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.
There are essentially no pharmacological tools available to modulate active absorption of Ca2+ in the intestines, in which TRPV6 plays a major role. Clinically, both enhancing and reducing Ca2+ absorption may be desirable, because increased absorption of Ca2+ is the most important risk factor for kidney stones and decreased Ca2+ absorption plays an important role in the pathomechanism of osteoporosis. The better understanding of how TRPV6 is regulated, could potentially lead to novel ways to pharmacologically enhance or reduce intestinal Ca2+ absorption.
| Kasimova, Marina A; Yazici, Aysenur; Yudin, Yevgen et al. (2018) Ion Channel Sensing: Are Fluctuations the Crux of the Matter? J Phys Chem Lett 9:1260-1264 |
| Hughes, Taylor E T; Lodowski, David T; Huynh, Kevin W et al. (2018) Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM. Nat Struct Mol Biol 25:53-60 |
| Hughes, Taylor E T; Pumroy, Ruth A; Yazici, Aysenur Torun et al. (2018) Structural insights on TRPV5 gating by endogenous modulators. Nat Commun 9:4198 |
| Carnevale, Vincenzo (2018) Protonation underlies tonic vs. use-dependent block. Proc Natl Acad Sci U S A 115:3512-3514 |
| Kasimova, Marina A; Yazici, Aysenur Torun; Yudin, Yevgen et al. (2018) A hypothetical molecular mechanism for TRPV1 activation that invokes rotation of an S6 asparagine. J Gen Physiol 150:1554-1566 |
| Yudin, Yevgen; Rohacs, Tibor (2018) Inhibitory Gi/O-coupled receptors in somatosensory neurons: Potential therapeutic targets for novel analgesics. Mol Pain 14:1744806918763646 |
| Howard, Rebecca J; Carnevale, Vincenzo; Delemotte, Lucie et al. (2018) Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport. Biochim Biophys Acta Biomembr 1860:927-942 |
| Granata, Daniele; Ponzoni, Luca; Micheletti, Cristian et al. (2017) Patterns of coevolving amino acids unveil structural and dynamical domains. Proc Natl Acad Sci U S A 114:E10612-E10621 |
| van Keulen, Siri C; Gianti, Eleonora; Carnevale, Vincenzo et al. (2017) Does Proton Conduction in the Voltage-Gated H+ Channel hHv1 Involve Grotthuss-Like Hopping via Acidic Residues? J Phys Chem B 121:3340-3351 |
| Badheka, Doreen; Yudin, Yevgen; Borbiro, Istvan et al. (2017) Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein ?? subunits. Elife 6: |
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