Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

Abstract

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.

Isoform-specific Inhibition of TRPC4 Channel by Phosphatidylinositol 4,5-Bisphosphate

“…Although PIP 2 and related phosphoinositides exert positive effects on the activity of many TRP channels, such as TRPV5 (32), TRPM4 (35,44), TRPM5 (45), TRPM7 (46,47), TRPM8 (33,34), and TRPC6 (7,48), it is inhibitory to few others, such as dTRPL (3) and TRPV1 (42, 49). Ironically, the inhibitory effect of PIP 2 on TRPV1 has been challenged by more recent studies showing the activating effect of PIP 2 on TRPV1 in inside-out patches (50,51). Therefore, among mammalian TRP channels, the PIP 2 -induced inhibition appears to be unique for TRPC4␣.…”

Mechanism of Ca2+-dependent desensitization in TRP channels

“…25,26 TRPV channels are more controversial: although it is agreed that PI(4,5)P 2 plays an essential role in their modulation, whether PI(4,5)P 2 inhibits or activates TRPV1 is still an open question. [27][28][29][30] TRPV5 can be activated by PI(4,5)P 2 , and PI(4,5) P 2 seems to play a role in diminishing its physiological inhibition by Mg 2+ . 31 Modulation by the remaining mammalian TRP channels has not yet been reported, but it is likely that additional examples of lipid modulation will be identified.…”

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

“…PI(4,5)P 2 has been proposed to inhibit TRPV1, mediating the sensitizing actions of bradykinin and nerve growth factor (Chuang et al, 2001;Cao et al, 2013a). In contrast, others (including us) have proposed that PI(4,5)P 2 activates TRPV1 (Stein et al, 2006;Lukacs et al, 2007Lukacs et al, , 2013aKlein et al, 2008), and the depletion of PI(4,5)P 2 by phospholipase C may play a role in Ca 2+ -dependent desensitization (Mercado et al, 2010;Lukacs et al, 2013b). However, a recent study measured the apparent affinity of TRPV1 for diC8-PI(4,5)P 2 and found that the activating effects occurred with a K 1/2 of only 0.03 mol % in the plasma membrane, raising the question of whether PI(4,5)P 2 levels in the plasma membrane ever get sufficiently low selectivity must involve multiple, specific inter actions between TRPV1 and the phosphoinositide ligand.…”

“…The crystal structure of GIRK2 bound to PI(4,5)P 2 shows a structured binding pocket in which several hydrogen bonds are formed between basic residues in the protein and both the 4 and 5 phosphates (Whorton and MacKinnon, 2011). Based on this structure, it seems reasonable to predict that GIRK2 might display some phosphoinositide Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels the response of pain receptor neurons to noxious stimuli, disagreement about whether PI(4,5)P 2 activates or inhibits TRPV1 continues (Chuang et al, 2001;Prescott and Julius, 2003;Stein et al, 2006;Lukacs et al, 2007Lukacs et al, , 2013a; Ufret-Vincenty et al, 2011;Cao et al, 2013a,b;Senning et al, 2014). There is also disagreement about whether PI(4,5)P 2 interacts directly with TRPV1 or acts via the integral membrane protein Pirt (Kim et al, 2008;Ufret-Vincenty et al, 2011).…”