Autoinhibition of TRPV6 Channel and Regulation by PIP2

Abstract

Summary Transient receptor potential vanilloid 6 (TRPV6), a calcium-selective channel possessing six transmembrane domains (S1-S6) and intracellular N and C termini, plays crucial roles in calcium absorption in epithelia and bone and is involved in human diseases including vitamin-D deficiency, osteoporosis, and cancer. The TRPV6 function and regulation remain poorly understood. Here we show that the TRPV6 intramolecular S4-S5 linker to C-terminal TRP helix (L/C) and N-terminal pre-S1 helix to TRP helix (N/C) interactions, mediated by Arg470:Trp593 and Trp321:Ile597 bonding, respectively, are autoinhibitory and are required for maintaining TRPV6 at basal states. Disruption of either interaction by mutations or blocking peptides activates TRPV6. The N/C interaction depends on the L/C interaction but not reversely. Three cationic residues in S5 or C terminus are involved in binding PIP2 to suppress both interactions thereby activating TRPV6. This study reveals “PIP2 - intramolecular interactions” regulatory mechanism of TRPV6 activation-autoinhibition, which will help elucidating the corresponding mechanisms in other TRP channels.

Auto-inhibitory intramolecular S5/S6 interaction in the TRPV6 channel regulates breast cancer cell migration and invasion

“…While the S4–S5 linker in TRPV6 is a critical regulatory element for its channel function 14 the functional of helix S5 which is in close proximity to the pore-forming S6 as revealed by structures 15 is not well understood. Among the three conserved positively charged residues (K524, R510, and R532) in the S4–S5 linker or S5 helix, R510 mediates the S4–S5 linker/TRP domain binding, K524 is critical for TRPV6 activation by PIP2 6 , 16 , but the role of R532 in S5 is unclear. Interestingly, the Catalogue of Somatic Mutations in Cancer (COSMIC) database predicted R510Q and R532Q as pathogenic mutations 17 .…”

“…Xenopus oocyte expression vector pBSMXT-MCS encoding human TRPV6 (accession number: Q9H1D0, containing 40 additional amino-acid residues at the start of the N-terminus compared to the one used in structural studies) cDNA was generated previously 6 . For mammalian cell expression, human TRPV6 cDNA was subcloned into a pCMV vector.…”

“…Secondary antibodies conjugated with HRP were purchased from GE healthecare. Biotinylation assays were performed as described previously 6 . Oocytes were washed with ice-cold PBS and incubated with 0.5 mg/ml sulfo-NHS-SS-Biotin (Pierce, Rockford, IL), followed by quenching 1 M NH 4 Cl.…”

“…The S6 helix was reported to undergo transition from an α helical configuration in the closed state to a π helical configuration in an open state, which enlarges the pore gate. We recently identified functionally autoinhibitory intramolecular interactions of the S4–S5 linker and pre-S1 domain with the TRP domain in TRPV6 and found that each interaction is mediated by a conserved residue pair and negatively regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) 6 . TRPV6 channel function substantially increased when either interaction was disrupted 6 .…”

“…We recently identified functionally autoinhibitory intramolecular interactions of the S4–S5 linker and pre-S1 domain with the TRP domain in TRPV6 and found that each interaction is mediated by a conserved residue pair and negatively regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) 6 . TRPV6 channel function substantially increased when either interaction was disrupted 6 . Structures of TRPV6 suggest the presence of more intramolecular interactions, which have yet to be investigated and may be relevant to TRPV6-dependent physiology and pathology.…”