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Abstract: FR-PO1025

Changing the Channel: Mutational Analysis of APOL1 Reveals Structural Insights Impacting G1 and G2 Associated Kidney Disease

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Schaub, Charles Michael, The Graduate Center of the City of New York, New York, New York, United States
  • Thomson, Russell P., Hunter College , New York, New York, United States
  • Giovinazzo, Joseph A., Hunter College , New York, New York, United States
  • Finkelstein, Alan, Albert Einstein College of Medicine, New York, New York, United States
  • Raper, Jayne, City University of New york, New York, New York, United States
Background

The human innate immunity factor Apolipoprotein L-1 (APOL1), is a cation selective channel that protects against African Trypanosomes. The channel has two putative transmembrane domains, requires acidification for activation, and opens upon subsequent neutralization. Natural variants of APOL1, G1 and G2, have been recognized as risk factors for kidney disease. Investigating the structure of APOL1 would assist in identifying mechanisms of channel formation responsible for kidney cell toxicity caused by the G1 and G2 variants.

Methods

Recombinant APOL1 (rAPOL1) was purified from E. coli BL21-DE3-RIPL cells expressing the pNIC-28 vector. The effects of single and multiple amino acid substitutions were first tested for trypanolytic activity, followed by channel formation and ion selectivity in planar lipid bilayers. Blue Native PAGE of APOL1 expressing FlpIn TREX 293 cells along with SDS-PAGE of rAPOL1 was performed to visualize oligomers of the APOL1 channel. Protease protection assays of rAPOL1 channels in LUVs were used to ascertain orientation and channel transmembrane domains via silver stain and mass spectrometry.

Results

At acidic pH, rAPOL1 G0 is a non-ideal cation channel. Substituting amino acids in the second predicted transmembrane domain not only inhibited in vitro trypanolytic activity, but also altered channel conductance and cation selectivity. Changes in the leucine zipper motif at the C-terminus abolished rAPOL1 activity in vitro and in planar lipid bilayers. Mutations within the C-terminus, that retain trypanolytic activity, indicate that rAPOL1 is an oligomer as a functional channel. Furthermore, oligomers of APOL1 have been visualized by Blue Native PAGE from the TREX 293 cells upon release from the ER. Fragments of the digested rAPOL1 channel protected by LUVs designate orientation and transmembrane regions.

Conclusion

These findings reveal that APOL1 requires oligomerization to form a functional channel, which is driven by residues in the C-terminus. In addition, we identified specific amino acids in the second transmembrane domain that govern cation selectivity.