Critical role of a K ⁺ channel in Plasmodium berghei transmission revealed by targeted gene disruption

Abstract

Regulated K ؉ transport across the plasma membrane is of vital importance for the survival of most cells. Two K ؉ channels have been identified in the Plasmodium falciparum genome; however, their functional significance during parasite life cycle in the vertebrate host and during transmission through the mosquito vector remains unknown. We hypothesize that these two K ؉ channels mediate the transport of K ؉ in the parasites, and thus are important for parasite survival. To test this hypothesis, we identified the orthologue of one of the P. falciparum K ؉ channels, PfKch1, in the rodent malaria parasite P. berghei (PbKch1) and examined the biological role by performing a targeted disruption of the gene encoding PbKch1. The deduced amino acid sequence of the six transmembrane domains of PfKch1 and PbKch1 share 82% identity, and in particular the pore regions are completely identical. The PbKch1-null parasites were viable despite a marked reduction in the uptake of the K ؉ congener 86 Rb ؉ , and mice infected with PbKch1-null parasites survived slightly longer than mice infected with WT parasites. However, the most striking feature of the phenotype was the virtually complete inhibition of the development of PbKch1-null parasites in Anopheles stephensi mosquitoes. In conclusion, these studies demonstrate that PbKch1 contributes to the transport of K ؉ in P. berghei parasites and supports the growth of the parasites, in particular the development of oocysts in the mosquito midgut. K ؉ channels therefore may constitute a potential antimalarial drug target. malaria ͉ pathogenesis ͉ mosquito ͉ drug target

Functional characterization of malaria parasites deficient in the K+ channel Kch2

“…Before transfection, pB3DPbKch2 was linearized with three restriction enzymes ( Kpn I, Not I, and Sca I). Schizont-stage parasites of P. berghei 2.34 ANKA strain were transfected with ~100 μg of linearized targeting plasmid using Amaxa Nucleofactor® device, program T-001 [7]. Transfected parasites were immediately injected (intra venous) into female Swiss-Webster mice (four-weeks old) and mice were treated with pyrimethamine in the drinking water (70 μg/ml), starting 24 hours after transfection.…”

“…The alkali ion 86 Rb + was used as a tracer for K + in an uptake protocol [7]. To detect differences in 86 Rb + transport between WT, PbKch1-null [7], and PbKch2-null parasites, infected mouse red blood cells were harvested and treated with saponin to allow functional access to the parasites plasma membrane [7].…”

“…In the rodent malaria parasite P. berghei , targeted gene disruption showed that Kch1 contributes to K + transport across the parasite’s plasma membrane and appears to critically influence parasite development in the mosquito vector [7]. The function of the second K + channel, Kch2, however, is unknown.…”

Electrophysiological characterization of potassium conductive pathways in Trypanosoma cruzi

“…Furthermore, sexual reproduction of the mosquito-stage of Plasmodium bergei seems to be impaired by gene disruption of pbKch1, the homologous gene for pfKch1 (PfK1) in this parasite [Ellekvist et al, 2008], suggesting that K In Trypanosoma cruzi, the causative agent of Chagas disease, K þ conductive pathways have been postulated to play a role in osmoregulatory process, contributing to 7% of the regulatory volume decrease observed under hyposmotic stress [reviewed in Rohloff and Docampo, 2008]. On the other hand, pH steady state and the compensatory response to extracellular acidification are K þ dependent in T. cruzi mammalian stages, both process being Ba þ2…”

A Trk/HKT-Type K + Transporter from Trypanosoma brucei

“…The malaria parasite Plasmodium falciparum possesses two putative K ϩ channel subunits with 6 TM domains and one P-loop (19,52). Disruption of an orthologous gene in Plasmodium berghei strongly impaired the development of the malaria parasites in the mosquito (18). However, these putative channels have not yet been proven to be permeable to K ϩ .…”