Abstract: SA-PO1065

ApoL1 Confers pH-Switchable Ion Permeability to Phospholipid Vesicles

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic

Author

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

Variants in ApoL1 confer risk of certain chronic kidney diseases. ApoL1 is thought to function as an ion channel but reports vary substantially. We sought to characterize ApoL1 ion permease activity with the hope that it may provide insight into ApoL1-associated kidney disease.

Methods

Recombinant His-tagged ApoL1 was purified by Ni-affinity and gel filtration. Ion permeability was assessed using vesicle-based, voltage dependent Cl and K efflux assays using ion selective electrodes. ApoL1 membrane association was measured by mixing protein with lipid, Na2CO3 extraction to remove peripherally-associated protein, and isolation of the remaining integral membrane protein by floating the vesicles through a sucrose cushion.

Results

Addition of Apol1 to vesicles yields robust Cl selective permeability. The activity is dependent on pH at which protein and membranes interact, with a sharp drop above pH 6.0. K permeability is minimal at any pH when protein and vesicles are mixed and assayed at the same pH. However, K permeability is detected when protein and vesicles are allowed to interact at low pH and then shifted to neutral pH for efflux assay. K permeability is greatest if protein and vesicles are mixed at pH 5.5-6.0 and then assayed at pH 7.5. pH switch not only activates the K permeability but also partially inactivates the Cl permeability. Both Cl and K permease activities are linearly dependent on mass of protein, and are dependent on lipid composition, requiring the presence of negatively charged phospholipids. The K permeability requires the presence of Ca ion. Membrane association assays demonstrate pH-sensitive membrane insertion which occurs at low pH, requires the presence of negatively charged phospholipids, and is stable when pH is shifted back to neutral after insertion takes place.

Conclusion

ApoL1 inserts into vesicles at low pH and confers Cl-selective permeability if pH remains low, or switches to K-selective permeability if the pH is neutralized. The data suggest a model in which ApoL1 in the acidic and low Ca environment of the endocytic pathway could insert into membranes and confer Cl selective permeability, and if subsequently targetted to the plasma membrane where it would be exposed to neutral pH and Ca, switch to K permeability. This model might account for the variety of ApoL1 effects that have been reported in both trypanosomes and cultured mammalian cells.