Cation channel conductance and pH gating of the innate immunity factor APOL1 are governed by pore-lining residues within the C-terminal domain

Abstract

The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei. Endocytosis and acidification of high-density lipoprotein (HDL)-associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated non-selective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic-recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted Helix-Loop-Helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with inter-species chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that while neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain (CTD), tyrosine-351 and glutamate-355, that influence pH gating properties, as well as a single residue, aspartate-348, which determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C-terminus as the pore-lining segment of this novel channel-forming protein.

APOL1 genotype-associated morphologic changes among patients with focal segmental glomerulosclerosis

“…Over the past decade, epidemiologic, translational, and basic science research has led to innumerable insights about APOL1-associated nephrotic syndrome. We have a greater understanding of at-risk populations and the natural history of individuals affected [4,5,14,15], "second hits" that potentiate APOL1's penetrance [7,23], and cell structures and pathways that are involved in the disease's pathogenesis and progression [24][25][26][27]. At the same time, there are still many questions left on the table-answering them could ultimately contribute to improved patient care.…”

Towards the NMR solution Structure and the Dynamics of the C-terminal Region of APOL1 and its G1, G2 Variants with a Membrane Mimetic

“…It is likely that additional protein domains and an extended bilayer membrane model is necessary for this behavior of the protein. The protonation status of D348 within the predicted transmembrane (TM) domain of APOL1-G0 C-terminus (D337-E335) is involved in pH gating function (46) and the G1 and G2 variants demonstrated increased cation channel activity in phospholipid vesicles (48). This predicted TM region in the C-terminus forms an α-helix with similar residue orientation in our NMR model.…”

“…Additionally, the amphipathic helical membrane addressing domain (MAD) located N-terminal to the SRA-interacting C-terminal region of APOL1 proteins has been postulated to be a pH sensor and acts as a “hinge” structure between the C-terminus of APOL1 and the rest of the protein (41,51). APOL1 is known to localize to the endosomal and lysosomal compartment (6,18,19,50,52) and exposure to low pH could stabilize the membrane localization (46,47), given the presence of bis(monoacylglycero)phosphate and phosphatidylinositols in the slightly negatively charged endolysosomal membranes (53,54). This conformation can expose the C-terminal region of APOL1 to the cytoplasm, where interaction with SNAREs and other effector proteins is feasible.…”

APOL1 risk variants and the development of HIV‐associated nephropathy

“…In a recent report, bioinformatic studies suggested that G1 and G2 altered ion channels' structural configuration in plasma membranes, resulting in K + efflux [70]. The influx of sodium and calcium by cation-selective pores could also enhance K + efflux [72,73]. Additionally, RRV-induced opening of the mitochondrial permeability transition pore has also been demonstrated to cause cell death [74].…”