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

Abstract

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.