Chunling Chen

Mice Lacking Sodium Channel β1 Subunits Display Defects in Neuronal Excitability, Sodium Channel Expression, and Nodal Architecture

Sodium channel β1 subunits modulate α subunit gating and cell surface expression and participate in cell adhesive interactions in vitro. β1(-/-) mice appear ataxic and display spontaneous generalized seizures. In the optic nerve, the fastest components of the compound action potential are slowed and the number of mature nodes of Ranvier is reduced, but Nav1.6, contactin, caspr 1, and Kv1 channels are all localized normally at nodes. At the ultrastructural level, the paranodal septate-like junctions immediately adjacent to the node are missing in a subset of axons, suggesting that β1 may participate in axo-glial communication at the periphery of the nodal gap. Sodium currents in dissociated hippocampal neurons are normal, but Nav1.1 expression is reduced and Nav1.3 expression is increased in a subset of pyramidal neurons in the CA2/CA3 region, suggesting a basis for the epileptic phenotype. Our results show that β1 subunits play important roles in the regulation of sodium channel density and localization, are involved in axo-glial communication at nodes of Ranvier, and are required for normal action potential conduction and control of excitability in vivo.

A Functional Null Mutation of SCN1B in a Patient with Dravet Syndrome

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Nav1.1 α subunits. Sodium channels are modulated by β1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Nav1.1 α subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of β1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b−/− versus Scn1b+/+ mice. Scn1b−/− CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a+/− model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b−/− mice seize spontaneously, the seizure susceptibility of Scn1b+/− mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

Reduced sodium channel density, altered voltage dependence of inactivation, and increased susceptibility to seizures in mice lacking sodium channel β2-subunits

Sodium channel β-subunits modulate channel gating, assembly, and cell surface expression in heterologous cell systems. We generated β2−/− mice to investigate the role of β2 in control of sodium channel density, localization, and function in neurons in vivo. Measurements of [3H]saxitoxin (STX) binding showed a significant reduction in the level of plasma membrane sodium channels in β2−/− neurons. The loss of β2 resulted in negative shifts in the voltage dependence of inactivation as well as significant decreases in sodium current density in acutely dissociated hippocampal neurons. The integral of the compound action potential in optic nerve was significantly reduced, and the threshold for action potential generation was increased, indicating a reduction in the level of functional plasma membrane sodium channels. In contrast, the conduction velocity, the number and size of axons in the optic nerve, and the specific localization of Nav1.6 channels in the nodes of Ranvier were unchanged. β2−/− mice displayed increased susceptibility to seizures, as indicated by reduced latency and threshold for pilocarpine-induced seizures, but seemed normal in other neurological tests. Our observations show that β2-subunits play an important role in the regulation of sodium channel density and function in neurons in vivo and are required for normal action potential generation and control of excitability.

Regulation of Persistent Na Current by Interactions between β Subunits of Voltage-Gated Na Channels

The β subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming α subunits, as well as their trafficking and localization. In heterologous expression systems, β1, β2, and β3 subunits influence inactivation and persistent current in different ways. To test how the β4 protein regulates Na channel gating, we transfected β4 into HEK (human embryonic kidney) cells stably expressing NaV1.1. Unlike a free peptide with a sequence from the β4 cytoplasmic domain, the full-length β4 protein did not block open channels. Instead, β4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of β1 or chimeric subunits including the β1 extracellular domain, however, favored inactivation. Coexpressing NaV1.1 and β4 with β1 produced tiny persistent currents, indicating that β1 overcomes the effects of β4 in heterotrimeric channels. In contrast, β1C121W, which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by β4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with β4, persistent current was slightly but significantly increased. Moreover, in β4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that β1 and β4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted β1 subunits.

Sodium Channel β2 Subunits Regulate Tetrodotoxin-Sensitive Sodium Channels in Small Dorsal Root Ganglion Neurons and Modulate the Response to Pain

Voltage-gated sodium channel (Nav1) β2 subunits modulate channel gating, assembly, and cell-surface expression in CNS neurons in vitro and in vivo. β2 expression increases in sensory neurons after nerve injury, and development of mechanical allodynia in the spared nerve injury model is attenuated in β2-null mice. Thus, we hypothesized that β2 modulates electrical excitability in dorsal root ganglion (DRG) neurons in vivo. We compared sodium currents (INa) in small DRG neurons from β2+/+ and β2−/− mice to determine the effects of β2 on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Nav1 in vivo. Small-fast DRG neurons acutely isolated from β2−/− mice showed significant decreases in TTX-S INa compared with β2+/+ neurons. This decrease included a 51% reduction in maximal sodium conductance with no detectable changes in the voltage dependence of activation or inactivation. TTX-S, but not TTX-R, INa activation and inactivation kinetics in these cells were slower in β2−/− mice compared with controls. The selective regulation of TTX-S INa was supported by reductions in transcript and protein levels of TTX-S Nav1s, particularly Nav1.7. Low-threshold mechanical sensitivity was preserved in β2−/− mice, but they were more sensitive to noxious thermal stimuli than wild type whereas their response during the late phase of the formalin test was attenuated. Our results suggest that β2 modulates TTX-S Nav1 mRNA and protein expression resulting in increased TTX-S INa and increases the rates of TTX-S Nav1 activation and inactivation in small-fast DRG neurons in vivo. TTX-R INa were not significantly modulated by β2.

Functional reciprocity between Na+ channel Nav1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth

Voltage-gated Na+ channel (VGSC) β1 subunits regulate cell–cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na+ current (INa) mediated by Nav1.6. In addition, β1 is required for high-frequency action potential firing. Transient INa is unchanged in Scn1b (β1) null CGNs; however, the resurgent INa, thought to underlie high-frequency firing in Nav1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Nav1.6 is reduced in Scn1b null cerebellar neurons. In place of Nav1.6 at the AIS, we observed an increase in Nav1.1, whereas Nav1.2 was unchanged. This indicates that β1 is required for normal localization of Nav1.6 at the AIS during the postnatal developmental switch to Nav1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with α subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and Nav1.6 such that β1-mediated neurite outgrowth requires Nav1.6-mediated INa, and Nav1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.

Voltage-Gated Na+ Channel β1 Subunit-Mediated Neurite Outgrowth Requires Fyn Kinase and Contributes to Postnatal CNS Development In Vivo

Voltage-gated Na+ channel β1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that β1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that β1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. β1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on β1-mediated neurite extension. These results suggest that β1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that β1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that β1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for β1 in vivo. We conclude that voltage-gated Na+ channel β1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

Sodium channel β1 subunits promote neurite outgrowth in cerebellar granule neurons

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel β1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or β1(-/-) mice were plated on top of parental, mock-, or β1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on β1-transfected monolayers, whereas β1(-/-) neurons showed no increase compared with control conditions. β1-Mediated neurite extension was mimicked using a soluble β1 extracellular domain and was blocked by antibodies directed against the β1 extracellular domain. Immunohistochemical analysis suggests that the β1 and β4 subunits, but not β2 and β3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for β1 during neuronal development and are the first demonstration of a functional role for sodium channel β subunit-mediated cell adhesive interactions.

Voltage-Gated Na+ Channel β1B: A Secreted Cell Adhesion Molecule Involved in Human Epilepsy

Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, β1 and β1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of β1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to β1B. We show that wild-type β1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of β1B with voltage-gated Na+ channels Nav1.1 or Nav1.3 is not detectable by immunoprecipitation and β1B does not affect Nav1.3 cell surface expression as measured by [3H]saxitoxin binding. However, β1B coexpression results in subtle alteration of Nav1.3 currents in transfected cells, suggesting that β1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of β1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that β1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.

Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Background: Voltage-gated Na+ channels are composed of α and β subunits. Results: We identified the cysteine residue in β2 responsible for disulfide linkage to α. Conclusion: α and β2 associate through a single disulfide bridge to achieve proper subcellular targeting in neurons. Significance: Understanding how Na+ channel complexes are formed in neurons is crucial for understanding the development of excitability. Voltage-gated Na+ channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na+ channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na+ channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.