Direct Activation of TRPV6 Channels by Phosphatidylinositol 4,5-Bisphosphate

Abstract

Transient Receptor Potential Vanilloid 6 (TRPV6) is an inwardly rectifying Ca2+ selective ion channel that plays a role in Ca2+ absorption in the intestines. The activity of these channels have been shown to depend on the presence of phosphatidylinositol 4,5-bisphosphate (PIP2). Increased in cytoplasmic Ca2+ concentrations inhibit these channels, protecting cells from toxic Ca2+ overload. Both Ca2+ Calmodulin (CaCaM) and depletion of PIP2 have been implicated in Ca2+ induced inactivation. In addition, it has been proposed that cytoplasmic ATP binds directly to the channels, and this binding is important for maintaining TRPV6 channel activity in whole-cell patch clamp experiments. To evaluate whether PIP2, CaCaM and ATP affects channel activity directly or indirectly, we have performed excised inside-out patch clamp measurements with these compounds. Channel activity upon excision showed marked current run-down, characteristic of PIP2 dependent channels. ATP reactivated the channels, but only in the presence of Mg2+. This could mean that MgATP provides substrate for lipid kinases present in the patch membrane, allowing the resynthesis of PIP2. To test this hypothesis, we have used wortmannin and LY294002 at concentrations where they inhibit phosphatidylinositol 4-kinases, and found that they both inhibit the effect of MgATP in excised patches. At lower concentrations, where they specifically inhibit PI3Kinases, they did not inhibit the effects of MgATP. We also found that PIP2 reactivates TRPV6 in excised patches, and this effect is inhibited by CaCaM. To further establish PIP2 as a direct activator of TRPV6, we purified the channel protein and reconstituted it in planar lipid bilayers. We found that the reconstituted protein showed inward rectification, and its activity depended on the presence of PIP2. Our data establish PIP2 as a direct activator of TRPV6 and raise doubt that intracellular ATP regulates the channel through direct binding.