25°C) that is distinctly lower than the sensitivity of the mammalian form ( >42°C) but consistent with thresholds measured in behavioral assays. Using in vivo calcium imaging with the genetically encoded calcium sensor GCaMP3, we show that TRPV 1 -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. [ABSTRACT FROM AUTHOR]

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)P2) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) 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)P2-specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P3-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)P2 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)P2, and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels. [ABSTRACT FROM AUTHOR]

Investigates the contribution of sialic acid residues to the potassium currents involved in the repolarization of mouse ventricular myocytes. Physiological effect of potassium current levels; Production of potassium current through the summary of three distinct currents; Contribution to voltage dependence of transient current component of potassium.

Investigates the role of sialic acid residues in the function of potassium channels involved in the repolarization of the mouse ventricle. Suggestion that the channels underlying currents in the mouse ventricle vary in regard to the number of sialic acid residues attached to them during posttranslational processing; Arrhythmogenic currents to the transient component resulting from improper glycosylation of Kv4 channels.