Journal: Communications biology

Article Title: Functional correlates of clinical phenotype and severity in recurrent SCN2A variants.

doi: 10.1038/s42003-022-03454-1

Figure Lengend Snippet: Fig. 1 Nav1.2 channel and the locations of the functionally studied mutations. Note the color-coded four domains (DI–DIV), the positive charges of segment 4 (S4) on DI–DIV, and the inactivation gate (DIII–DIV linker) marked in red. a Linear representation of the full-length Nav1.2 channel with the mutations studied here (red) or previously (gray)7. b Cryo-electron microscopy structure of Nav1.224. The mutations are distributed in the pore module, the four peripheral voltage-sensing domains, the intracellular loops, and the C-terminus. Residues affected by the mutations are shown as sticks.

Article Snippet: Co-transfections involving the β2 subunit were performed using 4 μg wild-type or K905N sodium channel α1 subunit, 3 μg human β2 subunit (NCBI Reference Sequence: NM_004588.5; Origene Technologies, Rockville, MD) and 1 μg eGFP.

Techniques: Cryo-Electron Microscopy

Journal: Communications biology

Article Title: Functional correlates of clinical phenotype and severity in recurrent SCN2A variants.

doi: 10.1038/s42003-022-03454-1

Figure Lengend Snippet: Fig. 2 Voltage dependence of activation and inactivation of wild-type (WT) and mutant Nav1.2 channel variants associated with early-infantile (EI) severe/variable/benign, or later-onset (LO) SCN2A epilepsy. a Representative Nav1.2 current traces. Note that only the first 10 ms of the current traces elicited in the voltage range between −80 and +20 mV are shown. The voltage protocol (inset) consisted of 40-ms depolarizing voltage steps, elicited at 1 Hz in 5-mV increments, in the voltage range between −80 and +60 mV, from a holding potential (HP) of −120 mV. Current traces of individual variants were normalized to the same amplitude. Horizontal scale bar: 5 ms. b–e Activation and inactivation curves were obtained by non-linear least-squares fits of Boltzmann equations (Eq. (1)) to normalized conductance (G/Gmax) data of wild-type Nav1.2 (black solid circle) and mutant Nav1.2 channels (colored open circles). See the parameters of the fits in Supplementary Table 3; n values, the number of independent experiments for inactivation and activation, respectively, are shown in parentheses. f Mean half-maximal voltages of activation and inactivation (V0.5,act and V0.5,inact, respectively) of wild-type Nav1.2 (black bar) and mutant Nav1.2 channels (colored open bars). Data for individual cells are shown in open circles. Relative to wild-type, hyperpolarising shifts of the V0.5,act values are consistent with increased channel opening and gain-of-function (GoF), whereas depolarizing shifts are consistent with reduced channel opening and loss-of-function (LoF); hyperpolarising shifts of the V0.5,inact values are consistent with reduced channel availability (LoF), whereas depolarizing shifts are consistent with increased channel availability (GoF). Data shown are mean ± SEM; n values for all variants are shown in b–e. *P < 0.05, one-way ANOVA, followed by Dunnett’s post-hoc test; the detailed statistical evaluation of biophysical characteristics of the variants is shown in Supplementary Table 3.

Article Snippet: Co-transfections involving the β2 subunit were performed using 4 μg wild-type or K905N sodium channel α1 subunit, 3 μg human β2 subunit (NCBI Reference Sequence: NM_004588.5; Origene Technologies, Rockville, MD) and 1 μg eGFP.

Techniques: Activation Assay, Mutagenesis

Journal: Communications biology

Article Title: Functional correlates of clinical phenotype and severity in recurrent SCN2A variants.

doi: 10.1038/s42003-022-03454-1

Figure Lengend Snippet: Fig. 4 Action potential firing of the axon initial segment hybrid neuron incorporating Nav1.2 variants in response to synaptic conductance input in DAPC experiments. a Representative action potential firing elicited with ge/gi ratios of 1, 1.5, 2, 2.5, and 3 in the early-infantile (EI)-severe, EI-variable, EI- benign and later-onset (LO) groups. b–e Input–output relationships in the presence of Nav1.2 channel variants (WT, black solid circle; mutants, colored open circles) associated with EI-severe (b), EI-variable (c), EI-benign (d), or later-onset (LO) (e) SCN2A epilepsy. Two-way ANOVA, followed by Dunnett’s post-hoc test, was used to compare the AP firing frequencies elicited by scaled excitatory to inhibitory conductance ratios (ge/gi) in the presence of Nav1.2 variants; asterisks indicate P < 0.05 (see individual P values in Supplementary Table 7). Note the increased firing activity in the early-infantile severe/ variable groups compared to wild-type, whereas later-onset variants result in an almost complete loss of firing. Data shown are mean ± SEM; n, the number of independent experiments between parentheses.

Article Snippet: Co-transfections involving the β2 subunit were performed using 4 μg wild-type or K905N sodium channel α1 subunit, 3 μg human β2 subunit (NCBI Reference Sequence: NM_004588.5; Origene Technologies, Rockville, MD) and 1 μg eGFP.

Techniques: Activity Assay

Journal: Communications biology

Article Title: Functional correlates of clinical phenotype and severity in recurrent SCN2A variants.

doi: 10.1038/s42003-022-03454-1

Figure Lengend Snippet: Fig. 6 The phenotypic spectrum of SCN2A-related disorders. Pink/red boxes indicate phenotypes associated with gain-of-function (GoF) of the Nav1.2 channel, and blue boxes loss-of-function (LoF) phenotypes; Onset refers to age of seizure onset. The phenotypic groups associated with more severe epilepsies are sometimes grouped as SCN2A developmental and epileptic encephalopathies (gray box), which encompasses both GoF and LoF phenotypes. The early-infantile (EI) phenotypes can be considered a spectrum of clinical severity associated with biophysical GoF. While the later-onset (LO) epilepsy phenotypes and ID/ASD without epilepsy are both associated with LoF, it is not clear whether these represent a spectrum of severity (related to partial or total loss of channel function), or are distinct disorders caused by different mechanisms of channel hypofunction (for example pure LoF vs dominant negative effect).

Article Snippet: Co-transfections involving the β2 subunit were performed using 4 μg wild-type or K905N sodium channel α1 subunit, 3 μg human β2 subunit (NCBI Reference Sequence: NM_004588.5; Origene Technologies, Rockville, MD) and 1 μg eGFP.

Techniques: Dominant Negative Mutation