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Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis.

ABSTRACT: Apolipoprotein L-1 (APOL1), the trypanolytic factor of human serum, can lyse several African trypanosome species including Trypanosoma brucei brucei, but not the human-infective pathogens T. brucei rhodesiense and T. brucei gambiense, which are resistant to lysis by human serum. Lysis follows the uptake of APOL1 into acidic endosomes and is apparently caused by colloid-osmotic swelling due to an increased ion permeability of the plasma membrane. Here we demonstrate that nanogram quantities of full-length recombinant APOL1 induce ideally cation-selective macroscopic conductances in planar lipid bilayers. The conductances were highly sensitive to pH: their induction required acidic pH (pH 5.3), but their magnitude could be increased 3,000-fold upon alkalinization of the milieu (pK(a) = 7.1). We show that this phenomenon can be attributed to the association of APOL1 with the bilayer at acidic pH, followed by the opening of APOL1-induced cation-selective channels upon pH neutralization. Furthermore, the conductance increase at neutral pH (but not membrane association at acidic pH) was prevented by the interaction of APOL1 with the serum resistance-associated protein, which is produced by T. brucei rhodesiense and prevents trypanosome lysis by APOL1. These data are consistent with a model of lysis that involves endocytic recycling of APOL1 and the formation of cation-selective channels, at neutral pH, in the parasite plasma membrane.

SUBMITTER: Thomson R

PROVIDER: S-EPMC4352821 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis.

Thomson Russell R Finkelstein Alan A

Proceedings of the National Academy of Sciences of the United States of America 20150217 9

Apolipoprotein L-1 (APOL1), the trypanolytic factor of human serum, can lyse several African trypanosome species including Trypanosoma brucei brucei, but not the human-infective pathogens T. brucei rhodesiense and T. brucei gambiense, which are resistant to lysis by human serum. Lysis follows the uptake of APOL1 into acidic endosomes and is apparently caused by colloid-osmotic swelling due to an increased ion permeability of the plasma membrane. Here we demonstrate that nanogram quantities of fu ...[more]

PMID: 25730870

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Project description:Key pointsWe report a novel method for the transient expression of SARS-CoV-2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes. Intracellular expression of SARS-CoV-2 E protein increases intra-Golgi pH. By targeting the SARS-CoV-2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH. This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID-19.AbstractThe envelope (E) protein of coronaviruses such as SARS-CoV-1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS-CoV-2 in mammalian cells by directed targeting using a carboxyl-terminal fluorescent protein tag, mKate2. The wild-type SARS-CoV-2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum-Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS-CoV-2 E to the plasma membrane, which enabled biophysical analysis using whole-cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two-electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+ , Cs+ and K+ . The E current is nearly time- and voltage-independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time- and voltage-dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS-CoV-2 in vitro and possible efficacy in treating COVID-19.

Dataset Information

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis.

Publications

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis.

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