Abstract
Apolipoprotein L1 (ApoL1) is a human serum protein conferring resistance to African trypanosomes, and certain ApoL1 variants increase susceptibility to some progressive kidney diseases. ApoL1 has been hypothesized to function like a pore-forming colicin and has been reported to have permeability effects on both intracellular and plasma membranes. Here, to gain insight into how ApoL1 may function in vivo, we used vesicle-based ion permeability, direct membrane association, and intrinsic fluorescence to study the activities of purified recombinant ApoL1. We found that ApoL1 confers chloride-selective permeability to preformed phospholipid vesicles and that this selectivity is strongly pH-sensitive, with maximal activity at pH 5 and little activity above pH 7. When ApoL1 and lipid were allowed to interact at low pH and were then brought to neutral pH, chloride permeability was suppressed, and potassium permeability was activated. Both chloride and potassium permeability linearly correlated with the mass of ApoL1 in the reaction mixture, and both exhibited lipid selectivity, requiring the presence of negatively charged lipids for activity. Potassium, but not chloride, permease activity required the presence of calcium ions in both the association and activation steps. Direct assessment of ApoL1-lipid associations confirmed that ApoL1 stably associates with phospholipid vesicles, requiring low pH and the presence of negatively charged phospholipids for maximal binding. Intrinsic fluorescence of ApoL1 supported the presence of a significant structural transition when ApoL1 is mixed with lipids at low pH. This pH-switchable ion-selective permeability may explain the different effects of ApoL1 reported in intracellular and plasma membrane environments.
Highlights
Apolipoprotein L1 (ApoL1) is a human serum protein conferring resistance to African trypanosomes, and certain ApoL1 variants increase susceptibility to some progressive kidney diseases
Based on the report of Thomson and Finkelstein [6], we looked for ApoL1-dependent potassium permeability with membrane association at low pH and an efflux assay at neutral pH, using chloride ionophore 1 (CI1) [11] to detect potassium permeability
We have investigated the biochemical properties of purified recombinant ApoL1 and generated novel data that expand our knowledge of this important protein
Summary
ApoL1, apolipoprotein L1; DIDS, 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid; DDM, dodecyl maltoside; val, valinomycin; CI1, chloride ionophore 1; PC, 90% phosphatidyl choline, 10% cholesterol; PA, 80% phosphatidyl choline, 10% phosphatidic acid, 10% cholesterol; PS, 80% phosphatidyl choline, 10% phosphatidyl serine, 10% cholesterol; PE, 80% phosphatidyl choline, 10% phosphatidyl ethanolamine, 10% cholesterol; ANOVA, analysis of variance; LC50, lethal concentration at which 50% of the population is killed. A second report by another group used full-length ApoL1 purified from human serum [8] These investigators demonstrated an activity that generated pores in phospholipid vesicles that allowed passage of calcein, a 623-molecular weight molecule that carries four negative charges at neutral pH. This activity required low pH, the presence of negatively charged phospholipid in the vesicle membranes, and low ionic strength to allow ApoL1 to physically associate with vesicles and form pores Another group has reported dramatically different results using recombinant ApoL1 refolded from bacterial. The transport properties of ApoL1 are unclear from the literature, with one report describing anion-selective permeability at low pH, one reporting permease activity allowing passage of rather large negatively charged molecules at low pH, and one reporting cation channel activity that requires low pH for membrane insertion but only functions at neutral pH. These data suggest a model in which ApoL1, through supporting pH-switchable ion-selective permeability, may mediate the different effects reported in both intracellular and plasma membrane environments
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