Activation of SGK1.1 Upregulates the M-current in the Presence of Epilepsy Mutations

Figures

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  FIGURE 1. Wild-type and activated SGK1.1 upregulate M-current. (A) Currents elicited in Xenopus oocytes after coinjection of cDNAs from Kv7.2/3 channel alone (first panel) or in combination with wild-type (second panel) or constitutively active (third panel) SGK1.1. (B) Peak current/voltage relationship. (C) Tail current (left) and tail currents measured at –30 mV after 0, + 20 or + 40 mV depolarizing pulses for the indicated construct combinations (right). Values represent mean ± SEM. Two-way ANOVA followed by Tukey’s correction for multiple comparisons, ****p < 0.0001. (D) Normalized conductance. Legends are indicated on (B).

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  FIGURE 2. SGK1.1 increases heteromeric Kv7.3/5 currents like heteromeric Kv7.2/3, supporting the need for heteromeric channel assembly to observe SGK1.1 effects. (A) Currents elicited in Xenopus oocytes after coinjection of cRNAs from Kv7.3/5 channel alone (first panel) or in combination with wild-type SGK1.1 (second panel). (B) Peak current/voltage relationship. (C) Tail current (left) and tail currents measured at –30 mV after 0, + 20 or + 40 mV depolarizing pulses. Values represent mean ± SEM (Multiple t-test Holm-Sidak correction method; *p < 0.05). (D) Normalized conductance. Legends are indicated on (B).

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  FIGURE 3. Constitutively active form SGK1.1 (S515D) upregulates Kv7.2 epilepsy mutation R207W in heteromeric assembly with Kv7.3 while WT SGK1.1 fails to. (A) Schematic representation of Kv7 channel structure. The basic organization of potassium channels is a tetramer with each monomer containing one pore-forming domain (PD) (transmembrane segments S1-S4) and a voltage sensor domain (VSD) (transmembrane segments S5-S6). Epilepsy mutants are represented as colored pentagons (R207W in red and A306T in yellow). (B) Currents elicited in Xenopus oocytes after coinjection of cDNAs from Kv7.2 (R207W)/3 channel alone (first panel) or in combination with wild-type (second panel) or constitutively active (third panel) SGK1.1. (C) Peak current/voltage relationship. (D) Tail current (left) and tail currents measured at –30 mV after 0, + 20 or + 40 mV depolarizing pulses for the indicated construct combinations (right). Values represent mean ± SEM (Two-way ANOVA, Tukey’s correction for multiple comparisons; ns, not significant; **p < 0.01; ***p < 0.0005; ****p < 0.0001). (E) Normalized conductance. Legends are indicated on (C).

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  FIGURE 4. Constitutively active form of SGK1.1 (S515D) upregulates Kv7.2 epilepsy mutation A306T in heteromeric assembly with Kv7.3 (A315T). (A) Currents elicited in Xenopus oocytes after coinjection of cDNAs from Kv7.2 (A306T)/3(A315T) channel alone (first panel) or in combination with wild-type (second panel) or constitutively active (third panel) SGK1.1. (B) Peak current/voltage relationship. (C) Tail current (left) and tail currents measured at –30 mV after 0, + 20 or + 40 mV depolarizing pulses for the indicated construct combinations (right). Values represent mean ± SEM (Two-way ANOVA Tukey’s correction for multiple comparisons; *p < 0.05; **p < 0.01). (D) Normalized conductance. Legends are indicated on graph (B).

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  FIGURE 5. Association between SGK1.1 (S515D) and heteromeric channel Kv7.2/3 is reduced in presence of epilepsy mutant Kv7.2 (R207W) but not Kv7.2 (A306T). (A) Representative immunoblot showing expression levels of Kv7.2 fused to YFP (top panel, 110 KDa) and GADPH (bottom panel, 50 KDa) from oocytes injected with the indicated constructs; N.I., water-injected oocytes. Asterisks denote migration of the indicated molecular mass marker. (B) Quantitative analysis of Kv7.2 expression levels in Xenopus oocytes. Values are mean ± SEM from at least three independent experiments (ANOVA Sidak’s test for multiple comparisons; ns, not significant; *p < 0.05; ***p < 0.0005). (C) Representative western blot showing expression levels of Kv7.2 fused to YFP (top panel, 110 KDa approximately) and GADPH (bottom panel, 50 KDa) from N2a Sgk1-KO cells transfected with the indicated constructs; N.T., non-transfected cells. Asterisks denote migration of the indicated molecular mass marker. (D) Quantitative analysis of Kv7.2 expression levels in N2a cells from two independent replicates (ANOVA Sidak’s test for multiple comparisons; ns, not significant). (E) Top, schematic representation of the CRISPR/Cas9 strategy…

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  FIGURE 6. Kv7.2/3 heteromeric channel is located in close proximity to Nedd4-2 and this association weakens in presence of activated SGK1.1. (A–C) Representative images of PLA analysis performed on N2a Sgk1-KO cells transfected with the indicated constructs. Cells transfected with SGK1.1 (S515D) and NR1 were used as negative control (dotted line at 0 in B,D). Bars correspond to 10 μM. (B–D) Quantification of PLA positive signals for each condition (ANOVA Kruskal Wallis test, ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.0005; ****p < 0.0001). Each dot represents an individual cell from at least three independent experiments.