Date of Degree
Biochemistry | Immunology of Infectious Disease | Other Biochemistry, Biophysics, and Structural Biology | Parasitology
Apolipoprotein L-1, Ion Channel, Electrophysiology, African Trypanosomes
The human innate immunity factor Apolipoprotein L-1 (APOL1) protects against Trypanosoma brucei brucei infection. Recent studies have shown recombinant APOL1 (rAPOL1) inserts into planar lipid bilayers at an acidic pH 5.6 and forms a cation-selective channel, which opens upon subsequent neutralization, pH 7.2. This corresponds with the pH changes APOL1 would encounter during endosome recycling, suggesting that APOL1 forms a pH-gated ion channel in the plasma membrane of the parasite, leading to uncontrolled ion flux and osmotic imbalance. However, structural and domain characteristics of the APOL1 channel are poorly understood, despite potential similarities to diphtheria and colicin toxins. Utilizing E. coli-derived rAPOL1, we tested the effect of numerous site-directed substitutions on channel properties in planar lipid bilayers and in trypanoltyic assays. With this approach, we revealed that a single residue in the C-terminus, aspartate-348, influences channel selectivity and pH gating. Furthermore, we hypothesized that the C-terminal heptad repeat motif is required for oligomerization and channel formation. Indeed, specific amino acid substitutions in this domain inhibit both channel formation and APOL1 mediated-trypanolysis. We then employed a scanning cysteine accessibility approach to elucidate membrane topology and transmembrane domains of the APOL1 channel. Taken together, these results highlight the importance of the C-terminus in APOL1 function, and lend constraints for possible structures of the functional channel.
Schaub, Charles M., "Mutational Analysis and Domain Characterization of the Apolipoprotein L-1 Ion Channel" (2020). CUNY Academic Works.
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