Kidney Week

Abstract: SA-OR029

The L530R Variation Found in Kidney Stone Patients Impacts the Structure and Function of TRPV5

Session Information

• Mineral Disease: Bones, Vessels, Stones
  November 04, 2017 | Location: Room 273, Morial Convention Center
  Abstract Time: 06:06 PM - 06:18 PM

Category: Mineral Disease

• 1201 Mineral Disease: Ca/Mg/PO4

Authors

• Wang, Lingyun, University of Alabama at Birmingham , Birmingham, Alabama, United States

Lingyun Wang, PhD

• Peng, Ji-Bin, University of Alabama at Birmingham , Birmingham, Alabama, United States

Ji-Bin Peng,

Background

TRPV5 is a Ca²⁺-selective channel that plays a key role in the reabsorption of Ca²⁺ ions in the kidney. Recently, a L530R variation in TRPV5 was found in some kidney stone formers. This variation introduces a positive charged residue into a hydrophobic region of the pore helix and may alter the Ca²⁺ transport activity of TRPV5. However, it is unclear to what extent this variation alters the structure and function of TRPV5.

Methods

To evaluate the function and expression of the TRPV5 variant, Ca²⁺ uptake in Xenopus oocytes and western blot analysis were performed. To assess the structural effects of L530R, TRPV5 was modeled using MODELLER based on the core structure of TRPV6 containing all the six transmembrane (TM) helices and the TRP domain. The L530R variation was introduced into TRPV5 using PyMOL. The modeled TRPV5 was embedded in a lipid bilayer composed of 299 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids using CHARMM-GUI, and water molecules were added on both sides of the bilayer. Two 400 ns molecular dynamics simulations were performed using AMBER14.

Results

The L530R variation abolished the Ca²⁺ uptake activity of TRPV5 in Xenopus oocytes. The variant protein was expressed with abnormal complex glycosylation. Simulation results show that the L530R variation breaks the hydrophobic interaction between L530 and L502, damaging the secondary structure of TM5. 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 phosphorus atoms 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 membrane where phosphorus atoms are located.

Conclusion

Our results indicate that the L530R variation damages the secondary structure of TM5 of TRPV5 and alters the interaction between TRPV5 and membrane lipids. This may lead to misfolding of TRPV5 or impaired translocation of the channel to the plasma membrane, and ultimately disrupt TRPV5-mediated Ca²⁺ reabsorption.

Funding

• NIDDK Support