De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy

Abstract

Malignant migrating partial seizures of infancy (MMPSI) is a rare epileptic encephalopathy of infancy that combines pharmacoresistant seizures with developmental delay. We performed exome sequencing in three probands with MMPSI and identified de novo gain-of-function mutations affecting the C-terminal domain of the KCNT1 potassium channel. We sequenced KCNT1 in 9 additional individuals with MMPSI and identified mutations in 4 of them, in total identifying mutations in 6 out of 12 unrelated affected individuals. Functional studies showed that the mutations led to constitutive activation of the channel, mimicking the effects of phosphorylation of the C-terminal domain by protein kinase C. In addition to regulating ion flux, KCNT1 has a non-conducting function, as its C terminus interacts with cytoplasmic proteins involved in developmental signaling pathways. These results provide a focus for future diagnostic approaches and research for this devastating condition.

Figure 1. MMPSI mutations increase amplitude of Kcnt1 currents

Equal amounts (20 ng) of cRNA for wild-type rKcnt1, Arg409Gln or Ala913Thr mutant constructs, or water alone were injected into Xenopus oocytes and two-electrode whole cell voltage clamp were performed 5 days post-injection. (a) Representative families of whole-oocyte currents evoked by stepping from −80 mV to +80 mV in 10 mV increments in oocytes expressing wild-type (WT) rKcnt1 or mutant channels and control oocytes. (b) Mean current-voltage relationship ± SEM for oocytes expressing wild-type rKcnt1 or mutant channels, and control oocytes (N= 5,5,5,7 respectively). Currents were measured at the end of the test pulse. (c) Relative currents (± SEM) at +80 mV for the four groups of oocytes in B. Both MMPSI mutations had significantly increased currents compared to wild-type Kcnt1 or water-injected oocytes as analyzed by ANOVA p<0.05 followed by Tukey’s test at p< 0.001. The two MMPSI mutants were not significantly different from each other.

Figure 2. MMPSI mutations mimic and occlude the effects of phosphorylation of rKcnt1 at Ser407

(a) Schematic diagram of rKcnt1-B, including the relative locations of 13 consensus PKC phosphorylation sites and the p.Arg409Gln and p.Ala913Thr mutations. Representative families of whole-oocyte currents evoked by stepping from −80 mV to +50 mV in 10 mV increments in oocytes expressing wild-type rKcnt1 (b) or S407A mutant channels (c) before and after application of the PKC activator TPA (100 nM). (d) Relative increases in currents at +80 mV for wild-type rKcnt1 or Serine to Alanine mutations of the 13 consensus PKC phosphorylation sites mutants, measured 20 minutes after application of TPA (N=3–5 for all mutants except for wild-type and Ser407Ala for which N= 8 each). Representative families of whole-oocyte currents evoked by stepping from −80 mV to +80 mV in 10 mV increments in oocytes expressing wild-type rKcnt1 (e) or Arg409Gln mutant channels (f) before and after application of TPA (100 nM). (g) Relative increases in currents (± SEM) at +80 mV for wild-type rKcnt1 the Arg409Gln and Ala913Thr mutations, 20 minutes after application of TPA (100 nM) (N=8 for wild-type and N=4 for mutants).

Figure 3. rKcnt1 mutations do not alter Na⁺ sensitivity but suppress channel subconductance states

(a) Inside-out recordings of an excised membrane patch containing several R409Q mutant channels with various concentrations of Na⁺ (20, 40, 60 or 80 mM) at the cytoplasmic face of the channels. The closed state (C) and openings to the level of one or more channels are indicated in red. Values of NPo (Number of channels × Open Probability) are shown at right. Patch was held at −80 mV. (b) Traces at left show excised inside-out single channel recordings from patches containing (WT) rKcnt1 and the Arg409Gln and Ala913Thr mutant channels and held at −80 mV with 20 mM Na⁺. The closed state and openings to one or more fully open channel levels are shown in red. Also shown is the level of the most prominent subconductance state (γ_sub). All corresponding point histograms at right show the distribution of openings from the closed state (C) to the first open state (O) or to the subconductance state (Sub). (c) Mean Na⁺ Concentration-response relationships for R409Q and A914T mutant channels. NP_O values are normalized to the NP_O obtained at 80 mM Na⁺. (n = 4 for each mutant, data are shown + SEM). (d) Group data for the mean proportion of time that (WT) rKcnt1 and the Arg409Gln and Ala913Thr mutant channels spent in the subconductance state over the fully open state (n = 12 for all conditions, data are shown + SEM).