The objective of this grant renewal proposal is to understand the molecular mechanism of regulation of sensory TRP channels by phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] or PIP2. TRPV1 channels are activated by heat, capsaicin, low pH and a plethora of other pain producing stimuli. They are sensitized to low-grade stimuli through activation of G-protein coupled receptors (GPCRs) by pro-inflammatory agents such as bradykinin. Sensitization plays an important role in thermal hyperalgesia, a disturbing symptom in neuropathic pain syndromes. GPCRs involved in sensitization usually activate phospholipase C (PLC). Both protein kinase C (PKC) and the reduction in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] have been implicated in sensitization of TRPV1. Strong pharmacological activation of TRPV1 with capsaicin on the other hand leads to desensitization of TRPV1, a phenomenon thought to underlie the use of capsaicin as a local analgesic. Intriguingly PLC activation also plays a crucial role in desensitization of TRPV1. The major goal of this proposal is to understand how two phenomena, sensitization and desensitization, with opposite results on channel activity, can be mediated by the same signaling pathway. The activity of TRPV1, similar to many other TRP channels, depends on the presence of PI(4,5)P2. In addition, PI(4,5)P2 also partially inhibits the channel in intact cell. W found that GPCR activation by bradykinin in sensory neurons leads to the selective decrease of PI(4,5)P2, but not its precursor PI(4)P. Since both PI(4)P and PI(4,5)P2 can support TRPV1, this would not significantly limit channel activity. The selective decrease in PI(4,5)P2 on the other hand synergizes with PKC to potentiate TRPV1 activity. This effect was mimicked by disruption of the tubulin cytoskeleton. During desensitization on the other hand, TRPV1 stimulation by saturating capsaicin concentrations leads to a strong depletion of both PI(4,5)P2 and PI(4)P leading to diminished channel activity. The cold and menthol sensitive TRPM8 is inhibited upon GPCR activation by bradykinin, presumably since the activity of this channel specifically depends on PI(4,5)P2. Our overall hypothesis is that differential regulation of PI(4,5)P2 and PI(4)P levels by different PLC isoforms determines specificity of regulation of phosphoinositide sensitive TRP channels, depending on their lipid specificity profile. In this proposal we will systematically examine this hypothesis using a wide range of approaches including studying recombinant and native channels in sensory neurons, using various fluorescence-based lipid sensors to monitor levels of phosphoinositides in live cells, single cell real time PCR, patch clamp and studying the nerve impulses evoked by capsaicin in the ex-vivo skin-nerve preparation. We propose the following specific aims:
Aim 1 Evaluate the role of PLC activation in sensitization of TRPV1 by pro-inflammatory agents.
Aim 2 Evaluate the role of PLC activation in desensitization of capsaicin-induced currents.
Aim 3 Evaluate the role of PLC activation in regulation of TRPM8 by GPCRs and Ca2+ influx.

Public Health Relevance

Chronic pain is an unmet medical need;associated costs have been estimated in the United States at $150 billion annually. Both intense heat and cold may cause pain, and hypersensitivity to these stimuli is a major symptom of neuropathic pain. We study the molecular regulation of thermosensitive ion channels, and our work has the potential to lead to better therapeutic strategies against pain, and better understanding of the mechanism of chemotherapy-induced neuropathic pain.

National Institute of Health (NIH)
Research Project (R01)
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Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
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Silberberg, Shai D
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Rutgers University
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Lukacs, Viktor; Yudin, Yevgen; Hammond, Gerald R et al. (2013) Distinctive changes in plasma membrane phosphoinositides underlie differential regulation of TRPV1 in nociceptive neurons. J Neurosci 33:11451-63
Cao, Chike; Zakharian, Eleonora; Borbiro, Istvan et al. (2013) Interplay between calmodulin and phosphatidylinositol 4,5-bisphosphate in Ca2+-induced inactivation of transient receptor potential vanilloid 6 channels. J Biol Chem 288:5278-90
Rohacs, Tibor (2013) Regulation of transient receptor potential channels by the phospholipase C pathway. Adv Biol Regul 53:341-55
Cao, Chike; Yudin, Yevgen; Bikard, Yann et al. (2013) Polyester modification of the mammalian TRPM8 channel protein: implications for structure and function. Cell Rep 4:302-15
Yudin, Yevgen; Rohacs, Tibor (2012) Regulation of TRPM8 channel activity. Mol Cell Endocrinol 353:68-74
Zakharian, Eleonora; Cao, Chike; Rohacs, Tibor (2011) Intracellular ATP supports TRPV6 activity via lipid kinases and the generation of PtdIns(4,5) P?. FASEB J 25:3915-28
Yudin, Yevgen; Lukacs, Viktor; Cao, Chike et al. (2011) Decrease in phosphatidylinositol 4,5-bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels. J Physiol 589:6007-27
Zakharian, Eleonora; Cao, Chike; Rohacs, Tibor (2010) Gating of transient receptor potential melastatin 8 (TRPM8) channels activated by cold and chemical agonists in planar lipid bilayers. J Neurosci 30:12526-34
Thyagarajan, Baskaran; Benn, Bryan S; Christakos, Sylvia et al. (2009) Phospholipase C-mediated regulation of transient receptor potential vanilloid 6 channels: implications in active intestinal Ca2+ transport. Mol Pharmacol 75:608-16
Rohacs, Tibor (2009) Phosphoinositide regulation of non-canonical transient receptor potential channels. Cell Calcium 45:554-65

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