Alteration in glycerol and metalloid permeability by a single mutation in the extracellular C‐loop of Leishmania major aquaglyceroporin LmAQP1

Abstract

SummaryThe Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of antimonite [Sb(III)], an activated form of Pentostam or Glucantime. Downregulation of LmAQP1 provides resistance to trivalent antimony compounds and increased expression of LmAQP1 in drug-resistant parasites can reverse the resistance. Besides metalloid transport, LmAQP1 is also permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. LmAQP1 also plays a physiological role in volume regulation and osmotaxis. In this study, we examined the role of extracellular C-loop glutamates (Glu143, Glu145 and Glu152) in LmAQP1 activity. Alteration of both Glu143 and Glu145 to alanines did not affect either the biochemical or physiological properties of the protein, suggesting that neither residue is critical for LmAQP1 activity. Alteration of Glu152 to alanine, aspartate and glutamine affected metalloid transport in the order, wild-type > E152Q > E152D > E152A. In fact, axenic amastigotes expressing E152A LmAQP1 accumulated negligible levels of either arsenite [As(III)] or Sb(III). Alteration of Glu152 significantly affected volume regulation and osmotaxis, suggesting that Glu152 is critical for the physiological activity of the parasite. More importantly, alteration of Glu152 to alanine did not affect glycerol permeability. Although the metalloids, As(III) and Sb(III), are believed to be transported through aquaglyceroporin channels as they behave as inorganic molecular mimic of glycerol, this is the first report where metalloid and glycerol transport can be dissected by a single mutation at the extracellular pore entry of LmAQP1 channel.

The LABCG2 Transporter from the Protozoan Parasite Leishmania Is Involved in Antimony Resistance

“…Parasites can decrease the uptake of, increase the efflux of, or inactivate the drug by sequestration, among other possible mechanisms. The LmAQP1 aquaglyceroporin is the only protein known to transport antimony inside L. major (31,32), and its downregulation subsequently leads to increased drug resistance (33). Concerning efflux, members of the eukaryotic ABCC subfamily are involved in Sb III and As III resistance by exporting these metal ions outside the cells or by sequestering them within intracellular vesicles (34).…”

Metalloid Transport Systems

“…Thus, engineering NIPs to reduce As(III) permeability is a plausible approach to creation of low arsenic crops. A recent report demonstrates the feasibility of that approach: a mutation of a single residue (Glu152) at extracellular entry point of the pore of the Leishmania aquaglyceroporin LmAQP1 reduced the rate of As(III) movement without affecting glycerol conduction [80]. This shows that in at least one AQP metalloid and glycerol transport can be separated by a single mutation.…”

Trypanosoma Brucei Aquaglyceroporins Facilitate the Uptake of Arsenite and Antimonite in a pH Dependent Way

“…Glu125 in loop C of Plasmodium falciparum aquaglyceroporin (PfAQP) is responsible for a high capability of water transport that is largely abolished by changing this amino acid [28]. Likewise, mutation of Glu152 to Ala, within the same loop in Aquaglyceroporin 1 from Leishmania (LmAQP1), affected water as well as metalloid permeation, but not the glycerol transport [29]. These findings demonstrate that a fine-tuning of selectivity and capability for substrate transport are not exclusive of the classical three regions mentioned above, and that the three TbAQPs here studied present enough differences at their C-termini as well as in their C loops to account for the observed differences in permeability [13].…”