A molecular determinant of phosphoinositide affinity in mammalian TRPV channels.

Transient Receptor Potential Vanilloid 6 (TRPV6) is a constitutively active, Ca2+ selective, inwardly rectifying ion channel. It constitutes the initial entry step in the transcellular Ca2+ transport process in intestinal epithelial cells1. Its genetic deletion in mice causes disturbances in extracellular Ca2+ homeostasis2,3. Ca2+ influx via TRPV6 in the epidydimis is required for male fertility in mice4. In the absence of divalent cations, TRPV6 also conducts monovalent currents, which are much larger than Ca2+ currents and thus are widely used to assess the activity of these channels. The activity of TRPV6, similarly to most TRP channels, depends on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]5,6. TRP channels are activated by diverse cellular and environmental signals7. Their only known common functional feature is modulation by phosphoinositides, mainly PI(4,5)P2. The vast majority of TRP channels are positively regulated by PI(4,5)P2, but negative effects may coexist on some TRP channels, such as TRPV1 or some TRPC-s see recent reviews for further details8,9,10. PI(4,5)P2 and its precursor PI(4)P are the most abundant phosphoinositides in the plasma membrane. While PI(4,5)P2 received the most attention, PI(4)P may also play distinct roles in regulation of some TRP channels11,12. Ion channels, including TRPs, are generally thought to be activated by phosphoinositides via direct interactions between the negatively charged head group of the lipid and (mainly) positively charged residues in the cytoplasmic domains of the channel protein. How TRP channels interact with this phospholipid has been the subject of intensive research and many different cytoplasmic regions have been implicated. Most efforts focused on the roles of various parts of the cytoplasmic C-terminus13,14,15,16. The apparent affinity of an ion channel to phosphoinositides is a major determinant of its inhibition by PI(4,5)P2 depletion upon activation of phospholipase C (PLC)10,17,18. It is not very well understood, however, what determines the affinity of mammalian ion channels for PI(4,5)P2. Earlier work on mammalian Kir channels found that PI(4,5)P2 interacting residues are generally conserved between family members, and the difference between high and low affinity channels could be traced to a subtle conformational change caused by a relatively conservative substitution of Leucin to Isoleucin19. Recently it was shown that a Kir channel from a marine sponge Amphimedon queenslandica displays very weak interactions with PI(4,5)P2, and lacks two key positively charged PI(4,5)P2 interacting residues that are highly conserved in mammalian Kir channels20. Reintroduction of the two positively charged residues substantially strengthened the interactions of the invertebrate Kir channel with PI(4,5)P220. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6, lack a positively charged residue in the TM4-TM5 loop, equivalent of which was shown to interact with PI(4,5)P2 in TRPV121, a channel with high affinity for phosphoinositides22. When we reintroduced this positive charge to TRPV6 (G488R), the mutant showed markedly slower rundown in excised patches and increased apparent affinity for dioctanoyl (diC8) PI(4,5)P2. The mutant channel was completely resistant to depletion of PI(4,5)P2 by the voltage-sensitive PI(4,5)P2 5′-phosphatase from Ciona intestinalis (ciVSP)23, and showed essentially no Ca2+-induced inactivation, presumably because of its increased affinity for PI(4,5)P2. Computational modeling showed that in the mutant TRPV6 the introduced 488R residue interacted with PI(4,5)P2.