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Biochem Biophys Res Commun
2017 Oct 21;4923:362-367. doi: 10.1016/j.bbrc.2017.08.102.
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The L530R variation associated with recurrent kidney stones impairs the structure and function of TRPV5.
Wang L
,
Holmes RP
,
Peng JB
.
???displayArticle.abstract??? TRPV5 is a Ca2+-selective channel that plays a key role in the reabsorption of Ca2+ ions in the kidney. Recently, a rare L530R variation (rs757494578) of TRPV5 was found to be associated with recurrent kidney stones in a founder population. However, it was unclear to what extent this variation alters the structure and function of TRPV5. To evaluate the function and expression of the TRPV5 variant, Ca2+ uptake in Xenopus oocytes and western blot analysis were performed. The L530R variation abolished the Ca2+ uptake activity of TRPV5 in Xenopus oocytes. The variant protein was expressed with drastic reduction in complex glycosylation. To assess the structural effects of this L530R variation, TRPV5 was modeled based on the crystal structure of TRPV6 and molecular dynamics simulations were carried out. Simulation results showed that the L530R variation disrupts the hydrophobic interaction between L530 and L502, damaging the secondary structure of transmembrane domain 5. The variation also alters its interaction with membrane lipid molecules. Compared to the electroneutral L530, the positively charged R530 residue shifts the surface electrostatic potential towards positive. R530 is attracted to the negatively charged phosphate group rather than the hydrophobic carbon atoms of membrane lipids. This shifts the pore helix where R530 is located and the D542 residue in the Ca2+-selective filter towards the surface of the membrane. These alterations may lead to misfolding of TRPV5, reduction in translocation of the channel to the plasma membrane and/or impaired Ca2+ transport function of the channel, and ultimately disrupt TRPV5-mediated Ca2+ reabsorption.
Fig. 1. The L530R variation abolishes the Ca2+ uptake activity of TRPV5. (A) The location of L530 in the modeled structure of the TRPV5 tetramer. The top view of the TRPV5 tetramer is shown on the left and only two monomers are shown in the side view (right panel) for clarity. The four monomers are shown in cyan, tan, yellow and orange, respectively. D542 in the Ca2+ selective filter, L530 where variation occurred, and Ca2+ ions are shown in red, blue and pink, respectively. (B) Ca2+ uptake in Xenopus oocytes expressing TRPV5 with L530, R530, or in control oocytes. Each group contained Ca2+ uptake values of 18 Xenopus oocytes from two frogs. NS means the difference is not significant. (C) Western blot analysis of TRPV5 variants (L530 and R530) expressed in Xenopus oocytes. Band B represents complex-glycosylated form of TRPV5, and band A represents core-glycosylated form. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. The L530R variation changes electrostatic potential at position 530 and damages the secondary structure of transmembrane helix 5. (A) The electrostatic potential of residue 530 (dotted circle) shifted positively when L530 was replaced by R530. (B) Transmembrane helix 5 (S5) broke into two helices in the R530 variant as a result of the disruption of the interaction between R530 and L502. The pore helix (P) and S5 are shown in orange and red, respectively. The carbon and nitrogen atoms of residue 530 are labeled in cyan and blue, respectively. For clarity, the hydrogen atoms of residue 530 are not shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. The L530R variation alters the interaction between residue 530 and lipid molecules. (A) Radial distribution functions (RDFs) of residue 530 with phosphorus (P) atoms (upper panel) and carbon (C) atoms (lower panel) of POPC lipids. (B) Simulation snapshots showing the coordination of residue 530 with the surrounding lipid molecules. The P, C, O (oxygen) and N (nitrogen) atoms of POPC are shown in tan, gray, red and blue, respectively. The labelling of atoms in residue 530 is the same as in Fig. 2B. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. The L530R variation causes a shift of the pore helix and residue D542 towards the membrane surface. (A) Comparison of the mass density of residue 530, pore helix and residue D542 between L530 and R530 systems. The dashed cyan lines indicate the density peaks of the P atoms of POPC lipids. Note the density values of P atoms of POPC and pore helix are reduced 40 times and 10 times, respectively, in order to be shown in the same scale. (B) Final structures showing the position of residue 530 (black dotted lines) and pore helix (in red) relative to membrane surface (cyan lines). The P atoms of POPC are shown as balls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
S1- Sequence alignment between human TRPV5 and rat TRPV6. The identical and similar residues between the two sequences are labeled in yellow and pink, respectively. The linker helices (LH1-LH2), TM helices (S1-S6), pre-S1, pore helix (P), and the TRP domain are indicated as cyan cylinders. Residues L502, L530 and the selective filter are shown in green, red, and blue, respectively. The identity between the two sequences is 83.1% which indicates that it is reliable for modeling the structure of hTRPV5 based on the structure of rTRPV6.
S2- Root mean square deviation (RMSD) for the C- alpha atoms of TRPV5 in L530 and R530 systems. The simulations were equilibrated after 200 ns, thus the trajectories after 200 ns were used for data analysis.
S3- The hydrophobic environment of L530 in the modeled TRPV5. Residue L530 is surrounded by leucine 502 (L502), alanine 505 (A505), and tyrosine 526 (Y526). The oxygen and carbon atoms of the residues are shown in red and cyan, respectively. The colors for each monomer of TRPV5 are the same as in Fig. 1A.
S4- Comparison of the secondary structure between the L530 and R530 systems. Upper and middle panels show the helix occupancy of each residue of the modeled TRPV5. The pore helix (P), TM helices (S1-S6) and the TRP domain are shaded in cyan boxes. The positions of residue 530 in the four monomers are shown with blue lines, and the position of L502 on monomers 1 and 4 are shown in yellow lines. Lower panels show the comparison of the helix occupancy for all helices, pore helix, S5 helix, residue L502 and residue 530. Helix occupancy for L502 was significantly decreased in monomers 1 and 4.
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