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

Abstract

The 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.

Figure 1

Predicted topology of LmAQP1. Amino acids labeled as white letters in black circles are conserved between LmAQP1 and PfAQP; residues marked as white letters in gray circles are similar to PfAQP; black and gray squares indicate conserved and similar residues, respectively, which directly interact with glycerol in PfAQP. This topology was plotted using the TeXtopo package (Beitz, 2000).

Figure 2

Water transport in Xenopus oocytes expressing altered LmAQP1. Oocytes were injected with water (control), wild-type, E143A/E145A, E152A, E152D, and E152Q LmAQP1-cRNA.

Figure 3

Solute permeability in Xenopus oocytes expressing altered LmAQP1. Oocytes were injected with water (control), wild-type, E143A/E145A, E152A, E152D, and E152Q LmAQP1-cRNA.

Figure 4

Metalloid uptake of L. donovani cells expressing wild-type or altered LmAQP1. Panels A and B depict As(III) uptake in L. donovani promastigotes and amastigotes, respectively; Panels C and D represent Sb(III) uptake in L. donovani promastigotes and amastigotes, respectively. Cells were transfected with wild-type, E143A/E145A, E152A, E152D, E152Q LmAQP1 or vector control.

Figure 5

Volume regulation in the two life-cycle stage of Leishmania expressing either wild-type or altered LmAQP1. (A) L. donovani promastigotes, and (B) L. donovani amastigotes expressing the vector, (●); wild-type (○); E143A/E145A (▼); E152A (△); E152D (■); and E152Q (□) LmAQP1 were subjected to a hypoosmotic shock, and the relative changes in cell volume were followed by monitoring the absorbance at 550 nm, as described in Experimental procedures.

Figure 6

Movement of L. donovani promastigotes in response to an osmotic gradient. Cells transfected with wild-type, E143A/E145A, E152A, E152D, and E152Q LmAQP1 or vector control were counted for their ability to move into capillary tubes containing ±100 mM glucose.