A sequence variation between two orthologues alters functional expression of the potassium channel Kesv of Ectocarpus siliculosus virus
The potassium channel Kesv encoded by the Ectocarpus siliculosus virus (Kesv 1) differs by seven amino acid residues from its host-derived homolog (Kesv 2), resulting from lysogenic integration. When expressed in Xenopus laevis oocytes, Kesv 1 displayed significantly higher ion conductance and functional expression than Kesv 2, as demonstrated by GFP fluorescence and voltage clamp measurements. This study provides the first structural and functional analysis of Kesv 2, uncovering key differences between the original and host-derived variant. The systematic residue substitutions - based on location- from Kesv 2 to the corresponding residues in Kesv 1 illustrated that two amino acid exchanges in close proximity to the pore region (Q61H and T66A), albeit not individually but in combination, significantly resulted in a loss-of-function phenotype in Kesv 1. AlphaFold predictions and subsequent molecular dynamics simulations did not reveal significant differences between Kesv 1 and Kesv 2 structural models, suggesting that the loss of function cannot be attributed to differences at the structural level. Instead, a reduced surface expression of Kesv 2, caused by the sequence modulations in the brown algal host, appears more plausible. Notably, the pharmacological profiling with Linopirdine and Sotalol highlights differences in drug sensitivity, establishing these minimalist channels (core channel structure without regulatory domains) as tractable models for dissecting novel fundamental principles of ion channel function and drug interaction, while highlighting key differences from more complex channel systems. Significance StatementPotassium channels are essential for cellular excitability, yet their large size and structural complexity limit our understanding of the core features underlying channel function. Here, we identified and established an orthologous model to compare the effects of evolutionarily acquired mutations in two voltage-sensing potassium channels-the viral potassium channel from Ectocarpus siliculosus virus (Kesv 1) and its host-homolog derivative (Kesv 2) as simplified model systems for understanding ion channel physiology and host-viral interactions. We provide the first functional characterization of Kesv 2 in Xenopus laevis oocytes using two-electrode voltage-clamp and site-directed mutagenesis, revealing that, despite sharing an identical SVGYG selectivity-filter motif and differing by only 7 residues, they exhibit distinct ion-conduction properties.