Identification of a large anion channel required for digestive vacuole acidification and amino acid export in Plasmodium falciparum

Abstract

Malaria parasites survive in human erythrocytes by importing and digesting hemoglobin within a specialized organelle, the digestive vacuole (DV). Although chloroquine and other antimalarials act within the DV, the routes used by drugs, ions, and amino acids to cross the DV membrane remain poorly understood. Here, we used single DV patch-clamp to identify a novel large conductance anion channel as the primary conductive pathway on this organelle in Plasmodium falciparum, the most virulent human pathogen. This Big Vacuolar Anion Channel (BVAC) is primarily open at the DV resting membrane potential and undergoes complex voltage-dependent gating. Ion substitution experiments implicate promiscuous anion flux with Cl- being the primary charged substrate under physiological conditions. Conductance and gating are unaffected by antimalarials targeting essential DV activities and are conserved on parasites with divergent drug susceptibility profiles, implicating an unexploited antimalarial target. A conditional knockdown strategy excluded links to PfCRT and PfMDR1, two drug-resistance transporters with poorly defined transport activities. We propose that BVAC functions to maintain electroneutrality during H+ uptake, allowing DV acidification and efficient hemoglobin digestion. The channel also facilitates amino acid salvage, providing essential building blocks for parasite growth. Direct transport measurements at the DV membrane provide foundational insights into vacuolar physiology, should help clarify antimalarial action and drug resistance, and will guide therapy development against the parasite's metabolic powerhouse.

Fig 1. A large conductance ion channel on the DV membrane.

(A) Schematic of a Plasmodium-infected erythrocyte showing endocytosis of erythrocyte cytosol, hemoglobin digestion in the DV, and multiple transport activities at the DV membrane. (B) Transmission electron micrograph of harvested DV showing intact membrane and luminal hemozoin crystals. Scale bar, 200 nm. (C) Patch-clamp pipette after expansion of the shank proximal to the pipette tip via positive internal pressure while heating. The heating filament is visible as a dark haze behind the pipette. The sharp, narrow bore tip is preserved, permitting DV capture and patch-clamp. (D) Recordings from a DV membrane patch with one functional ion channel. Imposed pipette potentials are indicated to the left of each trace; closed and open channel current levels are indicated with red and green dashes, respectively. Stable intermediate current levels reflect subconductance states. Buffer A with WOS additive in bath and pipette, S2 Table. (E) Current-voltage (i-V) relationship for the identified channel. Mean ± S.E.M. measured single channel current amplitudes, i, for transitions between fully closed and open levels at each imposed pipette potential (V_p). (F) Open probability at each V_p, calculated as the integrated current normalized to the maximum current associated with a fully open channel, i_max. Solid line, best fit to Eq. 2. The underlying data can be found at https://doi.org/10.5281/zenodo.15305314.

Fig 2. BVAC is permeant to diverse inorganic and organic anions.

(A) Left, single channel recordings with 100 mM CaCl₂ and 50 mM KCl as the primary charge carriers in bath and pipette solutions (Buffers B and C, respectively; S2 Table). V_p as indicated. Notice the negative, downgoing events that reflect channel openings at zero V_p and the smaller amplitudes at positive than equal negative potentials. The cartoon illustrates the recording arrangement. Right, i-V relationship showing a positive reversal potential (x-intercept), indicating either net anion influx or cation efflux at zero V_p. (B) Left, single channel recordings with 280 mM sorbitol in the pipette (Buffer D plus WOS) and 70 mM NaCl, 70 mM KCl in the bath (Buffer A plus WOS). Channel events are larger at positive V_p and upgoing at zero voltage. Right, current-voltage relationship showing negative reversal potential establishes preferential anion flux. (C) Bi-ionic experiments with indicated anions at 140 mM in the pipette and 140 mM Cl⁻ in the bath (top to bottom traces pipette/Bath solutions: Buffer E plus WOS/F plus WOS; Buffer G plus WOS/H plus WOS; Buffer I/F; Buffer J plus WOS/F plus WOS; Buffer K/F). V_p = −60 mV for all traces. Downgoing channel openings in each solution indicate that each anion is permeant; glutamate⁻ produces the smallest inward currents, reflecting a lower but nonzero BVAC permeability. Right, i-V relationships for these channels; modest shifts in reversal potential from 0 mV indicate relatively high permeability for each anion. The underlying data can be found at https://doi.org/10.5281/zenodo.15305314.

Fig 3. Conditional knockdowns establish essentiality of PfCRT and PfMDR1 and exclude links to BVAC.

(A) Ribbon schematic showing the modified pfcrt gene carrying a linker (blue), hemagglutinin tag (HA), DHFR degradation domain (DDD), and a 3′ untranslated glmS riboswitch. Removal of trimethoprim (TMP) destabilizes DDD and degrades PfCRT; glucosamine (GlcN) addition degrades the gene’s mRNA. (B) Immunoblot showing conditional knockdown of PfCRT, as quantified using isolated DVs and anti-HA antibody. Addition of GlcN or removal of TMP reduces PfCRT; together, these manipulations maximize knockdown. The wild-type negative control is included (WT). Bottom, aldolase loading control. (C) Mean ± S.E.M. % residual PfCRT upon tandem knockdown after loading control normalization, from total cell lysates (cell) or isolated DVs (DV). (D) Growth of indicated parasites over 5 days. Parental wildtype line (WT): control (black); GlcN pulse addition (red); chloroquine growth inhibition control (blue). CRT-KD: medium + TMP (black); TMP removal (red triangles); GlcN pulse (red circles). MDR1-KD: control medium (black); GlcN pulse (red circles). PfCRT or PfMDR1 knockdown abolishes parasite expansion (P < 0.05 for TMP removal or GlcN prepulse for CRT-KD, n = 4 independent trials; P < 0.05 for GlcN prepulse for MDR1-KD,n = 3). (E) Schematic of modified pfmdr1 with linker (blue), tandem HiBiT and FLAG tags (hbF), T2A ribosomal skip peptide (orange), a neomycin resistance gene (NeoR), and a 3′ untranslated glmS. (F) Anti-FLAG immunoblot showing PfMDR1 knockdown upon GlcN pulse treatment. The wild-type control and aldolase loading controls are included. (G) Mean ± S.E.M. % residual PfMDR1 after knockdown. Total membranes (cell) or enriched DVs (DV) were estimated from anti-FLAG immunoblots. NanoLuc luminescence was quantified using total cell lysates (lysate); PfMDR1 abundance was also measured using Nano-Glo blotting of total membranes after SDS-PAGE (NG-blot). (H) Single channel recordings after maximal knockdown of PfCRT or PfMDR1, as indicated to the left of each trace. Symmetric pipette and bath solutions: Buffer A with and without WOS (top and bottom traces); V_p = −60 mV. Channel activity was unchanged; both channels shown here exhibit functional dimerization as described in S3E Fig. (I) % of patches containing 1 or more channels for indicated parasites and treatments. PfCRT or PfMDR1 knockdown does not change BVAC abundance. ns, no statistically significant difference (P ≥ 0.69 for all comparisons, central Fisher’s exact test with melding confidence intervals) [67]. The underlying data can be found at https://doi.org/10.5281/zenodo.15305314.

Fig 4. An unexploited drug target.

Single channel open probabilities (P_o) at V_p of −20 mV without (ctrl) and with inhibitors present in both bath and pipette: CQ, 1 µM chloroquine; Mef, 1 µM mefloquine; ART, 1 µM artemisinin; CsA, 10 µM cyclosporin A; 3 µM XR-9576; VPL, 20 µM verapamil; 200 µM DIDS; Phdn, 100 µM phloridzin; Furo, 100 µM furosemide; Glyb, 200 µM glybenclamide. Pipette and bath solutions of Buffer A with WOS. Filled circles reflect single molecule recordings used for analysis. The underlying data can be found at https://doi.org/10.5281/zenodo.15305314.

Fig 5. Model showing two essential roles for BVAC.

Anion uptake via BVAC (yellow) maintains electroneutrality during DV acidification via active H⁺ uptake through pyrophosphatase and V-type ATPase pumps (pink and green transporters, respectively). Glutamate⁻ liberated by hemoglobin digestion is exported via BVAC, fueling parasite protein synthesis; the channel may also export neutral and positively charged amino acids.