Viktor Lukacs

Dual Regulation of TRPV1 by Phosphoinositides

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PIP2] regulates many ion channels. There are conflicting reports on the effect of PtdIns(4,5)P2 on transient receptor potential vanilloid 1 (TRPV1) channels. We show that in excised patches PtdIns(4,5)P2 and other phosphoinositides activate and the PIP2 scavenger poly-Lys inhibits TRPV1. TRPV1 currents undergo desensitization on exposure to high concentrations of capsaicin in the presence of extracellular Ca2+. We show that in the presence of extracellular Ca2+, capsaicin activates phospholipase C (PLC) in TRPV1-expressing cells, inducing depletion of both PtdIns(4,5)P2 and its precursor PtdIns(4)P (PIP). The PLC inhibitor U73122 and dialysis of PtdIns(4,5)P2 or PtdIns(4)P through the patch pipette inhibited desensitization of TRPV1, indicating that Ca2+-induced activation of PLC contributes to desensitization of TRPV1 by depletion of PtdIns(4,5)P2 and PtdIns(4)P. Selective conversion of PtdIns(4,5)P2 to PtdIns(4)P by a rapamycin-inducible PIP2 5-phosphatase did not inhibit TRPV1 at high capsaicin concentrations, suggesting a significant role for PtdIns(4)P in maintaining channel activity. Currents induced by low concentrations of capsaicin and moderate heat, however, were potentiated by conversion of PtdIns(4,5)P2 to PtdIns(4)P. Increasing PtdIns(4,5)P2 levels by coexpressing phosphatidylinositol-4-phosphate 5-kinase inhibited TRPV1 at low but not at saturating capsaicin concentrations. These data show that at low capsaicin concentrations and other moderate stimuli, PtdIns(4,5)P2 partially inhibits TRPV1 in a cellular context, but this effect is likely to be indirect, because it is not detectable in excised patches. We conclude that phosphoinositides have both inhibitory and activating effects on TRPV1, resulting in complex and distinct regulation at various stimulation levels.

Phospholipase C mediated modulation of TRPV1 channels

The transient receptor potential vanilloid type 1 (TRPV1) channels are involved in both thermosensation and nociception. They are activated by heat, protons, and capsaicin and modulated by a plethora of other agents. This review will focus on the consequences of phospholipase C (PLC) activation, with special emphasis on the effects of phosphatidylinositol 4,5-bisphosphate (PIP2) on these channels. Two opposing effects of PIP2 have been reported on TRPV1. PIP2 has been proposed to inhibit TRPV1, and relief from this inhibition was suggested to be involved in sensitization of these channels by pro-inflammatory agents. In excised patches, however, PIP2 was shown to activate TRPV1. Calcium flowing through TRPV1 activates PLC and the resulting depletion of PIP2 was proposed to play a role in capsaicin-induced desensitization of these channels. We will describe the data indicating involvement of PLC and PIP2 in sensitization and desensitization of TRPV1 and will also discuss other pathways potentially contributing to these two phenomena. We attempt to resolve the seemingly contradictory data by proposing that PIP2 can both activate and inhibit TRPV1 depending on the experimental conditions, more specifically on the level of stimulation of these channels. Finally, we also discuss data in the literature indicating that other TRP channels, TRPA1 and some members of the TRPC subfamily, may also be under a similar dual control by PIP2.

Decrease in phosphatidylinositol 4,5-bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels

Non‐technical summary  The transient receptor potential melastatin 8 (TRPM8) ion channel is a physiological sensor of environmental cold temperatures. This channel is also activated by menthol, which is responsible for the cooling sensation evoked by this compound. It is well known that we adapt to moderately cold temperatures, i.e. the same temperature feels less cold over time, and the cooling effects of menthol also wear off with time, presumably due to the calcium‐dependent desensitization of TRPM8 activity. The membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) is known to be required for TRPM8 activity. Our data support a model for desensitization, in which calcium influx through TRPM8 activates a phospholipase C enzyme, which breaks down PIP2, leading to decreased channel activity.

Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate Mediates Calcium-induced Inactivation of TRPV6 Channels

TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca2+ absorption in the intestines. These channels display high selectivity for Ca2+, but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca2+ concentrations. Here we studied the mechanism of this Ca2+-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca2+, but not Ba2+. We also show that Ca2+, but not Ba2+, influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC8 PI(4,5)P2 through the patch pipette inhibited Ca2+-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P2 also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P2, neither activated TRPV6 in excised patches nor had any effect on Ca2+-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P2 to PI(4)P by a rapamycin-inducible PI(4,5)P2 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca2+ entry into TRPV6-expressing cells. We propose that Ca2+ influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P2 contributes to the inactivation of TRPV6.

Distinctive Changes in Plasma Membrane Phosphoinositides Underlie Differential Regulation of TRPV1 in Nociceptive Neurons

Transient Receptor Potential Vanilloid 1 (TRPV1) is a polymodal, Ca2+-permeable cation channel crucial to regulation of nociceptor responsiveness. Sensitization of TRPV1 by G-protein coupled receptor (GPCR) agonists to its endogenous activators, such as low pH and noxious heat, is a key factor in hyperalgesia during tissue injury as well as pathological pain syndromes. Conversely, chronic pharmacological activation of TRPV1 by capsaicin leads to calcium influx-induced adaptation of the channel. Paradoxically, both conditions entail activation of phospholipase C (PLC) enzymes, which hydrolyze phosphoinositides. We found that in sensory neurons PLCβ activation by bradykinin led to a moderate decrease in phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), but no sustained change in the levels of its precursor PI(4)P. Preventing this selective decrease in PI(4,5)P2 inhibited TRPV1 sensitization, while selectively decreasing PI(4,5)P2 independently of PLC potentiated the sensitizing effect of protein kinase C (PKC) on the channel, thereby inducing increased TRPV1 responsiveness. Maximal pharmacological TRPV1 stimulation led to a robust decrease of both PI(4,5)P2 and its precursor PI(4)P in sensory neurons. Attenuating the decrease of either lipid significantly reduced desensitization, and simultaneous reduction of PI(4,5)P2 and PI(4)P independently of PLC inhibited TRPV1. We found that, on the mRNA level, the dominant highly Ca2+-sensitive PLC isoform in dorsal root ganglia is PLCδ4. Capsaicin-induced desensitization of TRPV1 currents was significantly reduced, whereas capsaicin-induced nerve impulses in the skin–nerve preparation increased in mice lacking this isoform. We propose a comprehensive model in which differential changes in phosphoinositide levels mediated by distinct PLC isoforms result in opposing changes in TRPV1 activity.

Promiscuous activation of transient receptor potential vanilloid 1 (TRPV1) channels by negatively charged intracellular lipids: the key role of endogenous phosphoinositides in maintaining channel activity.

Background: Regulation of TRPV1 by phosphoinositides is controversial. Results: ATP reactivates TRPV1 after run-down in excised inside-out patches by generating phosphoinositides; many other negatively charged lipids may also support TRPV1 activity. Conclusion: Despite its promiscuous activation, phosphoinositides are the key endogenous cofactors for TRPV1 activity. Significance: Our data may reconcile discordant data obtained in different experimental settings. The regulation of the heat- and capsaicin-activated transient receptor potential vanilloid 1 (TRPV1) channels by phosphoinositides is controversial. Data in cellular systems support the dependence of TRPV1 activity on phosphoinositides. The purified TRPV1, however, was recently shown to be fully functional in artificial liposomes in the absence of phosphoinositides. Here, we show that several other negatively charged phospholipids, including phosphatidylglycerol, can also support TRPV1 activity in excised patches at high concentrations. When we incorporated TRPV1 into planar lipid bilayers consisting of neutral lipids, capsaicin-induced activity depended on phosphatidylinositol 4,5-bisphosphate. We also found that TRPV1 activity in excised patches ran down and that MgATP reactivated the channel. Inhibition of phosphatidylinositol 4-kinases or enzymatic removal of phosphatidylinositol abolished this effect of MgATP, suggesting that it activated TRPV1 by generating endogenous phosphoinositides. We conclude that endogenous phosphoinositides are positive cofactors for TRPV1 activity. Our data highlight the importance of specificity in lipid regulation of ion channels and may reconcile discordant data obtained in various experimental settings.

Phosphoinositide Regulation of TRPV1

TRPV1 is a nonselective calcium permeable cation channel present in polymodal nociceptors that plays a crucial role in the development of inflammatory pain and thermal hypersensitivity. Plasmamembrane phosphoinositides are recognized as important regulators of TRPV1; the precise nature of their effect is, however, controversial. We and others have shown that phosphatidyl-inositol-(4,5)-bisphosphate [PI(4,5)P2] as well as other phosphoinositides activate TRPV1 in excised patches. Calcium influx via TRPV1 channels activates PLC and results in a robust depletion of both phosphatidyl-inositol-(4)- phosphate [PI(4)P] and PI(4,5)P2 in expression systems. Hydrolysis of PI(4,5)P2 under these circumstances is now accepted to be an important contributor to channel desensitization. However, the involvement of PI(4)P in the process remains an issue of debate. In addition, preceding data corroborated by our own observations suggest an additional indirect inhibitory effect of PI(4,5)P2, but not other phosphoinositides. In the present work we show that potentiation of whole-cell TRPV1 currents by bradykinin is partially impaired by dialyzing diC8-PI(4,5)P2 through the patch pipette in both expression systems and native cells (DRG neurons), supporting the notion that in addition to the well documented role of PKC-mediated phosphorylation in TRPV1 channel sensitization, PI(4,5)P2 depletion may also be involved in this phenomenon. Using mouse DRG neurons we confirm that in addition to PI(4,5)P2, PI(4)P is also an important activator of TRPV1. Both lipids are simultaneously depleted in response to TRPV1-activation, while dialysis of both lipids via the patch pipette reduced desensitization of TRPV1-positive neurons. In contrast Bradykinin receptor activation differentially regulated PI(4,5)P2 and PI(4)P abundance in DRG neurons, resulting in isolated PI(4,5)P2 depletion. These observations suggest important differences in phosphoinositides handling during calcium-activated and receptor-induced PLC activation, respectively, and may partially explain the differential TRPV1 regulation under these conditions.