Cryo Electron Microscopy of TRP Channels.

Cryo electron microscopy (cryo-EM) is a powerful technique that can be used to elucidate the structural architecture of a protein molecule in a physiologically relevant environment. In this method, purified protein is frozen in its aqueous buffer in a thin layer of vitreous ice in which the biological macromolecules are embedded in various orientations. Images of this frozen sample are collected with an electron microscope, and the data is processed using different software algorithms resulting in high-resolution structures of the protein. Proteins in the presence of various ligands or other macromolecular complexes can also be studied by this method. Here, we present a protocol for the purification and vitrification of TRP channels for single particle cryo-EM.

Structural insights on TRPV5 gating by endogenous modulators.

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.

Transient Receptor Potential (TRP) Channels.

Transient Receptor Potential (TRP) channels are evolutionarily conserved integral membrane proteins. The mammalian TRP superfamily of ion channels consists of 28 cation permeable channels that are grouped into six subfamilies based on sequence homology (Fig. 6.1). The canonical TRP (TRPC) subfamily is known for containing the founding member of mammalian TRP channels. The vanilloid TRP (TRPV) subfamily has been extensively studied due to the heat sensitivity of its founding member. The melastatin-related TRP (TRPM) subfamily includes some of the few known bi-functional ion channels, which contain functional enzymatic domains. The ankyrin TRP (TRPA) subfamily consists of a single chemo-nociceptor that has been proposed to be a target for analgesics. The mucolipin TRP (TRPML) subfamily channels are found primarily in intracellular compartments and were discovered based on their critical role in type IV mucolipidosis (ML-IV). The polycystic TRP (TRPP) subfamily is a diverse group of proteins implicated in autosomal dominant polycystic kidney disease (ADPKD). Overall, this superfamily of channels is involved in a vast array of physiological and pathophysiological processes making the study of these channels imperative to our understanding of subcellular biochemistry.

Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM.

The transient receptor potential vanilloid 5 (TRPV5) channel is a member of the transient receptor potential (TRP) channel family, which is highly selective for Ca2+, that is present primarily at the apical membrane of distal tubule epithelial cells in the kidney and plays a key role in Ca2+ reabsorption. Here we present the structure of the full-length rabbit TRPV5 channel as determined using cryo-EM in complex with its inhibitor econazole. This structure reveals that econazole resides in a hydrophobic pocket analogous to that occupied by phosphatidylinositides and vanilloids in TRPV1, thus suggesting conserved mechanisms for ligand recognition and lipid binding among TRPV channels. The econazole-bound TRPV5 structure adopts a closed conformation with a distinct lower gate that occludes Ca2+ permeation through the channel. Structural comparisons between TRPV5 and other TRPV channels, complemented with molecular dynamics (MD) simulations of the econazole-bound TRPV5 structure, allowed us to gain mechanistic insight into TRPV5 channel inhibition by small molecules.