Neuronal potassium channel activity triggers initiation of mRNA translation through binding of translation regulators

Abstract

Neuronal activity stimulates mRNA translation crucial for learning and development, but the mechanism linking translation to neuronal activity is not understood. In humans, learning and memory are severely disrupted by mutations in the potassium channel Slack (KCNT1, Slo2.2). We find that pharmacological stimulation of this channel and a constitutively active Slack mutation stimulate mRNA translation of a reporter for β-actin mRNA in cell lines and increases the synthesis of β-actin in the neurites of cortical neurons. Moreover, channel activation promotes the binding of two key mRNA translation regulators, FMRP (fragile X mental retardation protein) and CYFIP1 (cytoplasmic FMR1-interacting protein 1), to the channel itself, releasing both from eIF4E (eukaryotic initiation factor 4E), where they normally inhibit initiation of translation. This interaction provides a molecular mechanism for Slack activity-dependent regulation of translation and suggests that the effects of Slack mutations on this process may explain the severe intellectual disabilities associated with these mutations.

Fig. 1.. Slack-R455H mutation increases translation of β-actin reporter construct.

(A) Schematic of β-actin translation reporter construct: Fluorescent dendra2 protein flanked by 5′UTR and 3′UTR of β-actin contains nonfunctional myristylation (myr) tag. (B and C) Protein expression of dendra2 and Slack (n = 3 to 10 replicates), and (D) baseline fluorescence intensity of the dendra2_actin reporter [n = 5 plates, six FOV per plate, 3 to 22 cells per FOV, two-way analysis of variance (ANOVA) with post hoc comparisons] in human embryonic kidney (HEK) cells 24 hours after co-transfection of dendra2_actin with vector control, rat wild-type Slack (Slack-WT, WT), or Slack-R455H (R455H). a.u., arbitrary units. (E) qPCR of dendra2 and Slack mRNA levels (n = 3 replicates) and (F and G) actively translating protein of dendra2 and Slack using puromycin treatment and puromycin antibody IP (n = 5 replicates) 24 hours after co-transfection of dendra2_actin with Slack-WT or Slack-R455H. (H and I) Global translation levels (n = 3 replicates) after puromycin treatment 24 hours after transfection with Slack-WT or Slack-R455H. (J) Schematic of subcloned dendra2 reporter construct inserted into pEGFP-C1 vector without β-actin UTR. (K and L) Protein expression of dendra2 and Slack (n = 6 replicates) in HEK cells 24 hours after co-transfection of dendra2-C1 with vector control, Slack-WT, or Slack-R455H. [(C), (I), and (L)] Data are first normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [(C) and (L)] or Slack (I) levels and then to Slack-WT level [(C), (G), (I), and (L)]. (E) Gapdh was used as the housekeeping gene. (G) Data are normalized to Slack-WT level. Comparisons for all panels except D used unpaired t test. Data are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. See data S2 for exact P values. n.s., not significant.

Fig. 2.. Slack activation increases translation of β-actin reporter construct.

(A) Top: Schematic of FRAP experiment protocol. Bottom: Example cell from FRAP experiments (left, pre-photobleaching; center, post-photobleaching; right, post-recovery). (B to G) FRAP experiments with or without niclosamide (10 μM, 30 min) in HEK cells (n = 4 to 8 plates, 13 to 53 cells per plate). [(B) and (C)] Vector control or rat Slack-WT were co-transfected with dendra2_actin. [(D) and (E)] Only dendra2_actin was transfected into stable human Slack cells (hSlack-WT) or untransfected control cells. [(F) and (G)] Rat Slack-WT or Slack-R455H were co-transfected with dendra2_actin. [(B), (D), and (F)] FRAP time course. P values represent differences between the groups indicated, two-way repeated-measures ANOVA with post hoc comparisons with Holm-Šídák correction. [(C), (E), and (G)] Bar graph of 60-min recovery time point, ordinary two-way ANOVA with post hoc comparisons. Data are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. See data S2 for exact P values. WT, wild type.

Fig. 3.. Endogenous neuronal Slack channels regulate β-actin translation.

(A and B) Bar graph of FRAP experiment in primary cortical neurons (A) from Slack-WT mice or Slack^−/− mice with or without niclosamide (10 μM) (n = 9 to 22 cells) and (B) from Slack-WT or Slack-R455H mice (n = 61 to 91 cells). (C) Representative PLA images of individual neurons from Slack-WT (left) or Slack-R455H (right) mice. (blue, Slack; yellow, β-actin/PLA puncta; scale bars, 10 μm). (D) Quantification of puncta number from PLA images in the soma or neurites (n = 17 to 33 cells). All comparisons used unpaired t test. Data are expressed as means ± SEM. *P < 0.05; ***P < 0.001. See data S2 for exact P values.

Fig. 4.. Slack-R455H regulates translation initiation.

(A) Model where Slack regulates translation through elongation or termination stalling. Top: In cells without Slack, ribosomes stall on dendra2 mRNA during elongation or termination. This leads to low total translation levels but high ribosome binding. This requires a secondary mechanism that stalls ribosomes under baseline conditions, such as stalling through UPF3B or FMRP. Bottom: When mutant Slack is present, ribosome stalling is relieved, leading to increased total translation levels but decreased ribosome binding. (B) Model where Slack regulates translation initiation. Top: In cells without Slack, translation occurs normally but with low total translation levels and ribosome binding. This model does not require a secondary mechanism to explain the baseline condition. Bottom: Mutant Slack increases the rate of translation initiation. This leads to both increased ribosome binding and total translation levels. (C and D) Ribosome binding of ∆myr-dendra2 in HEK cells 24 hours after co-transfection of ∆myr-dendra2 and Slack-WT or Slack-R455H. Slack staining represents total Slack in cell membrane and serves as loading control. Dendra2 levels are normalized by Slack level and then to WT level. Slack levels are normalized to WT level (n = 3, unpaired t test). Data are expressed as means ± SEM. *P < 0.05. See data S2 for exact P values.

Fig. 5.. Slack-R455H mutation triggers the redistribution of the FMRP/CYFIP1 complex.

(A and B) IP of Slack (A) or eIF4E (B) from mouse cerebral cortex. (C) Quantification of Western blots in (A) and (B). Input, Slack IP, and eIF4E IP samples are normalized by GAPDH, Slack, and eIF4E levels, respectively, followed by normalization to Slack-WT level (n = 4 mice, paired t test). Data are expressed as means ± SEM. **P < 0.01. See data S2 for exact P values.

Fig. 6.. Slack channel activation triggers the redistribution of the FMRP/CYFIP1 complex.

(A) Western blotting showing protein expression in HEK cells transfected with vector control, rat wild-type Slack (Slack-WT), or Slack-R455H treated with or without niclosamide (nic; 5 μM, 30 min). (B and C) IP of Slack (B) or eIF4E (C) in HEK cells treated with/without niclosamide (5 μM, 30 min). (D) Quantification of Western blots in (A) to (C). Input, Slack IP, and eIF4E IP samples are normalized by GAPDH, Slack, and eIF4E levels, respectively, followed by normalization to Slack-WT level (n = 4 replicates, paired t test). Data are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. See data S2 for exact P values.

Fig. 7.. FMRP and CYFIP1 are required for Slack activity–dependent translation.

(A to D) Effect of knockdown of FMRP [(A) and (B)] or CYFIP1 [(C) and (D)] on dendra2_actin expression in HEK cells. Cell extraction was carried out 24 hours after triple transfection of dendra2_actin, rat Slack-WT, and fmr1 siRNA cocktail or cyfip1 siRNA or negative (Neg) siRNA control. Protein levels were normalized to GAPDH level, followed by normalization to neg siRNA level (n = 6 replicates, unpaired t test). (E to J) Effect of knockdown of FMRP [(E) and (F)] or CYFIP1 [(G) and (H)] or both [(I) and (J)] on niclosamide-induced translation of dendra2_actin in hSlack-expressing HEK cells. Cell extraction was carried out 48 hours after co-transfection of dendra2_actin with fmr1 siRNA cocktail, cyfip1 siRNA, or both fmr1 and cyfip1 (fmr1/cyfip1) siRNAs or negative siRNA control, with or without Slack activator niclosamide (nic; 10 μM, 90 min) treatment. n = 4 replicates for all experiments. [(F), (H), and (J)] Paired t tests. (K) Time course of FRAP experiment in hSlack-expressing HEK cells 48 hours after co-transfection of dendra2_actin with fmr1/cyfip1 siRNAs or negative siRNA control, with or without niclosamide (10 μM, 60 min). P values represent differences between indicated groups (n = 9 to 21 cells, two-way repeated-measures ANOVA with post hoc comparisons with Holm-Šídák correction). Data are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. See data S2 for exact P values.

Fig. 8.. Model of potential mechanism of Slack activity–dependent translation.

When Slack channels are inactive, the FMRP/CYFIP1 complex binds to eIF4E in a translation initiation suppression complex. When Slack channels are activated or when gain-of-function (GOF) channels are present, the FMRP/CYFIP1 complex moves from the eIF4E complex to Slack channels, allowing translation to resume.