Intracellular ATP supports TRPV6 activity via lipid kinases and the generation of PtdIns(4,5)P2

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.