Reed AP et al. (2016), Dominant-Negative Effect of a Missense Variant ...

XB-ART-53976

PLoS One 2016 Jan 01;115:e0156456. doi: 10.1371/journal.pone.0156456.

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Dominant-Negative Effect of a Missense Variant in the TASK-2 (KCNK5) K+ Channel Associated with Balkan Endemic Nephropathy.

Reed AP , Bucci G , Abd-Wahab F , Tucker SJ .

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TASK-2, a member of the Two-Pore Domain (K2P) subfamily of K+ channels, is encoded by the KCNK5 gene. The channel is expressed primarily in renal epithelial tissues and a potentially deleterious missense variant in KCNK5 has recently been shown to be prevalent amongst patients predisposed to the development of Balkan Endemic Nephropathy (BEN), a chronic tubulointerstitial renal disease of unknown etiology. In this study we show that this variant (T108P) results in a complete loss of channel function and is associated with a major reduction in TASK-2 channel subunits at the cell surface. Furthermore, these mutant subunits have a suppressive or 'dominant-negative' effect on channel function when coexpressed with wild-type subunits. This missense variant is located at the extracellular surface of the M2 transmembrane helix and by using a combination of structural modelling and further functional analysis we also show that this highly-conserved threonine residue is critical for the correct function of other K2P channels. These results therefore provide further structural and functional insights into the possible pathophysiological effects of this missense variant in TASK-2.

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Species referenced: Xenopus laevis
Genes referenced: kcnk5

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	Fig 1. T108P variant in TASK-2 causes a loss of function.(A) Representative whole-cell current traces at physiological extracellular pH 7.4 recorded from oocytes injected with equivalent amounts of mRNA for either WT TASK-2 or the T108P variant. Currents were recorded using 300 ms voltage steps from a holding potential of -80 mV delivered in 20 mV increments between -140 mV and +100 mV. (B) Similar currents recorded after extracellular. (C) Activation of WT TASK-2 currents at alkaline pH. Results shown as means ± s.e.m. (D) Averaged whole-cell currents from uninjected control oocytes and cells expressing either WT or T108P TASK-2 channels at the indicated external pH values (WT vs T108P, P<0.01 at pH 7.4 and pH 9, one-way ANOVA, post-hoc Tukey HSD test; n = 9 for all conditions).
	Fig 2. Dominant-negative effect of the T108P variant.Oocytes were coinjected with both WT TASK-2 and T108P mutant mRNA at different ratios and currents recorded at the indicated extracellular pH. At a 1:1 (WT:T108P) ratio the currents were markedly reduced. The effect is dose-dependent with further reductions at a ratio of 1:5 (WT:T108P). Results shown are means ± s.e.m. (WT vs 1:1, P<0.01 at pH 7.4 and pH 9; 1:1 vs 1:5, P<0.05 at pH 7.4 and pH9; 1:5 vs T108P, not significant at pH 7.4 or pH9, one-way ANOVA, post-hoc Tukey HSD test; n = 9 for all conditions).
	Fig 3. Structural modelling of the T108P variant.(A) Amino acid sequence alignment of the selectivity filter to M2 region containing the T108P variant for all 15 members of the human K2P family of K+ channels showing the highly-conserved nature of the mutated residue. T108 is indicated by an asterisk. (B) Homology model of TASK-2 created using the crystal structure of TREK-2. This reveals that T108 is located at the top of the M2 helix in the loop that connects M2 to the selectivity filter. T108 (yellow) on M2 hydrogen bonds with the backbone and side chain of E27 (green) located on the adjacent M1 helix (cyan).
	Fig 4. Effect of different amino-acid mutations at T108.Summary of mean currents recorded at different external pH values for oocytes injected with either WT TASK-2 or the indicated amino-acid mutations at T108. In comparison to WT, current amplitudes were markedly reduced for all mutants except T108S suggesting that H-bonding at this position is required for correct channel function. Results are shown as means ± s.e.m (WT vs T108P/A/V/G, P<0.01 at pH 7.4 and pH 9; T108P vs T108V, P<0.05 at pH 7.5 and pH 9; T108P vs T108G and T108A, P<0.01 at pH 7.5, P<0.05 at pH 9, one-way ANOVA, post-hoc Tukey HSD test; n = 9 for all conditions).
	Fig 5. T108P reduces trafficking to the cell membrane.Confocal microscopy of GFP-tagged WT and mutant TASK-2 channels. (A) WT TASK-2 and T108P tagged with GFP at the C-termini expressed in oocytes. The red fluorescent signal (Wheat Germ Agglutinin CF633) indicates the location of the cell membrane. WT channels tagged with GFP (green) exhibit a clear membrane-associated fluorescence, whereas the mutant T108P channels showed no membrane localization, and no GFP fluorescence in any other part of the oocyte. (B) Representative relative signal-intensity profiles for oocytes expressing WT or T108P mutant channels. Intensities were determined along the cross-sections indicated by the red lines in panel A.

References [+] :

Andres-Enguix, Functional analysis of missense variants in the TRESK (KCNK18) K channel. 2012, Pubmed