Malaria parasite mutants with altered erythrocyte permeability: a new drug resistance mechanism and important molecular tool

Abstract

Erythrocytes infected with plasmodia, including those that cause human malaria, have increased permeability to a diverse collection of organic and inorganic solutes. While these increases have been known for decades, their mechanistic basis was unclear until electrophysiological studies revealed flux through one or more ion channels on the infected erythrocyte membrane. Current debates have centered on the number of distinct ion channels, which channels mediate the transport of each solute and whether the channels represent parasite-encoded proteins or human channels activated after infection. This article reviews the identification of the plasmodial surface anion channel and other proposed channels with an emphasis on two distinct channel mutants generated through in vitro selection. These mutants implicate parasite genetic elements in the parasite-induced permeability, reveal an important new antimalarial drug resistance mechanism and provide tools for molecular studies. We also critically examine the technical issues relevant to the detection of ion channels by electrophysiological methods; these technical considerations have general applicability for interpreting studies of various ion channels proposed for the infected erythrocyte membrane.

Figure 1. Plasmodial surface anion channel activity at a range of membrane potentials detected with patch clamp

(A) Current–voltage profile from whole-cell recordings of an uninfected erythrocyte and a cell infected with a Plasmodium falciparum trophozoite (black and white circles, respectively). Notice that the infected cell has larger currents at each V_m. (B) Single plasmodial surface anion channel recordings in the cell-attached patch-clamp configuration. All traces were recorded from a single patch with imposed V_m values indicated to the right of each trace. For each trace, the closed channel level is indicated with horizontal dashes on each side. Channel openings are reflected by transitions away from this level; these transitions are upgoing at positive V_m and downgoing at negative V_m. Scale bar represents 100 ms (horizontal) and 2 pA (vertical). Notice that plasmodial surface anion channel openings are more frequent at negative V_m, consistent with greater absolute magnitudes of whole-cell currents at negative V_m in (A). Recordings were obtained with symmetrical molar salt solutions as described previously [15]. I: Current; V_m: Membrane potential.

Figure 2. Sequential diffusive pathway for nutrient acquisition by intracellular malaria parasites (A) and a model for plasmodial surface anion channel-mediated drug resistance (B)

(A) Nutrients and toxins may enter infected cells by passive uptake via PSAC on the erythrocyte membrane, a large-conductance nonselective channel on the PVM and multiple distinct carriers on the PPM. Metabolic waste products generated in the parasite cytosol (blue) exit the infected cell complex by flux in the opposite direction. According to our proposed model, various solutes share a single ion channel at the erythrocyte membrane and at the PVM, but use separate and specific transporters at the PPM. A nucleus and some organelles are shown within the parasite cytosol to exemplify the metabolic activity of the intracellular parasite. (B) In vitro selection with either leupeptin or blasticidin S generates separate PSAC mutants that have reduced uptake of the applied toxin but only modestly compromised uptake of nutrients and the other toxin. PPM: Parasite plasma membrane; PVM: Parasitophorous vacuolar membrane; PSAC: Plasmodial surface anion channel; RBC: Red blood cell membrane.

Figure 3. Possible mechanisms for functional polymorphisms in plasmodial surface anion channel activity

In each panel, a ‘wild-type’ channel is shown in the top bilayer, while mutant or polymorphic channels are shown on the lower membrane. Parasite-encoded channel subunits or enzymes are shown in blue, while human proteins are shown in green. Polymorphic residues are shown as red dots (plus arrowheads) on the channel protein or on modifying enzymes and can arise only on parasite-encoded proteins. (A) A parasite-encoded ion channel model can accrue multiple distinct single-nucleotide polymorphisms and yield a spectrum of channel phenotypes. (B) A human ion channel activated by a specific parasite enzyme (e.g., a kinase that phosphorylates multiple sites on the channel protein) can produce distinct patterns of activation if mutations in the enzyme yield differing affinities for the sites on the human target protein. (C) An ion channel consisting of both parasite and human subunits can accrue mutations via single-nucleotide polymorphisms in the parasite subunit. (D) Nonspecific activation of a human channel (e.g., via the action of ROS) cannot easily yield heritable changes in channel activity. P: Site of phosphorylation; ROS: Reactive oxygen species; SH: A thiol that can be oxidized to a disulfide, presented as an example target of ROS generated by parasite oxidative stress.

Figure 4. Key instrumentation in a patch-clamp set-up and an equivalent circuit for a pipette sealed on a cell membrane patch containing a single ion channel molecule

While a gap is shown between the pipette tip and the cell membrane to show the effect of finite R_seal, useful patch-clamp recordings require isolation of the membrane patch under the annulus of the pipette through covalent interactions between the pipette tip and cell surface. See the text for discussion of each component in this drawing. A/D: Analog-to-digital; C_cell: Equivalent total capacitance of the cell membrane; R_cell: Equivalent total resistance of the cell membrane; R_chn: Resistance of a single channel molecule; R_pip: Pipette resistance; R_seal: Seal resistance.