Erythrocytes infected with malaria parasites exhibit marked increases in permeability to organic and inorganic solutes. The plasmodial surface anion channel (PSAC), an unusual voltagedependent ion channel induced on the host membrane after infection, may play a central role in these permeability changes. Here, we identified a functional PSAC mutant through in vitro selection with blasticidin S. Resistance to blasticidin S was generated during culture and correlated with significant reductions in permeability to multiple solutes, consistent with uptake via a common pathway. Single channel recordings revealed marked changes in PSAC gating with the addition of a subconductance state not present in wild-type channels. The channel's selectivity profile and pharmacology also were significantly altered. Eventual loss of the mutant phenotype upon removal of selective pressure and slower growth of mutant parasites suggest that PSAC serves an important role in intracellular parasite survival. These findings provide solid evidence for the uptake of diverse solutes via PSAC and implicate one or more parasite genes in expression of this channel.drug resistance ͉ malaria ͉ nutrient acquisition ͉ fitness cost T he malaria parasite, Plasmodium falciparum, is a highly successful intracellular pathogen. In the human host, replication in erythrocytes leads to an exponentially increasing number of parasites and accounts for much of the clinical syndrome of malaria. To facilitate its rapid intracellular growth, the parasite remodels the erythrocyte cytoplasm (1), adds virulence factors to the surface of infected cells (2), and increases erythrocyte permeability to many small solutes. Increased uptake of solutes may be mediated by the plasmodial surface anion channel (PSAC) (3). This unusual ion channel has a singlechannel conductance of 20 pS in molar Cl Ϫ solutions and fast-flickering voltage-dependent gating. PSAC also has an atypical selectivity profile for anions (SCN Ϫ Ͼ I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ ) and effectively excludes Na ϩ despite permeability to bulky cations (4). Various antagonists have quantitatively identical effects on PSAC's open probability (5-7) and uptake of organic solutes including sugars (8), amino acids (9), purines (10), organic cations (11), and some vitamins (12), suggesting that PSAC functions as a shared route for the increased uptake of these diverse solutes after infection.Nevertheless, PSAC's role in solute transport is debated because other ion channels have been observed by some workers (13-16) and because a single ion channel with broad permeability to diverse solutes but stringent exclusion of Na ϩ is unprecedented. It is also debated whether PSAC and other putative channels are parasite-encoded proteins or modified human proteins (5). Addressing these issues is an important hurdle for transport studies in malaria and for drug development programs that seek to target the parasite-induced permeability changes.Blasticidin S is a fungal toxin (molecular weight 422.2) that kills most prokaryotic and eukaryoti...

Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency approximately 200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is approximately 80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.

Rates of reaction of (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-2) have been determined in 1:9 dioxane-water solutions containing 1.0 M KCl, 0.5 M KBr, and 0.1 M NaI over the pH range 4-13. These pH-rate profiles are more complicated than those for reaction of DE-2 in 0.2 M NaClO4 solutions and are interpreted in part by mechanisms in which halide ion attacks the diol epoxide as a nucleophile at intermediate pH, resulting in the formation of a trans-halohydrin. Reaction of DE-2 in these halide solutions at pH < ca. 5 occurs by rate-limiting carbocation formation, followed by capture of the intermediate carbocation by halide ion. The relative magnitudes of the rate constants for reaction of the intermediate carbocation with halide ions are estimated from product studies. The halohydrins are unstable intermediates and react quickly in subsequent reactions to yield tetrols in a ratio different than that formed from reaction of the carbocation with solvent. Nucleophilic attacks of 1.0 M Cl-, 0.5 M Br-, and 0.1 M I- on DE-2 are the principal reactions in the pH range ca. 6-9, leading to intermediate trans-halohydrins that hydrolyze to tetrols. At pH ca. 9-11, halohydrin formed from attack of halide ion on DE-2 reverts back to epoxide, leading to a negative break in the pH-rate profile. The main product-forming reaction of DE-2 at pH 11.3 is the spontaneous reaction. At pH > 12, the rate of reaction of DE-2 increases due to a second-order reaction of HO- with DE-2.