Opening of the human epithelial calcium channel TRPV6

Abstract

Ca2+-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues1–4. Altered TRPV6 expression is associated with a variety of human diseases5, including cancers6. TRPV6 channels are constitutively active1,7,8 and their open probability depends on the lipidic composition of the membrane, increasing significantly in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2)7,9. We previously solved crystal structures of detergent-solubilized rat TRPV6 in the closed state10,11. Corroborating previous electrophysiological studies3, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that tightly binds Ca2+. However, it has remained unknown how TRPV6 channels open and close their pores for ion permeation. Here we present cryo-EM structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining S6 helices at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This novel gating mechanism, which defines the constitutive activity of TRPV6, is unique for tetrameric ion channels and provides new structural insights for understanding their diverse roles in physiology and disease.

Molecular mechanism of TRPV2 channel modulation by cannabidiol

“…Many previous TRPV cryo-EM maps have shown strong lipid densities in hydrophobic pockets in the TMD [15][16][17]21,22 . The "vanilloid" pocket of TRPV1 is occupied by phosphatidylinositol (PI) in the apo structure in nanodiscs 15 (Figure 5A), and TRPV5 16 and TRPV6 17 both report obvious densities for annular lipids in this pocket in proteins prepared in both detergents and nanodiscs.…”

“…The density for CBD in this binding pocket in the CBD-bound TRPV2 in nanodiscs structure is prominent and clearly present in both half maps (Supplementary Figure 11). As of the time of writing this manuscript, no other TRPV channel structures [15][16][17][18][19][20][21][22][23] were reported to have lipid density in this pocket, so in combination with the good fit of the drug to the density, we are confident in assigning it to CBD. With the identification of the CBD binding pocket in the nanodisc structure, we re-examined the map of CBD-bound TRPV2 in detergent at the same pocket (Supplementary Figure 11).…”

“…Instead, Tyr525 forms a hydrogen bond with the backbone amide of Met640, as recently seen in the apo TRPV3 structure from the Lee group between the homologous residues Tyr575 and Met672 (PBD 6MHO) ( Supplementary Figure 12). As these bonds are broken in their 2-APB sensitized TRPV3 structure featuring a π-helix in S6 (PDB 6MHS), the Lee group 22 suggests that this interaction stabilizes the α-helix of S6 preventing it from forming a π-helix, which is implicated in channel opening 17,21,22 (Supplementary Figure 12).…”

“…The TRPV channel subfamily members (TRPV1-TRPV6) have strong structural homology 13 , yet the effects of lipids on these channels are widely divergent [14][15][16][17][18] . Structural studies of members of this family have shown that both modulating and structural lipids can bind the "vanilloid" pocket located in the transmembrane domain (TMD) between the S3 and S4 helices and the helical S4-S5 linker of each monomer [15][16][17] . This pocket has also been shown to be a drug binding site in some TRPV channels and different occupants of this pocket can transmit conformational changes into the pore which affect gating 15,16 .…”

Cryo-EM for Small Molecules Discovery, Design, Understanding, and Application

“…In the grand scheme of things, answers can be found at any resolution, it depends on the questions asked. For example, analyses of large conformational changes to understand the mechanism of action of a given protein can be done (and be very informative) even at resolutions of 4–8 Å or less (for example (Blees et al, 2017) (McGoldrick et al,2018) (Lu et al, 2017)). But for a SBDD process we need to understand, explain and eventually predict changes in atoms positions, atom-atom interactions, and main chain or side chain motions as small as 0.5–1.5 Å.…”