Na Channel β Subunits: Overachievers of the Ion Channel Family

Abstract

Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSCα subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independently of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.

Keywords: adhesion; development; excitability; voltage-gated Na+ channel; β subunit.

Figure 1

Topology of the voltage-gated Na⁺ channel α and β subunits. VGSCs contain a pore-forming α subunit consisting of four homologous domains of six transmembrane segments (1–6). Segment 4 contains the voltage sensor (Catterall, 2000). VGSCs also contain one or more β subunits. β1, β2, β3, and β4 contain an extracellular immunoglobulin (Ig) loop, transmembrane domain, and an intracellular C-terminal domain (Isom et al., 1994). β1B also contains an Ig loop, but has a different C-terminus lacking a transmembrane domain, and is thus a soluble, secreted protein (Patino et al., 2011). β1 contains a tyrosine phosphorylation site in its C-terminus (Malhotra et al., 2004) ψ, glycosylation sites. β1 and β3 are non-covalently linked to α, whereas β2 and β4 are covalently linked through disulfide bonds. Figure was produced using Science Slides 2006 software.

Figure 2

Functional architecture of β1/β1B. β1 contains residues responsible for interaction with α subunit in its intracellular and extracellular domains (Mccormick et al., ; Spampanato et al., 2004). Mutation sites responsible for causing genetic epilepsy with febrile seizures plus (GEFS + 1), temporal lobe epilepsy (TLE), and Dravet syndrome are located in the extracellular immunoglobulin loop (Meadows et al., ; Wallace et al., ; Audenaert et al., ; Scheffer et al., ; Patino et al., 2009). Alternative splicing site for β1B (Kazen-Gillespie et al., ; Qin et al., ; Patino et al., 2011), putative palmitoylation site (Mcewen et al., 2004), ankyrin interaction site (Malhotra et al., 2002), tyrosine phosphorylation site (Malhotra et al., 2004), N-glycosylation sites (ψ; Mccormick et al., 1998), α/β/γ-secretase cleavage sites (Wong et al., 2005), receptor protein tyrosine phosphatase β (RPTPβ) interaction (Ratcliffe et al., 2000), and putative fyn kinase interaction (Malhotra et al., , ; Brackenbury et al., 2008) are also marked. Figure was produced using Science Slides 2006 software.

Figure 3

β1-mediated neurite outgrowth requires γ-secretase activity. (A) Location of γ-secretase cleavage site on the intracellular domain of β1 (Wong et al., 2005). (B) Cerebellar granule neurons from postnatal day (P)14 wildtype mice were plated on top of monolayers of control or β1-expressing Chinese hamster lung cells, as described previously (Davis et al., 2004). Cultures were incubated with the either one of the γ-secretase inhibitors, L685458, or DAPT (both 1 μM); or control (DMSO) for 48 h (Kim et al., 2005). Cells were then fixed, processed for GAP43 immunocytochemistry, and neurite lengths measured, as described (Davis et al., 2004). Both L685458 and DAPT inhibited the increase in neurite length caused by β1 expressed in the monolayer. Data are mean + SEM (n = 300). Significance: ***P < 0.001, ANOVA with Tukey’s post hoc test.

Figure 4

Functional reciprocity between β1 and Na_v1.6. (A) Electrical excitability is impaired in Scn1b null cerebellar granule neurons. Action potential firing rate recorded from cerebellar granule neurons in brain slices from 12-day-old mice plotted as a function of injected current, normalized to action potential threshold for wildtype (filled circles) and Scn1b null (open circles). Data are mean ± SEM (n ≥ 15). Significance: *P < 0.05; **P < 0.01; ***P < 0.001; t-test. (B) β1-mediated neurite outgrowth is inhibited by the Scn8a null mutation. Neurite lengths of wildtype and Scn8a null cerebellar granule neurons grown on control Chinese hamster lung or β1-expressing monolayers (n = 300). Data are mean + SEM. Significance: ***P < 0.001, ANOVA with Tukey’s post hoc test. (C) Na_v1.6 expression is reduced at the axon initial segment of Scn1b null cerebellar granule neurons. Wildtype and Scn1b null cerebellar granule neurons cultured in vitro for 14 days labeled with anti-ankyrin_G (red) and Na_v1.6 antibodies (green). Scale bar, 20 μm. Arrows point to axon initial segment expressing ankyrin_G. (D) A model for Na⁺ current involvement in β1-mediated neurite outgrowth. Complexes containing Na_v1.6, β1, and contactin are present throughout the neuronal membrane in the soma, neurite and growth cone. Localized Na⁺ influx is necessary for β1-mediated neurite extension and migration. VGSC complexes along the neurite participate in cell–cell adhesion and fasciculation. β1 is also required for Na_v1.6 expression at the axon initial segment, and subsequent high-frequency action potential firing through modulation of resurgent Na⁺current. Electrical activity may further promote β1-mediated neurite outgrowth at or near the growth cone. Thus, the developmental functions of β1 and Na_v1.6 are complementary, such that (1) Na⁺ influx carried by Na_v1.6 is required for β1-mediated neurite outgrowth, and (2) β1 is required for normal expression/activity of Na_v1.6 at the axon initial segment. Fyn kinase and ankyrin_G are likely also present in all complexes, but are only shown once in each panel for clarity. The FGF-mediated, β1-independent neurite outgrowth pathway is also shown. Figure reproduced with permission (Brackenbury et al., 2010).