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Abstract: FR-PO915

Alterations in Plasma Membrane Ion Channel Structures Stimulate NLRP3 Inflammasome Activation in APOL1 Risk Milieu

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology


  • Jha, Alok, Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Kumar, Vinod, Feinstein Institute for Medical Research, Manhasset, New York, United States
  • Malhotra, Ashwani, Feinstein Institute Medical Research and NSLIJ, Manhasset, New York, United States
  • Skorecki, Karl, Rambam Health Care Campus, Haifa, Israel
  • Singhal, Pravin C., North Shore LIJ Health System, Great Neck, New York, United States

APOL1 has been demonstrated to act as ion transporter in cellular lipid membranes. APOL1 risk proteins have been shown to enhance K+ -efflux and inflammasome activation. However, the involved mechanism is not clear. We evaluated the role of basic configurations of APOL1 pores on K+-efflux and the activation of NLRP3 inflammasomes in APOL1 risk milieu.


Immortalized human podocytes were transduced with vector (V-PD) APOL1G0 (G0-PD), APOL1G1 (G1-PD), and APOL1G2 (G2-PD) employing lentivirus expression system and incubated with either vehicle or Glyburide (100 µM, a K+- efflux inhibitor) for 48 hours (n=4). Protein blots and cDNAs were probed for NLRP3, IL-1β, and cleaved Caspase-1. Bioinformatics studies were conducted to evaluate alterations in plasma membrane ion channel structures in G0-, G1-, and G2-podocytes, including pore domain and structures, membrane integration and orientation, electrostatic properties and pKa calculation, binding with phosphatidic acid, and grid formation and docking of Glyburide in podocytes expressing APOL1G0 and variants.


G1- and G2-podocytes enhanced the transcription of NLRP3. However, Glyburide inhibited this effect of APOL1G1 and APOL1G2. Superimposition of bioinformatic reconstructions of APOL1G0, G1, and G2 showed several aligned regions. The analysis of pore-lining residues revealed that Ser342 and Tyr389 are involved in APOL1G0 pore formation, however, conformations resulting from the Ser 384Met mutation in the case of G1 and deletion of the Tyr389 residue in the case of G2 are expected to alter pore characteristics, including K+ ion selectivity. Analysis of multiple membrane (lipid bilayer) models of interaction with the peripheral protein, integral membrane protein, and multimer protein revealed that for an APOL1 multimer model, G0 is not energetically favorable while the APOL1G1 and APOL1G2 moieties favor the insertion of multiple ion channels into the lipid bilayer. Protein-substrate/ligand interaction between Glyburide (a K+- efflux inhibitor) and APOL1G0/APOL1G1/APOL1G2 revealed that a relatively lower concentration of Glyburide could bind to APOL1G1 and APOL1G2 multimer.


The altered pore configurations carry the potential to facilitate K+ ion transport in APOL1 risk milieu.


  • NIDDK Support