Identification of Putative Potassium Channel Homologues in Pathogenic Protozoa

Citation: Prole DL, Marrion NV ( Identification of Putative Potassium Channel Homologues in Pathogenic Protozoa David L. Prole 0 Neil V. Marrion 0 0 1 Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom, 2 School of Physiology and Pharmacology, University of Bristol , Bristol , United Kingdom K+ channels play a vital homeostatic role in cells and abnormal activity of these channels can dramatically alter cell function and survival, suggesting that they might be attractive drug targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, leishmaniasis, trypanosomiasis and dysentery that are responsible for millions of deaths each year worldwide. The genomes of many protozoan parasites have recently been sequenced, allowing rational design of targeted therapies. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of K+ channels. These protozoan K+ channel homologues represent novel targets for anti-parasitic drugs. Differences in the sequences and diversity of human and parasite proteins may allow pathogen-specific targeting of these K+ channel homologues. - Funding: This work was funded by a Meres Senior Research Associateship from St. Johns College, Cambridge (to DLP). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Protozoan parasites are major contributors to worldwide disease [1]. They include apicomplexan parasites such as Plasmodium spp. (malaria), Toxoplasma gondii (toxoplasmosis), Cryptosporidium spp. (cryptosporidiosis, diarrhoea) and Babesia bovis (babesiosis), as well as the kinetoplastid parasites Trypanosoma spp. (sleeping sickness, Chagas disease) and Leishmania spp. (leishmaniasis). These parasites are together responsible for billions of infections and hundreds of thousands of deaths each year [1,2]. Other protozoan parasites causing widespread disease include Giardia intestinalis (giardiasis), Entamoeba histolytica (dysentery) and Trichomonas vaginalis (trichomoniasis). Current treatments for diseases caused by protozoa are often ineffective or poorly tolerated, and emergence of drug resistance is an imminent threat to their efficacy [35]. New therapeutic targets and drugs are therefore needed. K+ channels are a diverse family of transmembrane proteins, which form K+-selective pores and mediate K+ flux across membranes [6,7]. K+ channels are essential components in a multitude of homeostatic and signalling pathways and are present in animal cells [6], plants [8,9], fungi [10,11] and many bacteria [7,12]. Only a handful of organisms appear to lack K+ channels completely, and most of these are bacteria that are obligate parasites [7,12]. Many K+ channels are present in free-living protozoa such as Paramecium [13], but little is known about the existence and physiological role of K+ channels in pathogenic protozoa, many of which spend part of their life cycles as intracellular parasites. K+ channels are known to exist in Plasmodium spp. [1416] and K+-conductive pathways have also been observed in Trypanosoma cruzi [17], but the molecular identity of the channels underlying these latter K+ fluxes is unknown. Several subtypes of K+ channel exist, including voltage-gated (Kv), inward rectifier (Kir), two-pore (K2P), calcium-gated (KCa) and cyclic nucleotide-gated (KCNG) channels [6] (Figure 1A). These channels are all formed by a tetrameric arrangement of pore-forming domains, contributed to by each of four monomeric subunits in most channels, except K2P channels which exist as a dimer of subunits, with each subunit containing two domains that contribute to the pore (Figure 1A) [6,18]. The K+-conducting pore region is comprised of a tetrameric arrangement of re-entrant pore loops (P-loops), part of which forms the selectivity filter, together with the following pore-lining transmembrane domains (TMDs) from each subunit, which form the inner pore (Figures 1A and 1B). Diversity of K+ channels is increased by subunit heteromerization and by the association of auxiliary subunits, such as Kvb, KCNE, KChIP, BKb, and sulfonylurea (SUR) subunits, which alter the functional properties, trafficking, modulation and pharmacology of K+ channels [1922]. In many cell types K+ channels are found mainly in the plasma membrane, but they are also found in the membranes of intracellular organelles such as mitochondria [23,24], nuclei [2528], endosomes [29], endoplasmic reticulum [30], secretory vesicles [31,32] and intracellular vacuoles [3335]. Physiological roles of K+ flux include setting or altering membrane potentials, effecting osmolyte homeostasis, altering enzyme activity, promoting mitogenesis or apoptosis, and facilitating transmembrane transport processes [69,12,36]. Pharmacological or genetic perturbation of K+ channel activity has profound effects on cell function in many organisms, suggesting that parasite homologues of these channels might represent novel drug targets. Consistent with this, disruption of K+ channel function in Plasmodium falciparum and Plasmodium berghei is lethal to these parasites [16,37]. Figure 1. K+ channel families. (A) Topology diagrams of K+ channel subunits, showing locations of transmembrane domains (TMDs), functional domains and termini. Plus signs denote charged basic residues within the voltage sensor (S4) region of Kv and KCa1 channels. In contrast to other K+ channel subunits, KCa1.1 channel subunits have extracellular N-termini [145,146]. RCK denotes a Ca2+-binding regulator of conductance of K+ channels domain, which also binds a variety of other ionic ligands in different channels [6063]. CaM denotes calmodulin (CaM) bound to a CaMbinding site within the channel subunit [60,64]. CNBD denotes a cyclic nucleotide monophosphate (cNMP) binding site [65]; (B) A crystal structure of the KcsA pore domain is shown (PDB accession number 1K4C) [147], with only the TMDs and pore loops of two subunits depicted for clarity. Red circles represent a number of the K+ ions in the selectivity filter. doi:10.1371/journal.pone.0032264.g001 Recent advances in genomics have resulted in whole-genome sequencing of many pathogenic protozoa [1,3856]. In this study we examine the genomes of pathogenic protozoa comprehensively, using diverse K+ channel sequences from mammals, plants, fungi, bacteria and archaea, to search for the presence of predicted proteins that may fulfil roles as K+ channels. We show that genes encoding homologues of K+ channels exist in all pathogenic protozoa examined. Sequence divergence of putative protozoan channels from their human counterparts in regions that are known to be important for channel activation, ion conduction or drug binding may result in distinct pharmacological profiles. These parasite channels may therefore represent novel t (...truncated)