McTague A , Nair U , Malhotra S , Meyer E , Trump N , Gazina EV , Papandreou A , Ngoh A , Ackermann S , Ambegaonkar G , Appleton R , Desurkar A , Eltze C , Kneen R , Kumar AV , Lascelles K , Montgomery T , Ramesh V , Samanta R , Scott RH , Tan J , Whitehouse W , Poduri A , Scheffer IE , Chong WKK , Cross JH , Topf M , Petrou S , Kurian MA .

MR/L001497/1 Medical Research Council , RP-2016-07-019 Department of Health, Wellcome Trust , MRF_MRF-007-0003-STD-MCTAG MRF

                autosomal dominant nocturnal frontal lobe epilepsy 5
                epilepsy

           EPILEPSY, NOCTURNAL FRONTAL LOBE, 5; ENFL5

	Figure 1. Modeling the ion channel and gating apparatus of KCNT1(A) Side view of the homology model of the KCNT1 ion channel (residues 278–346) as a tetramer. F346 is present on the edge of the inner helix (in gold) and interacts with the inner helix of the adjacent subunit in the tetrameric arrangement. Membrane position is shown in spheres. (B) Top view of the tetramer arrangement of the ion channel and location of F346 on the inner helix. (C) F346 is part of the hydrophobic cavity (shown as surface), which mediates interactions between the inner membrane helices of the 2 subunits. F346 is shown in green; the surrounding hydrophobic residues are shown in red. (D) On mutation to leucine (F346L, in green), the hydrophobic interactions between the 2 subunits are likely to be reduced (black circle) because the side chain of leucine is much shorter than phenylalanine. (E) Model of a dimer of the gating ring (residues 373–1,044; residues 1,045–1,174 could not be modeled), which is a tetramer (dimer of the modeled dimer). Each subunit possesses 2 RCK domains: RCK1 (in blue) and RCK2 (in gold). F502 (in green) is present in the RCK1 domain, near the intersubunit interface (assembly interface). The RCK1-RCK2 intrasubunit interface is purple (residues from RCK1) and orange (residues from RCK2). The dimer interfaces formed by both RCK-1 and RCK-2 are indicated by an arrow. (F) F502 (green) and its neighboring hydrophobic residues (red), including W476, with which it could potentially form a pi-pi interaction. Distance between the centroid (spheres) of the 2 rings (F502 and W476) is 4.7 à , and the angle between the ring planes is 27.3°. (G) F502V could abolish the formation of the potential pi-pi interaction with W476 and is likely to reduce the hydrophobic interactions (black circle) because the side chain of valine is smaller than that of phenylalanine.
	Figure 2. Functional investigation of KCNT1 mutations in a xenopus oocyte model(A) Representative current traces obtained from oocytes expressing WT and EIMFS mutants (M896K, F502V, V271F, F346L, and L274I). Oocytes were held at −90 mV and stepped from −80 to 80 mV for 600 milliseconds every 5 seconds. Scale bars apply to all traces. (B) Current-voltage relationships for WT (n = 32), M896K (n = 15), F502V (n = 13), V271F (n = 9), F346L (n = 11), and L274I (n = 12). Currents were averaged and then normalized to the value at a test potential of 80 mV (Imax). (C) Comparison of current-voltage relationships between WT (solid circles, n = 32) and EIMFS mutations (M896K [squares, n = 15], F502V [triangles, n = 13], V271F [hexagons, n = 9], F346L [diamonds, n = 11], and L274I [inverted triangles, n = 12]). Currents were averaged and then normalized to the value at a test potential of 80 mV (Imax). (D) Average peak currents at 10 mV for WT (n = 44), M896K (n = 19), F502V (n = 16), V271F (n = 10), F346L (n = 11), and L274I (n = 12) channels. Peak currents for each mutant channel at 10 mV were compared to the peak currents for the WT channel at 10 mV. ***p < 0.001, ****p < 0.0001. (E) Comparison of pooled WT (n = 44) and EIMFS (n = 68) currents at 10 mV. ****p < 0.0001. EIMFS = epilepsy of infancy with migrating focal seizures; WT = wild-type.
	Figure 3. Effect of quinidine on xenopus oocytes expressing hKCNT1 channels(A) Representative current traces obtained from oocytes expressing WT and EIMFS mutants (M896K and F346L) with application of vehicle (ND96) and 300 μmol/L quinidine. Oocytes were held at −90 mV and stepped from −80 to 80 mV for 600 milliseconds every 5 seconds. Scale bars apply to all traces. (B) Current-voltage relationships for WT (n = 32), M896K (n = 15), F502V (n = 13), V271F (n = 9), F346L (n = 11), and L274I (n = 12) hKCNT1 channels in the presence of vehicle (ND96) and 300 μmol/L quinidine. Currents were averaged and then normalized to the value at a test potential of 80 mV (Imax). (C) Average percent inhibition at 80 mV of WT (n = 31) and EIMFS (M896K, n = 15; F502V, n = 13; V271F, n = 9; F346L, n = 11; and; L274I, n = 12) hKCNT1 channels by quinidine (300 μmol/L) depicting the variable degree of block by 300 μmol/L quinidine (1-way analysis of variance followed by Bonferroni post hoc analysis). *p < 0.1. EIMFS = epilepsy of infancy with migrating focal seizures; WT = wild-type.
	Figure 4. Schematic diagram of the location of mutations in KCNT1 in this and previously published studiesKCNT1 encodes sequence like a calcium-dependent potassium channel (SLACK), which forms tetramers (top left) or heteromers with KCNT2 or sequence like an intermediate conductance K channel (SLICK). The structure comprises 6 transmembrane domains with a pore-forming region, regulator of potassium conductance (RCK), and nicotinamide adenine dinucleotide–binding (NAD-B) domains. EIMFS phenotypes are shaded in purple, ADNFLE or NFLE in pink, others (Ohtahara syndrome, leukoencephalopathy, focal epilepsy, EOEE, West syndrome, unaffected) in orange. Mutations giving rise to >1 phenotype are shaded with a combination of the corresponding colors. Novel mutations identified in this study are outlined in green, those identified in previous studies in turquoise. ADNFLE = autosomal dominant nocturnal frontal lobe epilepsy; EIMFS = epilepsy of infancy with migrating focal seizures; EOEE = early-onset epileptic encephalopathy; NFLE = nocturnal frontal lobe epilepsy.

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