A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum

Abstract

A recent study on malaria-infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host- or parasite-derived and their molecular nature have not been resolved. Here we report a comparison of a malaria-induced anion channel with an endogenous anion channel in Plasmodium falciparum-infected human RBCs. Ion channel activity was measured using the whole-cell, cell-attached and excised inside-out configurations of the patch-clamp method. Parasitised RBCs were cultured in vitro, using co-cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (~15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I(-) > Br(-) > Cl(-). In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (~80 pS, outward conductance) was spontaneously active. The host membrane of malaria-infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria-infected RBCs were shown to have a low open-state probability (P(o)) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole-cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up-regulated by the malaria parasite P. falciparum.

Figure 1. Whole-cell and cell-attached single channel recordings of uninfected and infected RBCs

Current traces in uninfected RBCs in control (A) or after addition of PKA catalytic subunit (100 nm) to the pipette solution (B). Current traces in infected RBCs (C) bathing in NMDG-Cl solution (voltage pulses between −100 and +100 mV, 10 mV increments, 700 ms). D, corresponding I-V plots (mean ± s.e.m.) in control (○, n = 32), after addition of PKA (▵, n = 58), in infected cells with NMDG-Cl (•, n = 24) or NaCl (▪, n = 12) in the bath. E, typical current recordings at different potentials (-V_p) from infected RBCs. F, corresponding I-V plot (mean ± s.e.m., ○, n = 25). •, I-V plot obtained after multiplication of the I value by the corresponding mean P_o.

Figure 2. Excised inside-out single channel recordings of linear conductance anion channels in uninfected and infected RBCs

A, activation of an anion channel by exposure of the cytosolic side of the membrane to PKA (100 nm) and ATP (1 mm), after a 1–3 min lag period, at V_m of +100 mV. B, activation of the same channel by membrane deformation upon application of calibrated depression in the pipette (V_m of +100 mV). Substates corresponding to 1/3 and 2/3 of full amplitude are clearly visible. The right panel shows the distribution of current amplitude from 3 min recordings (#, number of events; o, open state; c, closed state; mb, millibars). C, examples of current recordings from infected RBCs showing fast gating at negative potentials. D, mean I-V plots (± s.e.m.) corresponding to A (○, n = 8), B (▪, n = 10) and C (•, n = 7).

Figure 3. Excised inside-out single channel recordings of outwardly rectifying anion channels in uninfected RBCs

A, example of current traces from uninfected RBCs showing outward rectification. B, mean I-V plots (± s.e.m.) corresponding to 10 recordings.