KCNK18 Biallelic Variants Associated with Intellectual Disability and Neurodevelopmental Disorders Alter TRESK Channel Activity

Figures

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  Figure 1. Pedigree, conservation, and graphical representation of the KCNK18 variants. (A). Pedigree of the family and Sanger sequencing chromatograms documenting co-segregation between compound heterozygosity for the two missense changes in KCNK18 and clinical traits transmitted in the family. (B). Multiple alignments of the amino acid stretches flanking the affected residues in TRESK orthologs showing conservation of both mutated residues (in red). Residues are referred to human KCNK18, NM_181840.1. (C). Schematic topology of the human K2P TRESK channel, showing location of Tyr163 (Y163) and Ser252 (S252) (red asterisks). Human TRESK is composed of 384 amino acid residues and shows the most characteristic features of K2P channels, including four transmembrane helices, two pore-forming domains, and intracellular N- and C-termini. A peculiar structural feature of TRESK (compared to other K2P channels) is the long intracellular loop between the second and third transmembrane segments (TMS), and the relatively short C-terminal tail [3]. In the image, representative domains are shown: PQIIIS and LQLP are calcineurin-binding motifs [5,17], while the serine cluster and Ser252 are indicated by…

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  Figure 2. p.Tyr163Asp and p.Ser252Leu do not significantly affect the basal current and ability of TRESK channels to control the cell’s resting potential. (A). Representative families of current traces for the indicated channel types. Cell membrane potential was held at −80mV. Each family of currents was evoked by voltage steps from −120 mV to 60 mV, with 20 mV increments. The recordings were performed 48 h after the injection of 1 ng of mRNA for each channel type. (B). Average IV relationships calculated from experiments as in A for the labelled channel types (color coded). The data points are mean ± standard error (0.001; n = 12). (C). Resting membrane potentials from individual oocytes un-injected (grey dots), injected with 1 ng of WT (black dots) or Y163D (green dots) or S252L (blue dots) or 0.5 ng of each Y163D and S252L (red dots) mRNA and recorded 48 h after injection. Note that the expression of all channel types similarly shifts the resting potential toward K+ reversal potential. The dots in C represent single cell recordings. The data are mean ± standard deviation.

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  Figure 3. KCNK18 p.Tyr163Asp and p.Ser252Leu impair the ionomycin-induced activation of TRESK channels. Representative data points showing ionomycin activation of TRESK WT, Y163D, S252L and Y163D + S252L currents as well as endogenous currents from un-injected oocytes. (A). Currents were evoked by 300 ms long voltage steps from 0 mV to −100 mV from oocytes injected with 1 ng of mRNA of the corresponding and indicated homomeric channel type and 0.5 ng for each subunit for the heteromeric channel Y163D/S252L. The sampled data represent the average of 50 ms long period of the steady-state currents recorded at −100 mV. Ionomycin activates the WT channels but has little effect on the oocytes that express the mutant channels or those that are uninjected. Note that the current decay upon reapplication of a solution containing 2 mM K+ in the recording chamber is due to the reduced K+ concentration rather than ionomycin washout. (B). Bar graph showing the mean of ionomycin activated currents (*** p < 0.001). All the groups were statistically significant compared to the uninjected group (WT vs. uninjected p < 0.001; Y163D vs. uninjected p = 0.02; S252L and Y163D + S252L vs. uninjected p < 0.01; n =…