Transient receptor potential vanilloid 6 (TRPV6) channels play an important role in Ca(2+) absorption in the intestines. Both phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and cytoplasmic ATP have been proposed to be important for maintaining TRPV6 activity. To evaluate whether PtdIns(4,5)P(2) and ATP affect channel activity directly or indirectly, we have used a dual approach, examining channel activity in excised patches and planar lipid bilayers. In excised inside-out patch-clamp measurements, ATP reactivated the human TRPV6 channels after current rundown only in the presence of Mg(2+). The effect of MgATP was inhibited by 3 structurally different compounds that inhibit type III phosphatidylinositol 4-kinases (PI4Ks). PtdIns(4,5)P(2) also activated TRPV6 in excised patches, while its precursor PtdIns(4)P had only minimal effect. These data demonstrate that MgATP provides substrate for lipid kinases, allowing the resynthesis of PtdIns(4,5)P(2). To determine whether PtdIns(4,5)P(2) is a direct activator of TRPV6, we purified and reconstituted the channel protein in planar lipid bilayers. The reconstituted channel showed high activity in the presence of PtdIns(4,5)P(2), while PtdIns(4)P induced only minimal activity. Our data establish PtdIns(4,5)P(2) as a direct activator of TRPV6 and demonstrate that intracellular ATP regulates the channel indirectly as a substrate for type III PI4Ks.
"Accordingly, TRPV5 À/À mice have disturbances in Ca 2þ reabsorption in the kidneys (Hoenderop et al., 2003), TRPV6 À/À mice have moderately impaired Ca 2þ absorption in the duodenum (Bianco et al., 2007) and the male TRPV6 À/À animals are infertile (Weissgerber et al., 2011). The activity of both of these channels depend on PI(4,5)P 2 (Lee et al., 2005b; Rohacs et al., 2005; Thyagarajan et al., 2008; Zakharian et al., 2011). Both channels are constitutively active, but undergo Ca 2þ -induced inactivation. "
[Show abstract][Hide abstract] ABSTRACT: Transient Receptor Potential (TRP) channels were discovered while analyzing visual mutants in Drosophila. The protein encoded by the transient receptor potential (trp) gene is a Ca(2+) permeable cation channel activated downstream of the phospholipase C (PLC) pathway. While searching for homologs in other organisms, a surprisingly large number of mammalian TRP channels was cloned. The regulation of TRP channels is quite diverse, but many of them are either activated downstream of PLC, or modulated by it. This review will summarize the current knowledge on regulation of TRP channels by PLC, with special focus on TRPC-s, which can be considered as effectors of PLC and the heat- and capsaicin-sensitive TRPV1, which is modulated by the PLC pathway in a complex manner.
[Show abstract][Hide abstract] ABSTRACT: The activity of the cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels diminishes over time in the presence of extracellular Ca(2+), a phenomenon referred to as desensitization or adaptation. Here we show that activation of TRPM8 by cold or menthol evokes a decrease in cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] levels. The decrease in PtdIns(4,5)P(2) levels was accompanied by increased inositol 1,4,5 trisphosphate (InsP(3)) production, and was inhibited by loading the cells with the Ca(2+) chelator BAPTA-AM, showing that it was the consequence of the activation of phospholipase C (PLC) by increased intracellular Ca(2+) concentrations. PtdIns(4,5)P(2) hydrolysis showed excellent temporal correlation with current desensitization in simultaneous patch clamp and fluorescence-based PtdIns(4,5)P(2) level measurements. Intracellular dialysis of PtdIns(4,5)P(2) inhibited desensitization both in native neuronal and recombinant TRPM8 channels. PtdIns(4)P, the precursor of PtdIns(4,5)P(2), did not inhibit desensitization, consistent with its minimal effect in excised patches. Omission of MgATP from the intracellular solution accelerated desensitization, and MgATP reactivated TRPM8 channels in excised patches in a phosphatidylinositol 4-kinase (PI4K)-dependent manner. PLC-independent depletion of PtdIns(4,5)P(2) using a voltage-sensitive phosphatase (ci-VSP) inhibited TRPM8 currents, and omission of ATP from the intracellular solution inhibited recovery from this inhibition. Inhibitors of PKC had no effect on the kinetics of desensitization. We conclude that Ca(2+) influx through TRPM8 activates a Ca(2+)-sensitive PLC isoform, and the resulting depletion of PtdIns(4,5)P(2) plays a major role in desensitization of both cold and menthol responses.
The Journal of Physiology 12/2011; 589(Pt 24):6007-27. DOI:10.1113/jphysiol.2011.220228 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly-(R)-3-hydroxybutyrates (PHB), linear polymers of (R)-3-hydroxybutyrate, are components of all biological cells in which short polymers (<200 monomer residues) are covalently attached to certain proteins and/or noncovalently associated with polyphosphates - inorganic polyphosphate (polyP), RNA, and DNA. The low concentrations, lack of unusual atoms or functional groups, and flexible backbones of this complexed PHB, referred to as cPHB, make them invisible to many analytical procedures; whereas other physical properties - water-insolubility, high intrinsic viscosity, temperature sensitivity, multiple bonding interactions with other molecules - make them requisite participants in vital physiological processes as well as contributors to the development of certain diseases.
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