Research briefPlasmodium falciparum: Growth response to potassium channel blocking compounds
Introduction
In a persistent human infection, Plasmodium falciparum parasites must successfully traverse widely variant environmental milieus; from the intracellular locale of the hepatocyte and RBC1, to the mosquito mid-gut and human circulatory system. The parasite’s ability to adapt to variations in its environment is dependent on transporter pumps, exchangers and ion channels, which together function to maintain the intra-parasite environment in the face of the often rapidly changing external conditions. Many classes of putative P. falciparum channels and transporters have been identified (Martin et al., 2005). The proper functioning of these channels and transporters is thought to be central for maintaining parasite viability (recently reviewed by Kirk (2004)).
Potassium (K+) channels are transmembrane proteins that gate open and closed to control the flow of K+ ions across cell membranes. K+ channels play critical roles in the regulation of the transmembrane electrochemical gradient, cell membrane potential and intracellular osmolarity and are essential for the survival of all known cells types. The modes and rate of channel activation (including voltage-dependence, Ca2+-sensitivity, H+-sensitivity, phosphorylation-dependence), level of ion conductance and sensitivity to K+ channel blocking compounds varies depending on the type of K+ channel. The Ca2+-activated K+ channels can be broadly classified into three types; big (BK), intermediate (IK) and small (SK) conductance K+ channels (see Hille for review (2001)).
The P. falciparum genome encodes two putative K+ channels (PfK1 and PfK2) that possess greatest homology to Ca2+-activated K+ channels (Ellekvist et al., 2004, Waller et al., 2008). Since these K+ channels appear to be essential for P. falciparum survival (Waller et al., 2008), the effect of various K+ channel blocking compounds upon the in vitro growth of P. falciparum asexual stage parasites was investigated using 72 h [3H]-hypoxanthine incorporation assays. Here, we report the novel anti-malarial effects for each of bicuculline methiodide and tubocurarine chloride and the novel lack of anti-malarial effects of apamine and verruculogen.
Section snippets
Parasite culture
Plasmodium falciparum 3D7 parasites were maintained by in vitro culture using standard culture conditions with 0.5% Albumax II (Invitrogen) supplementation and sorbitol synchronized as previously described (Waller et al., 2008). Parasitaemias were determined by microscopic examination of Giemsa-stained thin blood smears.
[3H]-hypoxanthine incorporations assays
Seventy two hours [3H]-hypoxanthine incorporation assays were performed using previously described methods (Waller et al., 2003) to determine the parasite’s growth response to
Results
Seventy-two hour [3H]-hypoxanthine incorporation assays were performed to determine the parasite’s growth response to various K+ channel blockers. Graphical plots of the growth inhibition data derived from the [3H]-hypoxanthine incorporations assays for each compound are shown in Fig. 1. The calculated 50% and 90% Inhibitory Concentration (IC50 and IC90) ± Standard Error of the Mean (SEM) are shown in Table 1. The IC50 and IC90 data for the anti-malarial quinine and quinidine (Table 1) were in
Discussion
The putative P. falciparum K+ channels PfK1 and PfK2 possess greatest homology to Ca2+-activated K+ channels (Waller et al., 2008). The compounds tested were selected based upon their ability to selectively block the three types (BK, IK, or SK) of Ca2+-activated K+ channels, and included apamine, bicuculline methiodide, charybdotoxin, haloperidol, tubocurarine chloride and verruculogen. The established anti-malarial quinine and quinidine, which possess demonstrated K+ channel blocking
Acknowledgments
We thank Ji-Mee Hwang for technical assistance during the initial stages of this investigation. KLW is supported by an Australian NHMRC Howard Florey Centenary Research Fellowship. This research was funded by the USAMRMC grant #DAMD17-02-1-0290.
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