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Kidney Week

Abstract: TH-PO808

Kidney Disease-Associated Variants of Apolipoprotein L1 Show Gain-of-Function in Cation Channel Activity

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Edwards, John C., St. Louis University, St. Louis, Missouri, United States
  • Bruno, Jonathan M., St. Louis University, St. Louis, Missouri, United States
Background

Variants in Apolipoprotein L1 (ApoL1) are responsible for increased risk of some progressive kidney diseases among people of African ancestry. ApoL1 is known to function as an amphitropic protein that can insert into phospholipid membranes and confer pH-regulated Cl- or K-selective permeability. Cl permeability is optimal when protein and vesicles are both mixed and assayed at pH 5.0. K permeability is optimal when protein and vesicles are mixed at pH 6.0 and assayed at pH 7.5. Whether these activities differ among the variants or contribute to disease pathogenesis is unknown.

Methods

Recombinant WT (G0) or each variant (G1, G2) were purified from E. Coli. We used a vesicle-based assay of voltage-driven ion flux. In brief, KCl-loaded vesicles were mixed with protein, extravesicular KCl removed, and voltage-driven efflux intiated by addition of either a K- or Cl-selective ionophore to assess Cl or K permeability, respectively. To assess membrane association, protein was mixed with vesicles under conditions that support K permeability, stripped with alkali, the membranes isolated by flotation through a sucrose cushion, and associated protein determined by quantitative western blotting.

Results

In each of 5 sets of purified protein, the K selective permease of G1 and G2 isoforms was significantly increased compared to G0. Combining all sets, initial efflux rates were 0.361 ± 0.028 for G0, 0.738 ± 0.081 for G1, and 0.688 ± 0.069 for G2 (%/sec, mean ± SEM; P<0.0025 for comparison of G0 with either G1 or G2). In contrast, we find no difference in the Cl selective permease activity among the isoforms. Compared to the WT, the two disease-associated variants show increased stable membrane association under conditions that support the K permease activity (amount bound: 56.2 ± 3.4 for G0, 76.6 ± 5.1 for G1, and 100.2 ± 11.8 for G2 (ng, mean ± SEM, n=8 for each; P<0.006 for G0 vs either G1 or G2; G1 vs G2 NS)).

Conclusion

Kidney disease-associated variants of ApoL1 show gain of function in the K permease activity, and show increased capacity to stably associate with membrane vesicles, suggesting that the increased activity may be due to more efficient membrane association and/or insertion. These data support a model in which enhanced potassium permeability may contribute to the progressive kidney diseases associated with high-risk ApoL1 alleles.

Funding

  • NIDDK Support