Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.

During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid‐osmotic model on the grounds of time‐resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid‐osmotic model can predict time‐dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model‐predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid‐osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum‐infected erythrocytes. The contribution of vesiculation to the malaria‐protective function of hemoglobin S is discussed.

To avoid clearance by the spleen, red blood cells infected with the human malaria parasite Plasmodium falciparum (iRBCs) adhere to the vascular endothelium through adhesive protrusions called ''knobs'' that the parasite induces on the surface of the host cell. However, the detailed relation between the developing knob structure and the resulting movement in shear flow is not known. Using flow chamber experiments on endothelial monolayers and tracking of the parasite inside the infected host cell, we find that trophozoites (intermediate-stage iRBCs) tend to flip due to their biconcave shape, whereas schizonts (late-stage iRBCs) tend to roll due to their almost spherical shape. We then use adhesive dynamics simulations for spherical cells to predict the effects of knob density and receptor multiplicity per knob on rolling adhesion of schizonts. We find that rolling adhesion requires a homogeneous coverage of the cell surface by knobs and that rolling adhesion becomes more stable and slower for higher knob density. Our experimental data suggest that schizonts are at the border between transient and stable rolling adhesion. They also allow us to establish an estimate for the molecular parameters for schizont adhesion to the vascular endothelium and to predict bond dynamics in the contact region.

Sickle cell trait, a common hereditary blood disorder, protects carriers from severe disease in infections with the human malaria parasite Plasmodium falciparum. Protection is associated with a reduced capacity of parasitized erythrocytes to cytoadhere to the microvascular endothelium and cause vaso-occlusive events. However, the underpinning cellular and biomechanical processes are only partly understood and the impact on endothelial cell activation is unclear. Here, we show, by combining quantitative flow chamber experiments with multiscale computer simulations of deformable cells in hydrodynamic flow, that parasitized erythrocytes containing the sickle cell haemoglobin displayed altered adhesion dynamics, resulting in restricted contact footprints on the endothelium. Main determinants were cell shape, knob density and membrane bending. As a consequence, the extent of endothelial cell activation was decreased. Our findings provide a quantitative understanding of how the sickle cell trait affects the dynamic cytoadhesion behavior of parasitized erythrocytes and, in turn, endothelial cell activation.