TPK and KCO type channels contain two and one ‘cation channel domain’ (pFAM PF07885), respectively (Figure 1), consisting of two transmembrane helices, a pore loop, and a selectivity filter. Four of these ‘pore module’ oligomerises represent the minimal structure of a functioning K channel. A multiple sequence alignment of Arabidopsis TPK/KCO channels revealed the existence of some important differences in modular structure between different family members: first, AtKCO3 is missing the N-terminal pore module; second, AtTPK4 has deletions outside of the pore modules in both N- and C-terminal domains that led to the removal of potential regulatory sequences such as the 14-3-3 binding motif and EF-hands; and third, the sequence of AtTPK4 was the most different from the consensus sequence of the entire TPK family (not counting AtKCO3) (Figure S1, Table S1).

Based on a very limited distribution of KCOs in plants and similarity, current evolutionary models assume that KCO3 is the product of a relatively late TPK4 gene duplication event [19,25,34]. To further elucidate phylogenetic relationships within the Arabidopsis TPK/KCO family, we separately analyzed the N- and C-terminal pore modules in terms of the sequence (Figure 1 and Figure S1), sequence similarity (Table S1), and phylogeny (Figure 2). It was found that all N-terminal pore regions were closely related, while the C-terminal copies were more divergent, suggesting ongoing evolutional processes on that domain (Figure 3). The C-terminal pore region of AtTPK4 was the most different from that of other TPK isoforms (Table S2). Furthermore, pore domain comparison confirmed our alignment results (Figure S1), showing that AtKCO3 lacks the N-terminal pore domain as was previously shown [34,35].

To test whether the observed differences between the first and second pores of Arabidopsis TPK family members are present in other species, the sequences of the TPK proteins from different taxa (photosynthetic bacteria, green and red algae, “low” plants, mono and di-cotyledons, SAR, metazoa, and viruses) were analyzed. Each pore sequence was extracted and examined individually. This showed that some pores of the TPK proteins exhibited significant similarity to the pores of voltage-dependent (PVG) channels such as AKT K⁺ channels, represented by the ion transport protein domain PF00520 (Pfam database classification) (Table S3) (E-value (≤0.001). Interestingly, both domains (‘ion channel’ PF07885 and ‘ion transport protein’ PF00520) belong to the voltage-dependent potassium channel superfamily (PLN03192). The similarity was especially evident in the region of the selectivity filter GYGD motif and could point to a common evolutionary origin [36]. However, the studied taxa differed greatly where the presence of such ‘voltage signatures’ (V-signatures) is concerned: Archaea, Bacteria, and SAR, organisms that predominantly have K⁺ channels with a single ion channel domain (i.e., only one pore loop) typically show V-signatures. The metazoa (multicellular animals), on the other hand, have mainly two-pore channels, neither of which shows a V-signature. Streptophyta (higher plants) have a distinctly different configuration with a V-signature in the first pore but not the second. Green and red algae have several intermediate patterns, where two-pore domain proteins show none, one, or two V-signatures. Amino acids, matching VDC signatures of the model species (e.g., Arabidopsis, rice, and Physcomitrella patens) are highlighted on the alignments (Figures S2–S4). The recent structural and evolutional revision of plant VG K⁺ channels suggests that these proteins form a completely different (from metazoan) channel group and originated from another (not metazoan) procaryotic ancestor [37].

Ion selectivity is defined by the GYGD motif, where Y and D residues are crucial for K⁺ selectivity and open state, respectively [38]. We found the exact match to the GYGD motif of TPK sequences from photosynthetic bacteria to high plants (Figures S2–S7). However, for bacteria and algae, we noticed a high degree of motif variation, where Y is often replaced by L or F. According to this observation, it could be suggested that such amino acid substitutions in the GYGD motif reduce the selectivity strength and provide the ability to transport other monovalent cations, most probably Na⁺ [38]. The second crucial amino acid in the GYGD motif is D, which is a crucial determinant of open state stability [38]. It was found that the first pore of KCO-like from photosynthetic bacteria KCO-like exhibited the variability of both sites, while for the second pore selectivity filter motif, it looks like GxxD (Figure S5). Unfortunately, for photosynthetic bacteria, there is only a limited number of sequences without VDC available, therefore, we cannot state that this conclusion would be the ultimate. In contrast with photosynthetic bacteria, the TPKs of green and red algae had variations only for Y (to F and L) in the first pore, but for the second pore, we found different variants from conserved GYGD to the complete absence of this motif (Figures S6 and S7).

A rather interesting example of the motif variation was found in low plants and monocotyledons (Figures S3 and S4). While the first pore GYGD was mostly conserved among these taxonomic plant groups, the second pore was conserved, but also not well-aligned/shifted. The functional outcome of such disturbance remains unclear. In the majority of Dicotyledons, in contrast, both pores have conserved a GYGD motif, with the only exception for a Glycine max (A0A0R0GFP1_N) for the first pore, and KCO3 with GFGD in Arabidopsis thaliana (Figure S2). However, it was reported that one of the Tobacco TPK isoforms may have variations (VHG and GHG instead of the GYG) in the second pore selectivity filter [39].