Characterization of two putative potassium channels in Plasmodium falciparum

In the course of a persistent human infection, Plasmodium falciparum parasites are exposed to dramatic changes in the extracellular milieu in terms of pH, osmolarity and ionic constituents. To survive such conditions transporter pumps, exchangers and ion channels operate to maintain the intra-parasite environment in the face of varying external conditions. Many classes of putative P. falciparum channels and transporters have been identified [1]. These are thought to be critical for parasite viability [2, 3] and offer potential new molecular targets for the development of novel anti-malarials.

One class of transporters, the potassium (K⁺) channels, are transmembrane proteins that gate open and closed to control the flow of K⁺ ions across cell membranes. K⁺ channels are critical in the regulation of the transmembrane electrochemical gradient, cell membrane potential and intracellular osmolarity and are essential for the survival of all known cells. K⁺ channels share several common features. They have 2, 4 or 6 membrane-spanning regions, two of which contribute to form the "pore region" through which K⁺ ions move (Figure 1A). A highly conserved GYG (or the less common GFG) signature motif within the pore region comprises the ion selectivity filter [4]. Functional channels result from a tetrameric complex of identical or closely related subunits, with the ion conduction pathway formed by the four-fold symmetry of the component subunits.

Here, a bioinformatic approach was used to identify two putative K⁺ channel genes (pfk1 and pfk2). This report details the cellular investigation of these putative K⁺ channel proteins, showing their differential expression and cellular localization in asexual blood stage parasites. The identification of only two putative K⁺ channels encoded within the P. falciparum genome, in conjunction with their differing expression and localization profiles suggests that these proteins may play unique and potentially critical roles in the maintenance of parasite viability.

Asexual P. falciparum parasites must rapidly respond to the widely variant ionic conditions, pH and osmolarities of their external milieus. In order to adapt to these environments, parasites most likely utilize ion channels and transporters to facilitate their regulation of intracellular ion concentrations. Allen and Kirk have shown that the transmembrane potential across the Plasmodium parasite plasma membrane is primarily generated by an electrogenic proton pump [21]. The proton efflux in this study was balanced by influx of K⁺, presumably through such surface channels. This K⁺ transport may be carried by PfK1 and/or PfK2. During blood-stage development, various parasite proteins are synthesized and exported into the RBC resulting in modification of normal RBC structure and function [20]. New permeation pathways (NPP) are hypothesized to facilitate the transport of low molecular mass solutes [2, 3] from the RBC membrane to the parasite and are likely to include many of the predicted parasite-encoded channels and transporters encoded in the parasite genome [1].

K⁺ channels are essential for the viability of all cells and are likely to be critical for malaria viability. The P. falciparum 3D7 genome encodes only two putative K⁺ channel genes, pfk1 and pfk2. The search criteria employed in this study specified that the selectivity filter motif be flanked by at least two predicted membrane-spanning segments. Although the number of transmembrane domains can vary with K⁺ channel type, in all cases, a single ion channel is created by the assembly of a combination of subunits that contribute four inner helices and four selectivity filter domains. Voltage-gated and Ca²⁺-activated K⁺ channels contain six (or seven, in the case of big conductance (BK) K⁺ channel) membrane spanning segments with the ion selectivity filter between the 5^th and 6^th membrane segment. Both PfK1 and PfK2 are predicted to have six membrane-spanning domains, and a pore region between the 5^th and 6^th segments based on hydropathy analysis and sequence alignments (Figure 1). The predicted pore-forming structures of PfK1 and PfK2 most resemble those of Ca²⁺-activated K⁺ channels. PfK1 also has a cluster of evenly spaced arginines in the 4^th membrane-spanning segment that suggests the possibility of voltage-sensitivity. PfK2 lacks these charged residues in the 4^th transmembrane segment and is thus more akin to intermediate conductance (IK) or small conductance (SK) channels. Full protein sequence alignments of PfK1 and PfK2 with other characterized K⁺ channels show less sequence similarity outside of the conserved pore-forming regions (data not shown), however their overall topologies are consistent with other voltage-gated and Ca²⁺-activated K⁺ channels.

A possible third P. falciparum K⁺ channel gene, PF14_0342, has been suggested from searches detailed by Martin et al [1]. This sequence is unlikely to encode a K⁺ channel due its lack of several key features that are common to many K⁺ channels. The predicted selectivity filter contains a GKG motif instead of GYG (Fig. 1C) and lacks appropriate flanking transmembrane regions.

Here, the differential expression and localization of two putative P. falciparum potassium channels have been demonstrated. Recent global gene expression studies show pfk1 mRNA detectable throughout the entire 48-hour asexual cycle [17‐19, 22]. In support of these data, PfK1 was detected by immunoblot in all RBC stages, though more protein was detected in mature forms. By IFA, PfK1 expression was detected from early trophozoites through schizont stage parasites. In ring stage parasites, the density of PfK1 may be too low, such that its detection was beyond the sensitivity of IFA. More intense labeling in mature-iRBCs (Figure 3) and also in multiply-iRBCs was consistently observed.

Like KARHP and MESA, PfK1 localizes to the RBC membrane. It is hypothesized that PfK1 is expressed by the parasite and exported into the RBC, where it is inserted into the RBC plasma membrane. Recent bioinformatic analyses of trafficked protein sequences have identified a conserved PEXEL/VTS motif that targets proteins for trafficking into the RBC [23, 24], in addition to the SS motif that directs proteins via the ER into the secretory pathway. In silico analysis [24, 25] of the PfK1 protein sequence for motifs indicated as potential export/transport signals failed to identify a VTS-like sequence, however manual searches yielded two potential VTS sequences, FFYRKLKNTFM and NFRRFLSSYKS (located 99 and 169 residues from the N-terminus, respectively). PfK1 also contains a predicted SS sequence (YDKRFSS RIKPK), located upstream of the VTS motifs and 20 residues from the N terminus of PfK1 (K. Haldar, personal communication).

PfK2 is primarily expressed in late schizonts and merozoites, in agreement with previous gene expression studies [18, 19, 23]. By IFA, PfK2 was detected in some, but not all, young ring stage parasites. Manual analysis of the PfK2 sequence revealed the presence of a possible SS cleavage motif and a low value VTS motif, but their configuration and location within the protein make it unclear whether these could function in the export of PfK2 (K. Haldar, personal communication).

From its localization, it is hypothesized that PfK1 encodes a parasite-induced ion channel that results in modification of the normal RBC to facilitate intracellular survival and maturation of the parasite by virtue of regulating K⁺ flux across the iRBC membrane, thereby effecting maintenance of the membrane potential and electrochemical gradient across the RBC plasma membrane and composition of the RBC cytosol.

PfK2, which is expressed predominantly in the merozoite, may function in the maintenance of membrane potential and the electrochemical gradient across merozoite membranes that are exposed to the human circulatory system. It is possible that PfK2 may act in concert with PfK1 during this time to adapt to environment changes the parasites are exposed to when released from the rupturing RBC. Invasion studies using the Apicomplexan parasite Toxoplasma gondii have shown that host cell invasion is accompanied by fluctuations of intracellular Ca²⁺ [26, 27], similar to those described during antigen-stimulated activation and proliferation of T cells. In those cells, effective Ca²⁺ signaling and ultimately downstream gene expression are modified by K⁺ channel activity to maintain the cell membrane potential [28]. By analogy, it is hypothesized that PfK2 is a merozoite-specific K⁺channel that functions during the process of merozoite invasion of RBCs, perhaps functioning in ways to similarly effect intracellular Ca²⁺ signaling and gene transcription in the invading merozoite.

Heterologous expression and patch-clamp studies are required to definitively assign PfK1 and PfK2 to a class of K⁺ channels. Functional expression of both native and codon-optimized PfK1 and PfK2 cDNAs in a variety of heterologous systems has so far proven unsuccessful (McBride and McDonald, unpublished data). Expression of active parasite-encoded K⁺ channels in heterologous systems would illuminate the potential function of PfK1 and PfK2. Such a system could be adapted for the screening of novel specific blockers in a high through-put format for identification of possible new anti-malarials.

To examine the importance of these K⁺ channels for parasite survival, attempts were made to knockout pfk1 and pfk2 expression. The ability to detect 'snap shots' of integration by PCR and inability to isolate parasite clones despite repeated attempts are consistent with the hypothesis that both pfk1 and pfk2 are critical for parasite viability. Failed attempts to disrupt various parasite genes thought to be necessary for parasite invasion, viability or propagation have been reported [10, 29‐31]. Further experimentation using either specific gene expression knockdown [10] or tetracycline-dependant conditional expression systems [32] may help unravel the precise functional roles of PfK1 and PfK2 in the regulation of K⁺ physiology in malaria-iRBCs.