1.A.91 The Plasmodial Surface Anion Channel (PSAC) Family

Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients as mediated by the plasmodial surface anion channel (PSAC), recently linked to parasite Cytoadherence-linked asexual protein 3.2 (CLAG3.2) gene. Although the CLAG3.2-encoded protein is integral to the host membrane, its contribution to solute transport was unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Sharma et al. 2015 identified a probable CLAG3.2 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 α-helical transmembrane segment, suggesting that the domain lines a water-filled pore. A single CLAG3.2 mutation (A1210T) in a leupeptin-resistant PSAC mutant fell 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 blocking affinity of PSAC inhibitors that bind at opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived leupeptin challenge significantly better than a transfection control. 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 development of therapies targeting PSAC. Plasmodium transporters have been reviewed (Staines et al. 2017).

Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake. Member proteins are unique to Plasmodium spp. Schureck et al. 2021 showed that RhopH is synthesized as a soluble complex of CLAG3, RhopH2, and RhopH3 with 1:1:1 stoichiometry. After transfer to a new host cell, the complex crosses a vacuolar membrane surrounding the intracellular parasite and becomes integral to the erythrocyte membrane through a PTEX translocon-dependent process. A 2.9 Å single-particle cryo-EM structure of the trafficking complex, revealed that CLAG3 interacts with the other subunits over large surface areas. This soluble complex is tightly assembled with extensive disulfide bonding and predicted transmembrane helices shielded. Schureck et al. 2021 proposed that the large protein complex is stabilized for trafficking but poised for host membrane insertiond through large-scale rearrangements, paralleling smaller two-state pore-forming proteins in other organisms.

The generalized reaction catalyzed by the PSAC is: 

Solutes (out) ↔ Solutes (in).


 

References:

Gupta, A., P. Balabaskaran-Nina, W. Nguitragool, G.S. Saggu, M.A. Schureck, and S.A. Desai. (2018). CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane. MBio 9:.

Meier, A., H. Erler, and E. Beitz. (2018). Targeting Channels and Transporters in Protozoan Parasite Infections. Front Chem 6: 88.

Pain, M., A.W. Fuller, K. Basore, A.D. Pillai, T. Solomon, A.A. Bokhari, and S.A. Desai. (2016). Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors. PLoS One 11: e0149214.

Schureck, M.A., J.E. Darling, A. Merk, J. Shao, G. Daggupati, P. Srinivasan, P.D.B. Olinares, M.P. Rout, B.T. Chait, K. Wollenberg, S. Subramaniam, and S.A. Desai. (2021). Malaria parasites use a soluble RhopH complex for erythrocyte invasion and an integral form for nutrient uptake. Elife 10:.

Shao, J., G. Arora, J. Manzella-Lapeira, J.A. Brzostowski, and S.A. Desai. (2022). Kinetic Tracking of Plasmodium falciparum Antigens on Infected Erythrocytes with a Novel Reporter of Protein Insertion and Surface Exposure. mBio e0040422. [Epub: Ahead of Print]

Sharma P., Rayavara K., Ito D., Basore K. and Desai SA. (2015). A CLAG3 mutation in an amphipathic transmembrane domain alters malaria parasite nutrient channels and confers leupeptin resistance. Infect Immun. 83(6):2566-74.

Staines, H.M., C.M. Moore, K. Slavic, and S. Krishna. (2017). Transmembrane solute transport in the apicomplexan parasite Plasmodium. Emerg Top Life Sci 1: 553-561.

Examples:

TC#NameOrganismal TypeExample
1.A.91.1.1

The plasmodial three-component surface broad specificity anion channel complex. One component is PSAC (Clag3.2, Clag3/2, RhopH1, of 1416 aas and at least two putative TMSs, one N-terminal and one C-terminal (residues 1199 to 1223)). The latter is an amphipathic α-helix thought to form the channel in the multisubunit complex (Sharma et al. 2015).  CLAG3 undergoes hetero-association, and its expression determines the channel phenotype quantitatively, leading to host erythrocyte permeability to ions and nutrients (Gupta et al. 2018). The isoforms traffic to and insert in the host membrane while remaining associated with two unrelated parasite proteins, RhopH2 (CLAG3.2 of 1414 aas (B0M163) and RhopH3   (CLAG3.3 (B0M0W2) of 897 aas and up to 4 TMSs, one N-terminal and up to three centrally located. Both the channel phenotypes and molecular changes are consistent with a multiprotein complex that forms the nutrient pore, supporting direct involvement of the CLAG3 protein in channel formation (Gupta et al. 2018). Inhibitors potentially useful theraputically at 5 μM concentrations include PRT1-20 and ISPA-28. Their use suggested that there may be two routes of nutrient entry via the PSAC (Pain et al. 2016). Reviewed by Meier et al. 2018. Malaria parasites use a soluble RhopH complex for erythrocyte invasion and an integral membrane channel form for nutrient uptake (Schureck et al. 2021). (See family description for more details.) The kinetics of CLAG3.2 insertion into the erythrocyte membrane has been studied (Shao et al. 2022).

Alveolata

PSAC (Clag3.2) of Plasmodium falciparum

 
1.A.91.1.2

Uncharacterized protein of 1454 aas and 2 or 3 TMSs (N- and C-terminal).

UP of Theileria orientalis

 
1.A.91.1.3

Rhoptry neck protein, Ron2, of 1350 aas and 2 TMSs, N- and C-terminal.

Ron2 of Babesia divergens

 
1.A.91.1.4

Rhoptry neck protein 2-like protein 2 (Precursor), related, of 1275 aas and 3 TMSs, 1 N-terminal, and two C-terminal.

Ron2 of Eimeria brunetti

 
1.A.91.1.5

Uncharacteerized protein of 1462 aas and 3 TMSs, one N-terminal, and two C-terminal.

UP of Hammondia hammondi