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. 2021 Feb 8;6(3):e141776.
doi: 10.1172/jci.insight.141776.

Sodium channel β1 subunits participate in regulated intramembrane proteolysis-excitation coupling

Alexandra A Bouza  1 , Nnamdi Edokobi  1 , Samantha L Hodges  1 , Alexa M Pinsky  1 , James Offord  1 , Lin Piao  2 , Yan-Ting Zhao  1 , Anatoli N Lopatin  2 , Luis F Lopez-Santiago  1 , Lori L Isom  1   2   3
Affiliations

Sodium channel β1 subunits participate in regulated intramembrane proteolysis-excitation coupling

Alexandra A Bouza et al. JCI Insight. .

Abstract

Loss-of-function (LOF) variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including developmental and epileptic encephalopathy and cardiac arrhythmia. β1 Subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming α subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Here, we investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase and show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA sequencing, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b-null cardiac tissue, which lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. We propose that human disease variants resulting in SCN1B LOF cause transcriptional dysregulation that contributes to altered excitability. Moreover, these results provide important insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multifunctionality of sodium channel β1 subunits.

Keywords: Cardiology; Cell Biology; Cell migration/adhesion; Sodium channels; Transcription.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. β1 subunits are substrates for BACE1 and γ-secretase intramembrane cleavage.
(A) Cartoon diagram of the proposed β1-mediated signal transduction cascade. (B) Schematic of β1 with BACE1 and γ-secretase cleavage sites. (C) Chinese hamster lung (CHL) cells stably expressing WT β1-V5 also endogenously express BACE1 and presenilin-1, the catalytic subunit of γ-secretase. (D) WT β1-V5 is cleaved by BACE1, and the β1-C-terminal fragment (β1-CTF) is found in the membrane fraction. (E) Treatment with γ-secretase inhibitor, DAPT, leads to a concentration-dependent accumulation of β1-CTF. (F) Quantification of E. Protein levels were normalized to the loading control and reported as fold change respective to the vehicle-treated group. Significance (P value less than 0.05) was determined using a 1-way ANOVA between each treatment and the negative control (vehicle treatment). (G) Scheduled treatments with DAPT and β-secretase inhibitor IV inhibit formation of respective cleavage products in a manner consistent with sequential cleavage. (H) Quantification of G. Protein levels were normalized to the loading control and reported as fold change respective to the vehicle-treated group. Significance (P value less than 0.05) was determined using a 1-way ANOVA between each treatment and the positive control (DAPT treatment alone). Data represent mean ± SEM. For each experiment, n = 3. See complete unedited blots in the supplemental material.
Figure 2
Figure 2. β1-ICD-V5 localizes to the nucleus.
(A) Full-length WT β1-V5 shows little to no nuclear localization, as determined by staining for the V5-epitope tag and DAPI. Strong colocalization is observed between staining for the V5-epitope tag of the β1-ICD and the nucleus (DAPI, yellow). (B) Quantification of intensity of V5 and DAPI staining across the transfected cell. Averaged data from 13–17 cells per condition are shown from 3 independent transfections. Data represent mean ± SD. Statistical significance was determined using Student’s t test.
Figure 3
Figure 3. β1-intracellular domain expression alters voltage-gated sodium channel gene expression.
(A) Immunocytochemistry for V5 for the enhanced GFP (eGFP) stable line and WT β1-ICD-V5-2A-eGFP (positive control) stable line. DAPI is shown in blue, eGFP in green, and V5 in red. (B) Immunoblot (anti-GFP) of CHL cells stably overexpressing eGFP only or WT β1-intracellular domain (β1-ICD0 with a V5 epitope tag and a 2A-eGFP sequence on the 3′ end. (C) RNA-Seq identifies VGSC genes are differentially expressed in presence of β1-ICD compared with control; n = 4. Gene transcripts were considered differentially expressed if they had a fold-change greater than or equal to 1.5 and a false discovery rate less than 0.05. (D) qPCR confirms some voltage-gated sodium channel (VGSC) genes are differentially expressed in the presence of β1-ICD compared with control; n = 3. Data represent mean ± SEM. Statistical significance was determined using Student’s t test.
Figure 4
Figure 4. β1-ICD modulates gene transcription in vitro and in vivo.
(A) Experimental design (n = 4 samples for each condition, all run in 1 RNA-Seq experiment). (B) Gene ontology (GO) groups overrepresented in analysis from CHL cells overexpressing the β1-ICD (left) and Scn1b-null cardiac ventricle (right). (C–E) Heat maps depicting genes altered in each RNA-Seq related to calcium ion binding (C), the immune response (D), and proliferation (E). (F) Percent of genes downregulated in each data set for calcium ion binding, immune response, and proliferation GO groups. (G) Percent of genes upregulated in each data set for calcium ion binding, immune response, and proliferation GO groups.
Figure 5
Figure 5. β1-ICD regulates potassium channel gene expression and potassium currents in cardiac ventricular myocytes.
(A) Experimental design of RNA-Seq experiments from CHL cells stably overexpressing the β1-ICD and from P10 Scn1b WT or Scn1b-null mouse cardiac ventricle. (B) RNA-Seq showed that β1-ICD expression downregulates potassium channel genes, whereas Scn1b-null mice show upregulated potassium channel gene expression in cardiac ventricle. (C) Representative potassium currents recorded from ventricular myocytes obtained from WT and Scn1b-null mice. To assess the I-V relationship, 5-second pulses were applied in +10 mV increments from –70 mV to +70 mV, following a 5-second prepulse to –120 mV from –70 mV holding potential. Scale bars: 5 nA and 2 seconds.
Figure 6
Figure 6. Comparison of current density and voltage dependence of inactivation of peak and end potassium currents.
(A and B) Scn1b deletion results in increased peak (Ipeak) (A) and end (Iend) (B) potassium current densities at depolarized potentials. *P ≤ 0.05, ***P ≤ 0.001 by Student’s t test (assuming equal variances). (A) n = 13 and n = 8 and (B) n = 13 and n = 10 for Scn1b WT and -null, respectively. Data represent mean ± SEM.
Figure 7
Figure 7. Comparison of current density, rate of decay, and availability of individual components of the potassium current.
(A and B) Mean current density (A) and time constant of current decay (B) measured at +60 mV for Ito f, Ito s, IK slow, and Iss currents in myocytes from Scn1b WT and -null mice. (A) At +60 mV current density of Ito f is unchanged, whereas that of Ito s, IK slow, and Iss is increased with Scn1b deletion. (B) At +60 mV, Scn1b deletion results in slower decay of IK slow (inset, Ito f data shown at higher magnification). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 by Student’s t test (assuming equal variances). (A and B) n = 15 and n = 10 for Scn1b WT and -null, respectively. Data represent mean ± SEM.
Figure 8
Figure 8. Excitation-contraction coupling in ventricular CMs from Scn1b-null mice.
(A) Representative example of excitation-contraction (E-C) coupling recording. Top: ICa triggered by voltage clamp depolarization; middle: whole-cell Ca2+ transient; bottom: Ca2+ transient time profile. (B) I-V curve shows dramatically decrease of ICa in CMs from Scn1b-null mice. (C) Ca2+ transient amplitude did not change in CMs from Scn1b-null mice compared with WT. (D) E-C coupling gain (ratio between the Ca2+ transient amplitude and the ICa) decreased in CMs from Scn1b-null mice. * P < 0.05; ** P < 0.01; *** P < 0.001. WT vs. Scn1b-null by Student’s t test. N, number of mice; n, number of cells. Data represent mean ± SEM.
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References

    1. O’Malley HA, Isom LL. Sodium channel β subunits: emerging targets in channelopathies. Annu Rev Physiol. 2015;77:481–504. doi: 10.1146/annurev-physiol-021014-071846. - DOI - PMC - PubMed
    1. Bouza AA, Isom LL. Voltage-gated sodium channel β subunits and their related diseases. Handb Exp Pharmacol. 2018;246:423–450. - PMC - PubMed
    1. Isom LL, et al. Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel. Science. 1992;256(5058):839–842. doi: 10.1126/science.1375395. - DOI - PubMed
    1. Isom LL, et al. Functional co-expression of the beta 1 and type IIA alpha subunits of sodium channels in a mammalian cell line. J Biol Chem. 1995;270(7):3306–3312. doi: 10.1074/jbc.270.7.3306. - DOI - PubMed
    1. Isom LL, Catterall WA. Na+ channel subunits and Ig domains. Nature. 1996;383(6598):307–308. - PubMed

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