PROJECT SUMMARY Transient Receptor Potential Vanilloid 6 (TRPV6) is a Ca2+ selective ion channel playing important roles in intestinal Ca2+ absorption, male fertility and cancer development. Its expression level in the intestines is regulated by the active form of vitamin D. TRPV6 is member of the highly diverse TRP ion channel family. The only known common functional feature among TRP channels is their dependence on, and modulation by phosphoinositides, mostly phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Most TRP channels require factors other than PI(4,5)P2 to open, which are often in complex interaction with PI(4,5)P2 regulation. This complexity hinders understanding of the molecular mechanism of how PI(4,5)P2 opens these channels. TRPV6 is constitutively active, thus devoid of these complexities, and therefore is an ideal model to gain molecular insight in its regulation by PI(4,5)P2. In the current proposal, we will study PI(4,5)P2 regulation of TRPV6 channels using a combination of computational and experimental approaches. We have built a homology model of TRPV6 based on the recent nearly full-length, side-chain resolution structure of the related TRPV1 channels. We have computationally docked PI(4,5)P2 to both TRPV6 and TRPV1. In aims 1 and 2 we will use a combination of electrophysiological techniques and further computational simulations to test predictions of our model, and to gain insight into how PI(4,5)P2 open these channels. TRPV6 channels are constitutively active, but undergo Ca2+ induced inactivation, similar to many other Ca2+ channels, presumably to avoid toxic Ca2+ overload. In the previous funding period we developed a mutant of TRPV6 that is resistant to inhibition by PI(4,5)P2 depletion, due to its high affinity for this lipid. We also identified a mutant that does not bind calmodulin (CaM), thus it is resistant to inhibition by this Ca2+ binding protein. In aim 3, we will use these two mutants, as well as fluorescence-based monitoring of CaM association with the channel, and depletion of PI(4,5)P2 in measurements performed simultaneously with patch clamp recording of channel activity. These experiments will dissect the roles of CaM and PI(4,5)P2 depletion in Ca2+-induced inactivation of channel activity.