Figure 1.
(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 [60]–[63]. CaM denotes calmodulin (CaM) bound to a CaM-binding 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.
Figure 2.
Multiple sequence alignment of protozoan K+ channel homologues with the pores of mammalian K+ channels.
Predicted pore-lining TMD regions are underlined. The GXG motif of human K+ channels is shaded in grey. Total number of residues in each protein is indicated in parentheses to the right of each sequence. L. braz. denotes L. braziliensis, and G.intest. denotes G. intestinalis. The proteins XP_001609692 and XP_001350669 encoded by the P. falciparum genome are identical to the previously described PfKch2 and PfKch1 proteins respectively [14], [16]. The proteins XP_001610013 and XP_668687 are identical to previously identified K+ channel homologues in B. bovis and Cryptosporidium hominis respectively [16]. The proteins labelled GYX have GYRD, GYSD or GYSE-containing selectivity filter regions, suggesting a lack of K+ selectivity or function.
Table 1.
Identity of K+ channel homologues in pathogenic protozoa.
Figure 3.
Protozoan K+ channels containing charged TMD4 regions.
(A) Topology diagram of Kv1.2, with the positively charged TMD4 shown in red; (B) Multiple sequence alignment of the TMD4 regions of human voltage-gated Kv1.2 and KCa1.1, plant voltage-gated KAT1 and the predicted TMD4 regions of those protozoan K+ channel homologues containing at least three basic residues within this region. Asterisks above the alignment indicate basic residues involved in voltage sensing in Kv1.2 channels.
Figure 4.
KCa channel homologues in Leishmania parasites.
(A) Phylogram showing the relationship between the sequences of human KCa channels and K+ channel homologues in Leishmania spp. (see Methods). Branch length scale bar and branch support values are shown (see Methods). Two main groups of Leishmania proteins (KCa1-like and KCa2/3-like) are indicated. Selectivity filter GYG-containing KCa2/3-like channels and their GYX-containing putative paralogues are also indicated; (B) Multiple sequence alignment of human KCa2.2 (small-conductance Ca2+-activated SK2 channels) with the GYG-containing KCa2/3-like homologues in Leishmania spp. Selectivity filter, TMD and P-loop regions are indicated above the alignment. Filled triangles above the alignment indicate KCa2.2 residues implicated in binding of inhibitory toxins. Those previously shown experimentally to alter toxin effects are indicated by red triangles, while additional residues implicated via molecular modelling are indicated by black triangles [78]. The yellow shaded region denotes the fragment of KCa2.2 that binds CaM [64] and open triangles indicate specific KCa2.2 residues known to be involved in binding CaM.
Figure 5.
K+ channel homologues in T. vaginalis contain domains similar to mammalian cyclic nucleotide-binding domains.
(A) Multiple sequence alignment of the C-terminal CNBD of human HCN2 (residues 516–668) with the putative CNBD-containing regions of protozoan KCNG homologues. The boundary between the C-linker and CNBD of HCN2, as well as the C-helix of the CNBD [65], are indicated. Residues of HCN2 that are shaded in yellow are those known to be directly involved in binding cNMP [65], [90], [148]. Asterisks below the alignment indicate absolutely conserved residues, while colons indicate conservation of physicochemical properties (ClustalW2). Predicted secondary structure was determined using SABLE (http://sable.cchmc.org) [144] and indicated by red underline (predicted alpha helical) or black underline (predicted beta-sheet). (B) Crystal structure of the CNBD of mouse HCN2 in complex with cAMP (a fragment of PDB accession number 1Q5O) [65]. Only the region encompassing the residues analogous to those of hHCN2 in the alignment in Figure 5A are shown (residues 490–641 of mHCN2, equivalent to residues 516–668 of hHCN2). Bound cAMP is shown in yellow, and side-chains of some key residues important for cAMP binding [90] are shown in red (E582, R591 and R632 of mouse HCN2, equivalent to E609, R618 and R659 respectively of hHCN2 – labelled with filled triangles in Figure 5A); (C) A representation of the coordination of cAMP by specific residues within the CNBD of mHCN2, made using LIGPLOT v4.5.3 [149]. Labels of mHCN2 residues interacting with cAMP that are conserved in parasite KCNG homologues are shown in magenta boxes.