Martin-Batista E et al. (2021), Activation of SGK1.1 Upregulates the M-current ...

XB-ART-58732

Front Mol Neurosci 2021 Jan 01;14:798261. doi: 10.3389/fnmol.2021.798261.

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

Martin-Batista E , Manville RW , Rivero-Pérez B , Bartolomé-Martín D , Alvarez de la Rosa D , Abbott GW , Giraldez T .

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In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 increases M-channel activity in the presence of two different epilepsy mutations found in Kv7.2, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying augmentation of M-channel activity by SGK1.1.

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Species referenced: Xenopus laevis
Genes referenced: nedd4 sgk1

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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).
	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).
	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).
	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).
	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 to knockout the Sgk1 gene. Insets indicate the sequence and hybridization sites for the guides, and PAM sequences (NGG) required for Cas9 to cut the DNA at the end of each targeted exon. Screening PCR primers are indicated as red arrows. Bottom, western blot detecting the presence of SGK1 protein expression in WT N2a cells or in selected single cell clones. Clone 16 was selected and used as Sgk1 knockout in this study. (F) PLA was performed on N2a Sgk1-KO cells transfected with the indicated constructs. Cells transfected with SGK1.1 (S515D) and NR1 were used as negative control (see F and dotted line at 0 in G). Bars correspond to 10 μM. (G) Quantification of PLA positive signals for each condition (ANOVA Kruskal Wallis test, ns, not significant; p < 0.05; *p < 0.0005; **p < 0.0001). Each dot represents an individual cell from at least three independent experiments.
	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.

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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).
	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).
	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).
	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).
	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 to knockout the Sgk1 gene. Insets indicate the sequence and hybridization sites for the guides, and PAM sequences (NGG) required for Cas9 to cut the DNA at the end of each targeted exon. Screening PCR primers are indicated as red arrows. Bottom, western blot detecting the presence of SGK1 protein expression in WT N2a cells or in selected single cell clones. Clone 16 was selected and used as Sgk1 knockout in this study. (F) PLA was performed on N2a Sgk1-KO cells transfected with the indicated constructs. Cells transfected with SGK1.1 (S515D) and NR1 were used as negative control (see F and dotted line at 0 in G). Bars correspond to 10 μM. (G) Quantification of PLA positive signals for each condition (ANOVA Kruskal Wallis test, ns, not significant; p < 0.05; *p < 0.0005; **p < 0.0001). Each dot represents an individual cell from at least three independent experiments.
	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.