Abstract
TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery.
Highlights
transient receptor potential vanilloid 5 (TRPV5) is a transient receptor potential channel involved in calcium reabsorption
The vast majority of this TRPV5 cryo-electron microscopy (cryo-EM) map is at high resolution (3–3.5 Å) as side chains are clearly visible in the transmembrane region of the channel (Supplementary Figure 1, Supplementary Figure 3)
This allowed for accurate model building in both the transmembrane domain (TMD) and the ankyrin repeat domain (ARD) (Fig. 1c, Supplementary Figure 3)
Summary
TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys[116] on the C-lobe of calcium-activated CaM and Trp[583] at the intracellular gate of TRPV5 Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery. While the role of the distal C-terminal binding site is well established in CaM-mediated inactivation of TRPV510 and the closely related TRPV611–13, the binding stoichiometry and the conformational changes that take place as a consequence of the binding of CaM to the channel have been unclear[3] The absence of this information prevents us from further understanding TRPV5 function and regulation in the kidney. Investigating TRPV5 modulation with cryoEM permitted us to gain insight regarding TRPV5 gating and potentially form the basis for rational drug design for the treatment and prevention of hypercalciuria and nephrolithiasis in future studies
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