Kim GE et al. (2014), Human slack potassium channel mutations increas...

XB-ART-51153

Cell Rep 2014 Dec 11;95:1661-1672. doi: 10.1016/j.celrep.2014.11.015.

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Human slack potassium channel mutations increase positive cooperativity between individual channels.

Kim GE , Kronengold J , Barcia G , Quraishi IH , Martin HC , Blair E , Taylor JC , Dulac O , Colleaux L , Nabbout R , Kaczmarek LK .

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Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na(+)-activated K(+) channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions.

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Species referenced: Xenopus laevis
Genes referenced: acta4 kcnt1
GO keywords: potassium channel activity

???displayArticle.disOnts??? early infantile epileptic encephalopathy
???displayArticle.omims??? DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 14; DEE14 [+]

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	Graphical Abstract Highlights Slack KCNT1 mutations are found in three different epilepsy syndromes Abnormal interactions between individual mutants greatly in- crease K+ current Cooperativity enhances K+ current even in a mutant with reduced channel conductance The same mutation can produce different forms of epilepsy in different individuals
	Figure 1. Slack Currents Are Increased by Mutations that Produce Epilepsy (A) Diagram of two subunits of a Slack channel with the point mutations described in this study. Numbering refers to the rat Slack sequence. Positions of the mutations within the C-terminal ‘‘gating ring,’’ containing two RCK domains (RCK1 and RCK2), are based on the low-resolution X-ray structure (Yuan et al., 2010). (B) The voltage-clamp protocol and representative current traces show that peak current amplitude (IKNa) is significantly increased in all mutants compared with the WT channel. Xenopus oocytes injected with cRNA encoding WT or mutant channels, or injected with water alone, were tested using a two-electrode voltage clamp on days 1, 2, or 4 following injection. Scale bars, 1 mA and 200 ms. (C) Bar graphs quantifying the increase in mean peak current amplitude at +20 mV for all mutant channels compared with WT Slack (mean ± SEM, #p < 0.01 against WT, two-tailed Student’s t test, n = 5; p < 0.05, p < 0.01, **p < 0.001 against WT, one-way ANOVA with Tukey’s post hoc test, n = 6–9 for all conditions).
	Figure 2. Channel Protein Expression Is Not Altered by Mutations (A) Plot of the relative increase in peak IKNa changes versus levels of Slack protein determined by densitometry of western blots of membrane fractions from oocytes homogenized immediately after the voltage-clamp experiments. A nonsignificant best fit (solid line) shows no relationship that can account for the in- creases in current (mean ± SEM). (B) The voltage dependence of channel activation is shifted leftward in the R455H and R907C mutants. Representative traces for the WT and R455H and R907C mutants, normalized to their peak IKNa at +60 mV, are shown. The shift in voltage dependence is made evident by the position of arrows lined up to the responses to test pulses at +40 mV. Scale bars represent 0.2 mm and 200 ms. (C and D) Mean peak IKNa at each of the command voltages, shown as an I/V plot for MMPSI (C) and ADNFLE (D) mutants. Current amplitudes were normalized to the peak IKNa at +60 mV in all cases. The increase in relative channel activity was seen at voltages R 60 mV and 40 mV for R455H (C) and R907C (D), respectively (mean ± SEM; p < 0.01, *p < 0.001 against WT, two-way ANOVA with Bonferroni’s post hoc test, n = 5–10 for all points).
	Figure 3. Single-Channel Recordings of WT and G269S Slack in Xenopus Oocytes (A and B) Representative on-cell single-channel ‘‘low-N’’ trace recorded at 80 mV (A) and the corresponding all-points amplitude histogram (B). C, closed state; O1, one channel opening; O2, two channel openings. SC1 and SC2 indicate the two prominent subconductance states. (C) Binomial distribution for six channels based on the single-channel Po in low-N patches (mean of eight low-N patches). (D) Representative on-cell high-N patch recorded at 80 mV. (E) All-points amplitude histogram of currents from (D). Experimental data show the increased frequency of multiple channel openings and dramatic reduction in dwell time in the fully closed state compared with the expected binomial distribution in (C). (F) Deviation from binomial distribution. The plot shows the ratio of the observed Po at each level, P(k)obs, to the predicted distribution obtained from the binomial distribution (C), P(k)binomial, for the first three levels. The ratios continued to increase with successive k for four, five, and six channels (not shown). (G and H) Representative on-cell, single-channel low-N trace of G269S channels at 80 mV (G) and the corresponding all-points amplitude histogram (H), representing 2 min of recording. (I) Binomial distribution for four channels based on the mean single-channel Po in low-N patches (n = 4 low-N patches). (J and K) Representative on-cell high-N patch containing four G269S channels (J) and the corresponding all-points amplitude histogram (K), showing a dramatically increased frequency of multiple channel openings compared with the expected binomial distribution (I). (L) Cooperativity ratio comparing P(k)obs for G269S versus WT Slack. The ratios were calculated from the means of four and five high-N patches for G269S and WT Slack, respectively. The ratios show a deviation from the expected value of one for each successive k, demonstrating increased positive cooperativity relative to WT Slack. The ratio for four open channels is 53 (not shown).
	igure 4. Single-Channel Recordings of Slack Mutants R455H and A945T in Xenopus Oocytes (A and B) Representative on-cell single-channel trace recorded at 80 mV from a patch expressing Slack R455H (A) and the corresponding all-points amplitude histogram (B). (C) Binomial distribution for five R455H channels based on the mean single-channel Po from four low-N R455H patches. (D and E) Representative high-N trace (D) and all-points amplitude histogram (E) from a patch containing five Slack R455H channels. (F) Cooperativity ratios (comparing P(k)obs for R455H versus WT Slack) were calculated from the means of three and five high-N patches for G455R and WT Slack, respectively. The ratio shows a significant deviation from the expected value of one for all five channel openings. (G and H) Representative low-N recording (G) and corresponding all-points amplitude histogram (H) from a patch containing A945T channels 80 mV. (I) Binomial distribution for six channels based on the mean single-channel Po in low-N A945T patches (n = 5 low-N patches). (J and K) Representative high-N recording (J) and corresponding all-points amplitude histogram (K) from patches containing six A945T channels. (L) Cooperativity ratio as calculated from the means of three and five high-N patches for A945T and WT Slack, respectively.

References [+] :

Allen, De novo mutations in epileptic encephalopathies. 2013, Pubmed