PMC

Interactions of TRPV5 with PI(4,5)P₂. a View of the lower S6 helix and TRP domain of PI(4,5)P₂-bound TRPV5. The PI(4,5)P₂-bound TRPV5 is shown in green and the PI(4,5)P₂ in orange. b View of the lower S6 helix and TRP domain of the lipid-bound TRPV5 structure (pink). c The 5’ phosphate of PI(4,5)P₂ (orange) interacts with R302 of the N-linker and K484 of the S4-S5 linker in the PI(4,5)P₂-bound TRPV5 structure. d The lipid-bound structure showing the N-linker, S4-S5 linker, S6 helix, and TRP domain in pink

TRPV5 activation by PI(4,5)P₂ and inactivation by CaM. A schematic representation of the proposed binding of both PI(4,5)P₂ and CaM to TRPV5, inducing either channel activation or inactivation. A dimer of TRPV5 is shown as a gray diagram, PI(4,5)P₂ is shown in orange, CaM is shown in pink and the blue circles indicate the flow of calcium ions

CaM-bound structure of TRPV5. a Side and bottom view of the CaM-bound TRPV5 density map at 4.4 Å resolution. Density for TRPV5 is shown in gray, annular lipids shown in khaki, and CaM shown in hot pink. b Cartoon representation of the CaM-bound TRPV5 model in a side and bottom view. TRPV5 is shown in blue and CaM is shown in hot pink. c Pore diagram of CaM-bound TRPV5. Constriction residues are labeled and shown as sticks. d Plot of pore radii of CaM-bound TRPV5 as a function of distance through the pore is shown in blue. For reference, the lipid-bound TRPV5 pore graph is shown in pink. The dotted line indicates the radius of a calcium ion

Comparison between lipid-bound and PI(4,5)P₂-bound TRPV5. a The pore diagram of PI(4,5)P₂-bound TRPV5 is shown in green. b Lipid-bound TRPV5 (pink) superimposed onto the PI(4,5)P₂-bound (green) pore diagram shows a slight shift in the S6 helices and important gating residues originating at the π-helix. c Plot of pore radii of lipid-bound TRPV5 (pink) and PI(4,5)P₂-bound TRPV5 (green) as a function of distance through the pore. The dotted line indicates the radius of a calcium ion. d Extracellular view of the tetrameric selectivity filter of lipid-bound (pink) and PI(4,5)P₂-bound TRPV5 (green). e Intracellular view of the tetrameric lower gate of lipid-bound (pink) and PI(4,5)P₂-bound TRPV5 (green). f Zoomed-in view of the overlayed TMDs of lipid-bound (pink) and PI(4,5)P₂-bound TRPV5 (green). DiC₈ PI(4,5)P₂ is shown in orange sticks

Lipid-bound TRPV5 structure in detergent. a A schematic representation of the TRPV5 domains per channel monomer. Dashed lines indicate regions for which a model could not be built. b Density map of lipid-bound TRPV5 at 3.9 Å resolution. Density for TRPV5 is shown in gray and the densities attributed to annular lipids are shown in khaki. c Cartoon representation of the lipid-bound TRPV5 model. The TRPV5 tetramer is depicted as pink cartoons and annular lipids as khaki sticks. d Cartoon representation of the lipid-bound TRPV5 pore highlighting the three constriction points in the selectivity filter and lower gate. e Plot of pore radii of lipid-bound TRPV5 as a function of distance through the pore. The dotted line indicates the radius of a dehydrated calcium ion

PI(4,5)P₂-bound TRPV5 structure and PI(4,5)P₂-bound TRPV6 modeling. a The initial reconstruction of PI(4,5)P₂-bound TRPV5 in nanodiscs before masking and 3D classification. TRPV5 density is shown in gray, annular lipids are shown in khaki, and PI(4,5)P₂ is shown in orange. b PI(4,5)P₂-bound TRPV5 cryo-EM density in nanodiscs after focused 3D classification. TRPV5 density is shown in gray, annular lipids are shown in khaki, and PI(4,5)P₂ is shown in orange. c Zoomed-in view of the TRPV5 PI(4,5)P₂ -binding pocket. The PI(4,5)P₂-binding site in the TRPV5 channel is located between the N-linker (R302, R305), S4-S5 linker (K484), and the S6 helix (R584) of the channel. d A model produced by molecular dynamics (MD) simulations of the predicted interaction between the homologous TRPV6 channel and PI(4,5)P₂. e Neutralization of key PI(4,5)P₂ interacting residues increases sensitivity to depletion of PI(4,5)P₂ in TRPV6. The TRPV6 currents in oocytes measured before and after incubation with 35 μM wortmannin for 1 h. Current values after wortmannin treatment are normalized to current values before treatment. P-values for significance values are shown after rounding to the first non-zero digit (analysis of variance). f Neutralization of key PI(4,5)P₂ interacting residues increases sensitivity to depletion of PI(4,5)P₂ in TRPV5. The TRPV5 currents in oocytes measured before and after incubation with 35 μM wortmannin for 1 h. Current values after wortmannin treatment are normalized to current values before treatment. P-values for significance values are shown after rounding to the first non-zero digit (analysis of variance). The sample size (n) indicates the number of individual oocytes tested, from at least two different oocyte preparations. Error bars represent ±SEM

Mechanism of TRPV5 inactivation by CaM. a Structure of rat CaM bound to TRPV5 C-terminus fragments. Both the N-lobe and C-lobe were visualized along with N640-K652 and H699-T709 of TRPV5. CaM is shown in hot pink, and TRPV5 is shown in blue. Calcium ions bound to the N-lobes and C-lobes of CaM are shown as green spheres. b CaM interaction with chain A of TRPV5. c CaM C-lobe interaction with W702 on the TRPV5 C-terminus. d CaM N-lobe makes multiple hydrophobic interactions along N640-K652. e The base of the TRPV5 pore is shown with K116 of CaM bound at W583, causing a steric blockage of calcium flow. f The excised inside out patch clamp experiments show that wild type TRPV5 is inhibited upon addition of Ca²⁺ activated CaM. Representative current trace is at −100 mV; the applications of 25 μM diC₈ PI(4,5)P₂, 0.2 μM CaM and 3 μM free Ca²⁺ are indicated by the horizontal lines. g The excised inside out patch clamp experiments show that Ca²⁺-activated CaM did not inhibit the W583L TRPV5 mutant. Representative current trace is at −100 mV; the applications of 25 μM diC₈ PI(4,5)P₂, 0.2 μM CaM and 3 μM free Ca²⁺ are indicated by the horizontal lines. h Summary of the data, current amplitudes after CaM were divided by current values before CaM applications, P = 0.011 (two-sample t-test). The sample size (n) indicates the number of individual patches tested, each obtained from a different oocyte. Error bars represent ±SEM