The Ionophores CCCP and Gramicidin but Not Nigericin Inhibit Trypanosoma brucei Aquaglyceroporins at Neutral pH

Abstract

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei parasites. The T. brucei aquaglyceroporin isoform 2, TbAQP2, has been linked to the uptake of pentamidine. Negative membrane potentials and transmembrane pH gradients were suggested to promote transport of the dicationic antitrypanosomal drug. Application of ionophores to trypanosomes further hinted at direct inhibition of TbAQP2 by carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Here, we tested for direct effects of three classical ionophores (CCCP, nigericin, gramicidin) on the functionality of TbAQP2 and the related TbAQP3 at conditions that are independent from the membrane potential or a proton gradient. We expressed TbAQP2 and TbAQP3 in yeast, and determined permeability of uncharged glycerol at neutral pH using stopped-flow light scattering. The mobile proton carrier CCCP directly inhibited TbAQP2 glycerol permeability at an IC50 of 2 µM, and TbAQP3 to a much lesser extent (IC50 around 1 mM) likely due to different selectivity filter layouts. Nigericin, another mobile carrier, left both isoforms unaffected. The membrane-integral pore-forming gramicidin evenly inhibited TbAQP2 and TbAQP2 in the double-digit micromolar range. Our data exemplify the need for suitable controls to detect unwanted ionophore side effects even when used at concentrations that are typically recommended to disturb the transmembrane ion distribution.

Evolution, function and roles in drug sensitivity of trypanosome aquaglyceroporins

“…2) (Alghamdi et al ., 2020). However, consideration of structural features of the pore do support TbAQP2 acting as a channel for larger and more structurally flexible solutes including pentamidine (Petersen and Beitz, 2020). In the endocytosis model, ubiquitination of TbAQP2 at the flagellar pocket is central for subsequent ubiquitination-mediated intracellular trafficking and delivery to intracellular organelles such as the lysosome.…”