Colombo S, Reddy HP, Petri S, Williams DJ, Shalomov B, Dhindsa RS, Gelfman S, Krizay D, Bera AK, Yang M, Peng Y, Makinson CD, Boland MJ, Frankel WN, Goldstein DB, Dascal N.

R01 NS031348 NINDS NIH HHS , R00 NS104215 NINDS NIH HHS

	Figure 1. Gnb1K78R/+ mice exhibit developmental delay as neonates and motor and cognitive deficits as adults. (A) Representative photo of a Gnb1K78R/+ pup (left) and a WT littermate (right) at P6 on the B6NJ background. (B) Pup weight at P0 on the B6NJ background. WT (black circles): n = 18 female and 20 male mice; Gnb1K78R/+ (red circles): n = 9 female and 14 male mice; female *p < 0.05 vs. WT and male **p < 0.01 vs. WT. Mann–Whitney U-test with 10,000 permutations. (C) Developmental milestones between P4 and P10 on the B6NJ background include body weight, surface righting reflex, negative geotaxis 90° and 180° from the downward-facing position, and vertical screen holding: n = 32 WT and 20 Gnb1K78R/+. Separation-induced USV between P5 and P11: n = 28 WT and 20 Gnb1K78R/+. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Mann–Whitney U-test with 10,000 permutations. (D) Open field test on the B6NJ background. Distance traveled: n= 18 WT and 13 Gnb1K78R/+; **p < 0.01. Two-way repeated measures ANOVA. Representative movement traces of a WT (left) and a Gnb1K78R/+ (right) mouse walking for 1 h. (E) Morris water maze test on the B6NJ background. n = 15 WT and 14 Gnb1K78R/+. Learning: **p < 0.01 for acquisition latency; *p < 0.05 for reversal latency. Two-way repeated measures ANOVA. Memory: ***p < 0.001 for Target vs. Left, Target vs. Right, and Target vs. Opposite during the acquisition probe trial; *p < 0.05 for Target vs. Left, **p < 0.01 for Target vs. Right, and Target vs. Opposition during the reversal probe trial. One-way repeated measures ANOVA with Bonferroni correction. Representative heat maps of a WT (top) and a Gnb1K78R/+ mouse during the reversal probe trial. All graphs represent mean ± SEM. PND, postnatal day.
	Figure 2. Excitability phenotypes in Gnb1K78R/+ mice. (A) Electroconvulsive threshold tests. Minimal clonic forebrain seizure endpoint in Gnb1+/+, Gnb1K78R/+, and Gnb1Del71/+ mice (WT n = 16 male, 13 female; Gnb1K78R/+ n = 12 male and 8 female; Gnb1Del71/+ n = 10 male, 15 female). (B) Representative traces from video-EEG recordings of cortically implanted Gnb1K78R/+ and WT littermates on the F1H background showing SWDs in Gnb1K78R/+ mice. FR, front right; FL, front left; BL, back left. (C) Quantification of SWDs per 24 h. n = 14 WT and 19 Gnb1K78R/+. WT = 11.71 ± 6.527 vs. Gnb1K78R/+ = 1,724 ± 100.6. (D) The mean duration of SWDs. n = 19 Gnb1K78R/+ mean ± SEM: 3.397 ± 0.239. (E) The fundamental frequency of peak spectral power. n = 19 Gnb1K78R/+ mean ± SEM: 7.342 ± 0.101. All graphs represent mean ± SEM. ****p < 0.0001 n.s., non-significant by two-way ANOVA with Dunnett's test for multiple comparisons (a) and Mann–Whitney U-test with 10,000 permutations (b).
	Figure 3. Excitability phenotypes in Gnb1K78R/+ cortical neurons in microelectrode arrays (MEA). (A) Raster plots showing WT and Gnb1K78R/+ firing across the 16 electrodes of a well of a representative plate, between 50 and 150 s of a 3-min recording at DIV36. The black bars indicate spikes, and the red bars indicate bursts. (B) Graphs representing spontaneous activity on MEA of Gnb1K78R/+ cortical neurons (red) and WT cortical neurons (black) from DIV15 to DIV27. Graphs show the combined analysis of nine plates from DIV15 to DIV27. n = 144 WT wells and 148 Gnb1K78R/+ wells from nine plates (8 biological replicates = 8 independent primary cultures). The combined MWU permutated p-values of each plate generated using the Fisher's method are shown.
	Figure 4. Pharmacological response of the pre-clinical models. (A) SWD manual counts on EEG recordings before and after acute treatment of adult mice with the AEDs ethosuximide (ETX; 200 mg/kg IP), valproate (VPA; 200 mg/kg IP), and phenytoin (PHT; 30 mg/kg IP). n = 17 WT and 21 Gnb1K78R/+ for ETX, 12 WT, and 21 Gnb1K78R/+ for VPA, and 5 WT and 7 Gnb1K78R/+ for PHT. IP, intra-peritoneal. For each time point, mean SWD counts have been compared using a t-test. *p < 0.05; **p < 0.01, ***p < 0.001; ****p < 0.0001. (B) Graphs representing spontaneous activity on MEA from DIV15 to DIV31 of untreated Gnb1K78R/+ cortical neurons (red), and Gnb1K78R/+ cortical neurons treated with 250 μM (purple), 750 μM (green) or 2 mM (blue) of ETX chronically from DIV3 to DIV31, normalized to WT cortical neurons (black). MFR, number of bursts per minute, mean burst duration, and mean IBI are shown from left to right. n = 30 wells from five biological replicates for all genotypes/conditions. (C) Statistics for the graphs shown in B. All p-values correspond to K78R/+ treatment compared to the WT vehicle using a Mann–Whitney U-test with 1,000 permutations. All graphs represent mean ± SEM. DIV, days in vitro; n.s., non-significant.
	Figure 5. GABAB-evoked GIRK currents are larger in K78R/+ hippocampal and inhibitory cortical neurons. (A) Representative traces showing whole-cell voltage clamp recordings from inhibitory and excitatory WT (black) and K78R (red) cortical neurons. Overlaid traces recorded from individual neurons showing currents evoked by three 20 s sequential applications of increasing baclofen concentrations. Application period and concentrations are indicated on the top left recordings. Plots show the amplitudes of baclofen-evoked currents normalized to capacitance for each neuronal subtype, in single neurons. The box plot indicates the range, interquartile range, and median value; the green lines show mean values. Number of cortical excitatory neurons = 29 WT, 34 Gnb1K78R/+; number of cortical inhibitory neurons = 21 WT, 22 Gnb1K78R/+; from six litters. (B) Hippocampal neurons have the same layout as described above. Number of hippocampal excitatory neurons = 35 WT, 33 Gnb1K78R/+; number of hippocampal inhibitory neurons = 11 WT, 12 Gnb1K78R/+; from six litters. Comparisons between K78R and WT groups were performed using an unpaired t-test if the data passed the Shapiro–Wilk normality test or using the Mann–Whitney test if normality was not satisfied.
	Figure 6. K78R is a GoF for GIRK1/2 and GIRK2 activation but, at high expression levels, shows an LoF for GIRK1/2. (A) Representative records of GIRK1/2 currents in Xenopus oocytes injected GIRK1 and GIRK2 RNAs (0.05 ng each), and the indicated amounts of Gβ RNA. The amount of Gγ RNA was 1/5 of Gβ. The dotted line shows zero current. (B) Summary of GIRK1/2 activation by GβWTγ and GβK78Rγ in the experiment shown in (A), n = 8 to 13. The dotted line shows that the basal level of current in oocytes injected with GIRK1/2 RNA without coexpression of Gβγ. At 0.1 ng Gβ RNA, there was an outlier point (~17 μA). The exclusion of the outlier did not greatly affect the statistical significance (p = 0.0243, t = 2.411, df = 23). (C) Changes in surface expression of GβWT and GβK78R as a function of RNA dose, in the presence of GIRK1/2, measured in giant excised membrane patches (GMP). Data shown are the net fluorescent signal produced by the expressed Gβ, after subtraction of the average signal observed in naïve (uninjected) oocytes, n = 8 to 18. (D) Dose-dependent activation of GIRK1/2 vs. actual surface expression of Gβγ as measured in GMP. (E) Representative images of membrane patches at different Gβ RNA doses in the presence of GIRK1/2. (F) Representative records of GIRK2 currents in Xenopus oocytes injected with GIRK2 RNA (2 ng; C-terminally YFP-labeled GIRK2 was used) and the indicated amounts of Gβ RNA. The amount of Gγ RNA was 1/5 of Gβ. The dotted line shows zero current. (G) Summary of the experiment is shown in (F), n = 6 to 14. (H) Changes in surface expression of GβWT and GβK78R as a function of RNA dose, in the presence of GIRK2, measured in GMP. Analysis as in (C), n = 11 to 14. (I) Dose-dependent activation of GIRK2 vs. actual surface expression of Gβγ as measured in GMP. The results were fitted to the Hill equation with a fixed Hill coefficient of 4. The fit yielded dissociation constant (Kd) of 118 and 162 AU and maximal currents of 3.4 and 3.18 μA for GβWTγ and GβK78Rγ, respectively. (J) Representative images of membrane patches at different Gβ RNA doses in the presence of GIRK2. All graphs represent mean ± SEM. For each Gβ dose, raw data for GβWT and GβK78R were compared using a two-tailed t-test or Mann–Whitney rank sum test. AU, arbitrary units.
	Figure 7. Ethosuximide blocks GIRK channels expressed in Xenopus oocytes. (A) Representative records showing the effect of ETX on Ibasal of GIRK1/2 (top panel), and GIRK2 activated by coexpressed Gβγ (bottom panel). Sequential application of increasing doses of the drug inhibited the current. Approximately 2.5 mM of Ba2+ was added at the end of the protocol for complete inhibition of GIRK. (B) Dose-dependence curves of ETX inhibition of GIRK1/2 and GIRK2 channels activated by saturating (5 ng RNA) doses of either GβWTγ or GβK78Rγ and Ibasal of GIRK1/2. The semitransparent rectangle indicates the therapeutic range of ETX concentrations. Each symbol is mean ± SEM, n = 6 to 19. The solid lines show fits to the Hill equation. Details of fits are shown in Supplementary Figure 5B. All graphs represent mean ± SEM.

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