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Kidney Week

Abstract: TH-PO744

Kidney Disease Associated Variants of ApoL1 Show Gain-of-Function in the pH-regulated Cation Channel Activity

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Author

  • Edwards, John C., St. Louis University, Saint Louis, Missouri, United States
Background

Variants in the protein ApoL1 are largely responsible for the increased risk of progressive kidney disease in people of African ancestry. ApoL1 is an amphitropic membrane protein that inserts into membranes a low pH where it confers anion-selective permeability. After titration to neutral pH, the anion permeability is suppressed and a non-selective cation permeability is activated. We used vesicle based ion flux and membrane association assays to assess for functional difference among variants.

Methods

Recombinant ApoL1 isoforms were expressed in E. coli. KCl-loaded phospholipid vesicles were mixed with protein and assayed for efllux in presence of a large KCl gradient. Efflux was initiated by addition of selective ionophore (valinomycin to assess Cl permeability, or Cl ionophore 1 to assess K permeability), and detected with a chloride selective electrode. The initial rate of efflux after addition of ionophore minus the rate with control vesicles is taken as the ApoL1-dependent ion permeability. To assess membrane association, vesicles and protein were mixed at pH 6, then titrated to pH 7.5, stripped of peripherally bound protein, separated by flotation through a sucrose cushion and detected by western blotting.

Results

All variants show Cl-selective permease activity at low pH that is essentially identical. In contrast, the K-permeability of the disease-associated variants G1 and G2 is substantially higher than the WT (G0). All three variants were purified simultaneously, with assays performed on the same day with the same reagents. Three independent sets of purified protein showed the same pattern of activity. Combining data from the three sets, G1 and G2 showed 2.08 ± 0.17 and 2.03 ± 0.28 fold greater activity than G0 (mean ± SEM, P<0.001 for each). Membrane association experiments demonstrated consistently increased membrane binding by G1 and G2 compare to G0 with 3.02 ± 0.49 and 3.30 ± 0.36 fold more binding than G0, respectively (P<0.001 for each).

Conclusion

Kidney disease associated variants of ApoL1 show consistently greater cation channel activity and greater membrane insertion than the wild type protein. The data suggest increased membrane insertion and hence greater channel activity in kidney cells could be a mechanism contributing to disease progression.