On the mode of action of phlorizin as an antimalarial agent in in vitro cultures of plasmodium falciparum

Kutner, Shirley; Breuer, William; Ginsburg, Hagaï; Cabantchik, Z. I.

doi:10.1016/0006-2952(87)90389-3

Cited by 62 publications

(39 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Phloridzin blocking in HB3-3rec was indistinguishable from that measured in the untransfected HB3 parent (Fig. 4A and B), consistent with conserved affinity for this inhibitor in diverse parasite lines (41,42). In contrast, the A1210T transfection mutant yielded channels with significantly reduced half-maximal affinity for phloridzin (K 0.5 ϭ 35, 47, and 1,260 M for HB3, HB3-3rec, and HB3-c3A1210T, respectively; P Յ 0.001 for comparison of HB3-c3A1210T to both other lines at 100 M inhibitor and no significant difference between HB3-3rec and HB3 [n ϭ 4 to 8 each]).…”

Section: Resultssupporting

confidence: 71%

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

et al. 2015

View full text Add to dashboard Cite

Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted ␣-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.T he human malaria parasite Plasmodium falciparum remodels its host erythrocyte by exporting many proteins, generating membranous structures in the host cytosol, and increasing erythrocyte permeability to many solutes. Studies by multiple groups have determined that anions, sugars, purines, organic cations, and some vitamins have increased permeability after infection (1-4). The increase in permeability is primarily mediated by a parasitederived ion and nutrient channel known as the plasmodial surface anion channel (PSAC) (5). Importantly, both PSAC single-channel properties and the relative increases in solute permeabilities are conserved in divergent malaria parasites (6). Because Babesia parasites do not induce PSAC-like activity in erythrocytes that they invade (7), this channel is thought to be restricted to the genus Plasmodium.The clag multigene family, also conserved in and restricted to malaria parasites (8), has recently been linked to PSAC activity (9-11). Two paralogs on parasite chromosome 3, known as clag3.1 and clag3.2, appear to play a critical role, as identified by genetic mapping experiments with ISPA-28, an isolate-specific PSAC antagonist that blocks channels on the Dd2 laboratory clone but is ineffective against channels from other lines. These genes undergo epigenetic switching to encode a single protein that is initially packaged in specialized organelles known as rh...

show abstract

Section: Resultssupporting

confidence: 71%

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Prior to these findings, workers in the PSAC field accepted that parasite killing by phloridzin was primarily mediated via its actions on PSAC; there was concordance between its K 0.5 for channel inhibition (Fig. 2E) and its IC 50 for parasite growth inhibition (17). Because the selected mutants had growth rates unaffected by 100 M phloridzin despite an unchanged K 0.5 for PSAC inhibition, this concordance appears to be only coincidental.…”

Section: Discussionmentioning

confidence: 85%

“…These antagonists are believed to kill parasites by preventing PSAC-mediated uptake of essential nutrients. This approach selected for unambiguous and stable resistance to growth inhibition by 100 M phloridzin, a nonspecific antagonist of PSAC (17). Osmotic lysis and electrophysiological studies with phloridzin revealed that resistance did not result from either an altered mechanism or an affinity for PSAC inhibition.…”

mentioning

confidence: 99%

Plasmodial Surface Anion Channel-independent Phloridzin Resistance in Plasmodium falciparum

Desai

Alkhalil

Kang

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

The plasmodial surface anion channel (PSAC) is an unusual ion channel induced on the human red blood cell membrane after infection with the malaria parasite, Plasmodium falciparum. Because PSAC is permeant to small metabolic precursors essential for parasite growth and is present on red blood cells infected with geographically divergent parasite isolates, it may be an ideal target for future antimalarial development. Here, we used chemically induced mutagenesis and known PSAC antagonists that inhibit in vitro parasite growth to examine whether resistance mutations in PSAC can be readily induced. Stable mutants resistant to phloridzin were generated and selected within 3 weeks after treatment with 1-methyl-3-nitro-1-nitrosoguanidine. These mutants were evaluated with osmotic lysis and electrophysiological transport assays, which indicate that PSAC inhibition by phloridzin is complex with at least two different modes of inhibition. Mutants resistant to the growth inhibitory effects of phloridzin expressed PSAC activity indistinguishable from that on sensitive parasites, indicating selection of resistance via mutations in one or more other parasite targets. Failure to induce mutations in PSAC activity is consistent with a highly constrained channel protein less susceptible to resistance mutations; whether this protein is parasiteor host-encoded remains to be determined.We recently used the cell-attached and whole cell patch clamp methods to identify an unusual ion channel on the red blood cell (RBC) 1 membrane of infected cells (1). This channel, the plasmodial surface anion channel (PSAC), has a number of unusual functional properties that distinguish it from known human channels. First, it accounts for increases in permeability to a range of solutes identified in many previous studies. PSAC is permeant to both anions (2) and organic solutes such as sugars (3), amino acids (4), purines (5), some vitamins (6), and a number of organic cations (7). Amazingly, although broadly permeant, it effectively excludes Na ϩ by Ͼ100,000-fold relative to Cl Ϫ . This combination, unparalleled in other known channels, permits osmotic stability of the infected RBC in serum while permitting the acquisition of diverse essential nutrients (8). PSAC achieves its stringent exclusion of Na ϩ through the combined effects of extracellular pore mouth cationic amines to repel extracellular cations and at least one weak binding site in its pore to favor bulky dehydrated ions over smaller ions (9).Another unusual feature of PSAC is its small single channel conductance of ϳ20 pS in 1.15 molar Cl Ϫ solutions (10); most other channels with broad selectivity profiles exhibit larger single channel conductances. A channel's chord conductance, calculated from measured current amplitudes at a range of imposed membrane potentials, quantitatively corresponds to the number of ions passing through the open channel per unit time. In terms of Eyring rate theory, this channel's small conductance reflects the one or more energetic barriers that each solute mo...

show abstract

“…In addition to inhibition by phloridzin (25), furosemide, NPPB [nitro-2-(3-phenyl-propylamino)benzoic acid] (23), and glybenclamide (24)-nonspecific antagonists of channels and carriers-PSAC is unexpectedly blocked by dantrolene, a specific antagonist of sarcoplasmic reticulum Ca 2ϩ channels without measurable activity against five other classes of anion channels (G. Lisk and S. A. Desai, submitted for publication). A small secondary screen of dantrolene derivatives subsequently achieved further specificity for PSAC by identifying a nitrophenyl-furan derivative that has higher affinity for PSAC inhibition and no measurable activity against the sarcoplasmic reticulum Ca 2ϩ channels (22).…”

Section: ͼ Brmentioning

confidence: 99%

The Plasmodial Surface Anion Channel Is Functionally Conserved in Divergent Malaria Parasites

Lisk

Desai

2005

Eukaryot Cell

View full text Add to dashboard Cite

The plasmodial surface anion channel (PSAC), a novel ion channel induced on human erythrocytes infected with Plasmodium falciparum, mediates increased permeability to nutrients and presumably supports intracellular parasite growth. Isotope flux studies indicate that other malaria parasites also increase the permeability of their host erythrocytes, but the precise mechanisms are unknown. Channels similar to PSAC or alternative mechanisms, such as the upregulation of endogenous host transporters, might fulfill parasite nutrient demands. Here we evaluated these possibilities with rhesus monkey erythrocytes infected with Plasmodium knowlesi, a parasite phylogenetically distant from P. falciparum. Tracer flux and osmotic fragility studies revealed dramatically increased permeabilities paralleling changes seen after P. falciparum infection. Patchclamp of P. knowlesi-infected rhesus erythrocytes revealed an anion channel with striking similarities to PSAC: its conductance, voltage-dependent gating, pharmacology, selectivity, and copy number per infected cell were nearly identical. Our findings implicate a family of unusual anion channels highly conserved on erythrocytes infected with various malaria parasites. Together with PSAC's exposed location on the host cell surface and its central role in transport changes after infection, this conservation supports development of antimalarial drugs against the PSAC family.

show abstract

On the mode of action of phlorizin as an antimalarial agent in in vitro cultures of plasmodium falciparum

Cited by 62 publications

References 12 publications

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

Plasmodial Surface Anion Channel-independent Phloridzin Resistance in Plasmodium falciparum

The Plasmodial Surface Anion Channel Is Functionally Conserved in Divergent Malaria Parasites

Contact Info

Product

Resources

About