Pore size of the malaria parasite's nutrient channel

Abstract

The malaria parasite, Plasmodium falciparum, requires large amounts of nutrients to sustain its rapid growth within the human red blood cell. A recently identified ion channel on the surface of the intraerythrocytic parasite may provide direct access to these nutrients in the red blood cell cytosol. Evidence supporting this role was obtained by incorporating this channel into planar lipid bilayers. In bilayers, this channel has conductance and gating properties identical to the in situ channel, passes soluble macromolecules of up to 1400 Da, and functions as a high capacity, low affinity molecular sieve. These properties, remarkably similar to those of a pore on Toxoplasma gondii (another protozoan parasite causing human disease), suggest a novel class of channels used by these intracellular parasites to acquire nutrients from host cytosol.

Figure 1

Incorporation of the nutrient channel into planar lipid bilayers. (A) Channel activity seen after addition of parasite vesicles to planar lipid bilayers. Dashed and solid lines represent channel open and closed states, respectively. Bathing solution was designed to simulate RBC cytosol and contained (both sides) 100 mM KCl with buffer A (20 mM Hepes/2.0 mM MgCl₂/1 mM Na₂ATP, pH 7.3 with NaOH). V_b was +30 mV. (B) The channel’s current–voltage relationship. Main figure shows unitary current amplitudes (measured by eye) plotted against V_b. The cis bathing solution was the same as that used in A. The trans solution contained either 100 mM CsCl + buffer A (•) or 240 mM glycine + buffer A (○). The decrease in slope conductance (from 155 to 86 pS) and the minimal change in E_rev (from +9 to +5 mV) upon replacement of CsCl with glycine confirm that these channels were equally permeable to cations, anions, and glycine, consistent with similar measurements on intact parasites (3). The slope conductances and the E_rev values were identical to those measured by patch clamp of intact parasites (3), confirming that this is the same channel. Channel events in CsCl (Left Inset) and glycine (Right Inset) at V_b = −50 mV. Scale, 4 pA/100 ms. (C) Subconductance states. Solid and dashed lines represent closed and fully open states, respectively. The dotted line represents a subconductance level seen previously (3). It is not a separate channel because there are two full transitions (indicated by arrows) that would require the unlikely simultaneous opening and closing of two independent channels. Bath solutions are the same as those in A. V_b = +30 mV.

Figure 2

Voltage dependence of the channel. (A) Typical channel activity at a range of V_b values indicated. The bathing solution on both sides of the bilayer was 160 mM KCl + buffer A. The channel’s closed and fully open states are indicated by solid and dashed lines,respectively, at each voltage. Notice that the channel is mostly open at ±25 mV and less so at −70 and +60 mV. The intermediate level shown in the sweep recorded at +60 mV is a subconductance state. (B) Average normalized current as a function of V_b. At each V_b, recordings from the channel in A were integrated (average of 45 s each) and normalized to the value for a fully open channel. Although different from classical open probability determinations, this calculation incorporates transport during subconductance states and indicates the net flux per unit time. Notice that near V_b = 0, the channel passes ≥96% of its maximal current. The solid line is given by i_ave/i_max = 1.01/{[1 + exp((−40 − V_b)/13)] · [1 + exp((V_b - 58)/9)]}, representing a fit to the product of two Boltzman equations (11).

Figure 3

Pore size of the nutrient channel. (A) Open amplitudes of single channels at V_b = +25 mV after addition of PEG. The bathing solution on both sides of the bilayer is 100 mM KCl with buffer A without (control) or with 20% (wt/vol) PEG (of indicated size). These channels are primarily open at this voltage (also seen in Fig. 2). Notice that the current amplitude increases with PEG size but reaches a maximum by PEG 1450. (B) Slope conductance of single channels (γ) vs. PEG diameter measured in water (15). •, Average slope conductances ± SEM measured in 100 mM KCl with buffer A and 20% (wt/vol) PEG 200, 400, 600, 1000, 1450, and 3350 (n = 3 single channels each, ≥ 4 V_b values for each conductance). The dashed lines represents the average slope conductance measured in symmetric bathing solutions of 55 mM KCl/11 mM Hepes/1.1 mM MgCl₂/1 mM Na₂ATP, pH 7.3 (lower dashed line, n = 2 single channels) and 160 mM KCl/32 mM Hepes/3.2 mM MgCl₂/1 mM Na₂ATP, pH 7.3 (upper dashed line, n = 4 single channels). The solid line is a linear fit of the data for PEG 600, 1000, and 1450. It intercepts the upper dashed line at diameter of 23 Å, the estimated pore size of the channel.

Figure 4

Channel conductance vs. KCl concentration. Single channel slope conductances were determined in symmetric bathing solutions of buffer A with 55, 100, 160, 300, 455, 583, and 2000 mM KCl (n = 2, 5, 4, 2, 1, 3, and 3 single channels, respectively; ≥4 V_b values for each conductance). The average conductances with SEMs are here shown against the KCl activity (KCl concentration multiplied by tabulated activity coefficients; ref. 17). Notice that channel conductance does not saturate with increasing KCl concentrations.