Transient Receptor Potential (TRP) channels are calcium permeable ion channels that play roles in a multitude of biological processes. Despite their diversity of activation mechanisms, phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PIP2] have emerged as common regulators of these ion channels. Most TRP channels have been shown to require PIP2 for activity. TRPV6 is an epithelial Ca2+ channel responsible for active Ca2+ absorption in the intestine. The expression level of TRPV6 is regulated mainly by the active form of vitamin D3. Once expressed, TRPV6 is constitutively active, but its activity is limited by Ca2+-induced inactivation. We have shown earlier that the activity of this channel depends on the presence of PIP2, and that depletion of this lipid by phospholipase C (PLC) activation plays a major role in Ca2+-induced inactivation. Knowledge on the molecular mechanism of PIP2 activation of TRP channels is very limited. Our hypothesis is that PIP2 activates TRPV6 through binding to positively charged residues in the cytoplasmic regions, and this binding causes a conformational change in transmembrane domain 6 (TM6) leading to opening of the channel. TRPV6 is an ideal candidate to study the mechanism of activation by PIP2, because, unlike other TRP channels, it is constitutively active; its activity only depends on PIP2.
In aim 1 we will systematically mutate conserved positively charged amino acids in the cytoplasmic domains of TRPV6, to identify PIP2 interacting residues. We will test the effects of the mutations on the sensitivity of the channel to PIP2 using electrophysiological and biochemical techniques.
In Aim 2 we will use Cys-scanning mutagenesis to identify gating structures in TRPV6 that open upon PIP2 binding. Intracellular ATP has been proposed to directly bind to TRPV6 and its absence has been associated with channel rundown in whole-cell patch clamp experiments. We show that in excised patches ATP re-activates TRPV6 only in the presence of Mg2+. Our hypothesis is that MgATP provides substrate for lipid kinases and thus allows PIP2 re-synthesis. We will test this hypothesis in aim 3 by applying hydrolysable and non-hydrolysable analogues of ATP with and without Mg2+ in excised patches and on reconstituted channels in planar lipid bilayers. We will also test the effects of lipid kinase inhibitors in excises patches on TRPV6 activity induced by MgATP. Calmodulin has been proposed to be involved in Ca2+-induced inactivation of TRPV6, but the direct effects of CaM have not been demonstrated in excised patches. We show robust calcium-dependent inhibition of TRPV6 by CaM in excised patches. It is likely that Ca-CaM and phosphoinositide depletion act in concert to inhibit channel activity upon increased cytoplasmic Ca2+ concentrations.
In aim 4 we will study the relationship between CaM and PIP2 regulation of TRPV6, using the combination of electrophysiology, biochemistry and molecular biology.

Public Health Relevance

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Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
6R01GM093290-04
Application #
8784840
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Nie, Zhongzhen
Project Start
2011-04-01
Project End
2015-03-31
Budget Start
2013-07-01
Budget End
2014-03-31
Support Year
Fiscal Year
2013
Total Cost
$201,976
Indirect Cost
$74,947
Name
Rutgers University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
078795851
City
Newark
State
NJ
Country
United States
Zip Code
07103
Carnevale, Vincenzo; Rohacs, Tibor (2016) TRPV1: A Target for Rational Drug Design. Pharmaceuticals (Basel) 9:
Rohacs, Tibor (2016) Phosphoinositide signaling in somatosensory neurons. Adv Biol Regul 61:2-16
Elokely, Khaled; Velisetty, Phanindra; Delemotte, Lucie et al. (2016) Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin. Proc Natl Acad Sci U S A 113:E137-45
Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A et al. (2016) A molecular determinant of phosphoinositide affinity in mammalian TRPV channels. Sci Rep 6:27652
Badheka, Doreen; Borbiro, Istvan; Rohacs, Tibor (2015) Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel. J Gen Physiol 146:65-77
Rohacs, Tibor (2015) Phosphoinositide regulation of TRPV1 revisited. Pflugers Arch 467:1851-69
Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor (2015) Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides. Sci Signal 8:ra15
Rohacs, Tibor (2014) Phosphoinositide regulation of TRP channels. Handb Exp Pharmacol 223:1143-76
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
Lukacs, Viktor; Rives, Jan-Michael; Sun, Xiaohui et al. (2013) 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. J Biol Chem 288:35003-13

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