Sequence analysis of the Plasmodium K⁺ channels. (A) Alignment of the P. berghei putative K⁺ channel PbKch1 and the orthologue P. falciparum K⁺ channel PfKch1. The ORFs of PbKch1 and PfKch1 encode 986 and 1,940 aa, respectively. Identical or similar amino acids are colored red. The channels belong to the superfamily of six transmembrane-segment K⁺ channels. Except for the S4 segment, the membrane topology of the channel was predicted by the TMHMM algorithm. The S4 segment of six transmembrane-segment K⁺ channels is notoriously difficult to predict because of a number of charged residues, usually arginines, and this particular segment was therefore identified by eye. The amino acid identity in the parts of the proteins comprising the six transmembrane domains (indicated by gray boxes) is 82%. The amino acids of the pore regions are identical. (B) Alignment of the S5–S6 linker of PfKch1 and PbKch1 with corresponding linkers from different hypothetical and known K⁺ channels. The pore loop contains the canonical K⁺ channel signature sequence ((T/S)XXTXGYG). B. bovis, C. hominis, and T. annulata: Hypothetical proteins from Babesia bovis [Protein Data Bank (PDB) ID code XP_001610013], Cryptosporidium hominis (PDB ID code XP_668687), and Theileria annulata (PDB ID code XP_952459). nBK, Caenorhabditis elegans BK channel (PDB ID code NP_001024261); hBK, human BK channel (PDB ID code NP_001014797); hShaker, human voltage-gated shaker-related K⁺ channel (PDB ID code NP_002223); hKCNQ1, human cardiac KCNQ channel (PDB ID code CAO03369). Amino acids in the K⁺ channel signature sequence are highlighted in bold; amino acid identity with PfKch1 is colored gray.

Characterization of ⁸⁶Rb⁺ transport in the PbKch1-null parasite. (A) Uptake of ⁸⁶Rb⁺ in isolated P. berghei parasites. Erythrocytes from mice infected with WT or PbKch1-null parasites were treated with saponin, thereby allowing functional access to the parasite's plasma membrane. The saponin-treated parasites were incubated with trace amounts of ⁸⁶Rb⁺, and samples were taken at the indicated time points. PbKch1-null parasites (triangles) showed decreased uptake compared to WT parasites (squares). Each point represents mean value ± SEM (n = 3). (B) Pharmacological characterization of ⁸⁶Rb⁺ uptake in isolated parasites. Uptake was assessed at a fixed time point (t = 10 min) in the presence of either suspending buffer alone (control), 10 mM Ba²⁺, 20 mM TEA, or 0.5 mM quinine. The height of each bar represents mean value ± SEM (n = 4). When denoted by *, ⁸⁶Rb⁺ uptake was statistically different from ⁸⁶Rb⁺ uptake in untreated WT parasites (P < 0.05, unpaired t test).

Transmission results of the WT and PbKch1-null parasite. Mosquitoes were fed on PbKch1-null infected mice or WT-infetced mice. Of 89 mosquitoes fed on WT-infected mice (n = 3), 81 contained oocysts (115 median oocyst per mosquito). Of 112 mosquitoes fed on PbKch1-null infected mice (n = 3) only two contained oocysts (1 and 22 oocysts, respectively).

Disruption strategy for generation of PbKch1-null parasites. (A) (Upper) The genomic locus. (Lower) The disrupted gene. The black line represents the locus (PDB ID code PB_RP3798) containing the gene encoding PbKch1 (bar). The deduced six transmembrane domains (indicated by the small interconnected bars below the gene) of PbKch1 are encoded by the core region. The 5′ and 3′ regions are the recombination sites used to replace, by double crossover, the core region of the gene with the Tg-DHFR ORF (gray color). The Tg-DHFR confers resistance to the compound pyrimethamine, which can be used to select PbKch1-null parasites. (B) Validation of the PbKch1-null genotype. Gene-specific primers amplify different PCR products from DNA purified from WT P. berghei and PbKch1-null P. berghei. Fragments 1 and 2 (same as in A) can be amplified only from WT parasites, not from PbKch1-null parasites. The primers used to generate fragments 3 and 4 (same as in A) target the Tg-DHFR ORF from outside the recombination sites. These fragments can be amplified only from PbKch1-null parasites, not from WT parasites, nor from parasites containing circular (nongenomic) copies of the targeting plasmid. Lanes 1–4: PCR products amplified from WT parasite DNA. The presence of fragments 1 (1,022 bp) and 2 (667 bp), but not 3 and 4, confirms the undisrupted genotype. Lanes 5–8: PCR products amplified from PbKch1-null parasite DNA. The presence of fragments 3 (968 bp) and 4 (625 bp), but not 1 and 2, confirms that the core region has been correctly replaced by the Tg-DHFR ORF, and that there is no contamination from WT parasite DNA. Standard (St) lanes contain BstEII-digested Escherichia coli λ-phage DNA with base-pair length of selected markers shown to the right. (C) RT-PCR with RNA from WT parasites (lanes 1–3) and PbKch1-null parasites (lanes 4–6). Primer pairs were the same as those used above to assess the presence of the uninterrupted PbKch1 gene and the interrupted gene where the core region of PbKch1 has been replaced by the Tg-DHFR ORF. Fragment 2 (667 bp) could be amplified from WT RNA (lane 1), but not from PbKch1-null RNA (lane 4). Fragment 3 (968 bp) could be amplified from PbKch1-null RNA (lane 5), but not from WT RNA (lane 2). PCR amplification not preceded by the reverse-transcriptase step gave no bands from WT RNA (lane 3) or PbKch1-null RNA (lane 6), which confirms the absence of genomic DNA contamination in the RNA preparations. Standard (St) marker is as mentioned in B.

Phenotype characterization of the PbKch1-null parasite. (A) Growth kinetics of the PbKch1-null parasite. Parasitemia of mice infected with WT parasites (squares) or PbKch1-null parasites (triangles) is shown. Each point represents mean value ± SEM. (B) Survival of mice infected with WT parasites (stippled curve) or PbKch1-null parasites (continuous curve). Median survival time was 15 days for mice infected with WT parasites and 21 days for mice infected with PbKch1-null parasites (P < 0.001, χ² test).