Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Apr 21;24(16):4030-42.
doi: 10.1523/JNEUROSCI.4139-03.2004.

Mice lacking sodium channel beta1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture

Chunling Chen  1 , Ruth E Westenbroek, Xiaorong Xu, Chris A Edwards, Dorothy R Sorenson, Yuan Chen, Dyke P McEwen, Heather A O'Malley, Vandana Bharucha, Laurence S Meadows, Gabriel A Knudsen, Alex Vilaythong, Jeffrey L Noebels, Thomas L Saunders, Todd Scheuer, Peter Shrager, William A Catterall, Lori L Isom
Affiliations

Mice lacking sodium channel beta1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture

Chunling Chen et al. J Neurosci. .

Abstract

Sodium channel beta1 subunits modulate alpha subunit gating and cell surface expression and participate in cell adhesive interactions in vitro. beta1-/- 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 Na(v)1.6, contactin, caspr 1, and K(v)1 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 beta1 may participate in axo-glial communication at the periphery of the nodal gap. Sodium currents in dissociated hippocampal neurons are normal, but Na(v)1.1 expression is reduced and Na(v)1.3 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 beta1 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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Generation of β1 (-/-) mice by homologous recombination. A, SCN1B targeting vector. B, Amino acid sequence of β1. Gray, Exon 3; underline, transmembrane domain; asterisks, cysteine residues that define the Ig fold domain. C, Southern blot analysis of ES cell clones. Digestion with KpnI, followed by Southern blotting with the 5′ probe, yielded a 4.8 kb endogenous band and a 3.8 kb targeted band (left). A similar experiment using the 3′ probe yielded a 5.9 kb endogenous band and a 7.7 kb targeted band (right). D, PCR analysis of tail DNA isolated from β1 (+/+), (+/-), and (-/-) mice, as indicated. The arrows indicate positions of endogenous and targeted (neomycin) PCR products. E, Northern blot analysis of total brain RNA isolated from β1 (+/+), (+/-), and (-/-) mice, as indicated. Molecular weight markers are shown in kilobases.
Figure 2.
Figure 2.
β1(-/-) mice show growth retardation and spontaneous seizures. A, Growth retardation in β1 (-/-) mice.β1 (-/-) (left) and β1 (+/+) littermates at P19. The scale is shown in inches. B, Generalized myoclonic seizure in a β1 (-/-) mouse. Electrocorticographic monitoring of P19 β1 (-/-) mice reveals abnormal interictal discharges and seizures during waking behavior. A spontaneous seizure episode of 55 sec duration displayed here shows abrupt bilateral onset and steady acceleration in the frequency of cortical spike discharges, with a sudden return to normal cortical activity at the termination of the seizure. The dotted lines represent 16 sec of continuous seizure activity.
Figure 3.
Figure 3.
Western blot analysis of brain membranes from β1 (-/-) mice. A, Sodium channel subunits are not upregulated in β1 (-/-) brain membranes. Equal aliquots of β1 (-/-) and β1 (+/+) mouse brain membranes were analyzed by Western blot using anti-pan-α, anti-β2, anti-β3, or anti-β4 antibodies (as indicated), followed by HRP-conjugated goat anti-rabbit IgG. Molecular weight markers are shown in kilodaltons. B, Western blot analysis of proteins involved in the axo-glial apparatus. Thirty micrograms of β1 (-/-) or β1 (+/+) mouse brain membranes were analyzed by Western blot using primary antibodies, as indicated, followed by HRP-conjugated goat anti-rabbit IgG. Dilutions of primary antibodies were: contactin (Cn), 1:2000; Caspr 1,1:500; neurofascin 155 (Nf155), 1:2500; ankyrinG (AnkG), 1:250. Molecular weight markers are shown in kilodaltons.
Figure 4.
Figure 4.
Sciatic nerve nodes of Ranvier in β1 (-/-) mice. Sciatic nerves were isolated from β1 (+/+) and β1 (-/-) P17-P19 mice and prepared for immunocytochemistry, as described in Materials and Methods. Immunofluorescent staining obtained from antibodies directed against pan-sodium channels, caspr 1, contactin, and Kv1.2 appeared to be unchanged in the mutant mice compared with control. A, β1 (+/+) mice: β1 subunits (red), caspr 1 (green). B, β1 (-/-) mice: β1 subunits (red), caspr 1 (green). There is no staining for β1 subunits. C,β1(+/+) mice: pan-sodium channels (red), caspr 1 (green). D,β1(-/-) mice: pan-sodium channels (red), caspr 1 (green). E,β1(-/-) mice: caspr 1 (green). F,β1(-/-) mice (identical section as in E): contactin (red). G, β1 (+/+) mice: caspr 1 (green) and Kv1.2 (red). H, β1 (-/-) mice: caspr 1 (green) and Kv1.2 (red). Scale bars, 10 μm.
Figure 5.
Figure 5.
Optic nerve nodes of Ranvier in β1 (-/-) mice. Optic nerves were isolated from β1 (+/+) and β1 (-/-) P17-P19 mice and prepared for immunocytochemistry, as described in Materials and Methods. Immunofluorescent staining obtained from antibodies directed against pan-sodium channels, caspr 1, contactin, and Kv1.1 appeared to be unchanged in the mutant mice compared with control. A, β1(-/-) mice: pan-sodium channels (green), caspr 1 (red). B, β1(-/-) mice: caspr 1 (green), Kv1.1 (red). C, β1(-/-) mice: Nav1.6 (red), caspr 1 (green). D, β1(-/-) mice: pan-sodium channels (green). E, β1 (-/-) mice: contactin (red) (same slide as in D). F, β1(-/-) mice: merged images of D and E. Scale bar: A-F, 5 μm. G, Sodium channels and contactin do not associate in the absence of β1 subunits. Whole-brain homogenates from β1 (+/+) or β1 (-/-) mice, as indicated, were immunoprecipitated with anti-contactin antibody or nonimmune IgG [lane IgG, containing β1 (+/+) homogenate], and immunoblotted with anti pan-sodium channel antibody, as described in Materials and Methods. Molecular weight markers are shown in kilodaltons. RB, Nonimmunoprecipitated aliquot of rat brain homogenate containing 500 fmol of sodium channel included as a positive Western blot control.
Figure 6.
Figure 6.
Conduction velocities in the optic nerve. A-D show CAPs at P15-P17 from β1 (-/-) mice (A, B) and β1 (+/+) littermates (C, D). Records were taken at room temperature (A,C; 25 ±1 °C) and at 37 °C (B, D). The solid lines illustrate the original sweeps. CAPs were fitted to the sum of three Gaussian curves between the arrows, and the fit is superimposed (dotted line just discernable). Also shown are the three individual curves (dotted lines) from which times to peak were measured. E, Bar graph showing conduction velocities for each component (mean ± SEM). Differences between β1 (-/-) (open bars) and β1 (+/+) (filled bars) were significant for peaks 1 and 2 (p ≤ 0.005 at both temperatures) and borderline for peak 3 (p = 0.04 at 25°C).
Figure 7.
Figure 7.
β1 (-/-) mice have fewer optic nerve nodes of Ranvier. A, Optic nerves of P17-P19 β1 (-/-) and β1 (+/+) littermates were cryosectioned and stained for pan-sodium channels (red) and caspr 1 (green). The panels in A show typical FOV. Nodes of Ranvier were defined by a region of high-sodium channel density in the nodal gap, bordered by caspr 1-positive paranodes. Individual nodes had a normal appearance in both β1 (-/-) and β1 (+/+) animals, but there were fewer such sites in the null mutant. B, Over 30 FOV were analyzed for both β1 (-/-) (red bar) and β1 (+/+) (blue bar) mice, and the results are summarized in the bar graph. β1 (-/-) optic nerves had significantly fewer nodes than β1 (+/+) (p < 0.001). C, Left, Ultra structural analysis of optic nerve cross-sections shows similar numbers of myelinated axons per unit area for β1 (+/+) (blue bars) and β1 (-/-) (red bars). However, a greater percentage of myelinated axons per nerve cross-section in β1 (-/-) optic nerves (right, red bar) appear to be degenerating (p = 0.067, Student's t test). Error bars in both panels represent SDs.
Figure 8.
Figure 8.
β1 (-/-) mice show disruptions in nodal architecture in the CNS and PNS. Sciatic nerves, optic nerves, and spinal cords were isolated from P19 β1 (-/-) and β1 (+/+) littermates and prepared for transmission electron microscopy as described in Materials and Methods. A, E, β1 (+/+) sciatic nerve. B, F, β1 (-/-) sciatic nerve. C, β1 (+/+) spinal cord. D,β1 (-/-) spinal cord. G, Optic nerve cross-section fromβ1 (+/+) mouse. H, Optic nerve cross-section from β1(-/-) mouse showing an example of a degenerating axon. Large arrows indicate normal paranodal myelin loops [for β1 (+/+)] or paranodal loops that are everted or pulled away [for β1 (-/-)]. Small arrows indicate electron dense transverse bands. Scale bars are as indicated for each image.
Figure 9.
Figure 9.
Voltage-dependent properties of β1 (+/+) and β1 (-/-) mice. A, Sodium current traces in response to depolarizations to -30 mV (inward current) or +20 mV (outward current) from a holding potential of -80 mV from β1 (+/+) (left) or β1 (-/-) (right) mice. B, Mean normalized conductance-voltage curves (squares) and inactivation curves (circles) for wild-type (WT; open symbols) and β1 (-/-) (filled symbols) mice. To generate conductance-voltage relationships, 20 msec depolarizations from a holding potential of -80 mV to the indicated potentials were applied. Peak current versus voltage curves were measured and fit with[(V - Vrev) × Gmax]/{1 + exp[(V - V.5)/k]}, where V was the test pulse voltage, Vrev, the reversal potential, V.5 the half activation voltage, and k a slope factor. Normalized conductance-voltage curves for each experiment were determined as 1/{1 + exp[(V - V.5)/k]}, and the means ± SEM of these curves are plotted. Inactivation curves were measured using a 20 msec long prepulse to the indicated potentials, followed by a test depolarization to +10 mV. Mean normalized peak test pulse current is plotted versus prepulse potential.
Figure 10.
Figure 10.
β1 (-/-) mice show abnormal expression of Nav 1.1 and Nav 1.3 in the hippocampus. A, β1 (+/+) mice stained with anti-β1ex. B, β1 (-/-) mice illustrating the lack of anti-β1ex staining and the specificity of the null mutation. C, β1 (+/+) mice stained with anti-Nav1.1. D,β1(-/-) mice stained with anti-Nav1.1 illustrating lack of staining of many neurons in the CA2/CA3 region and in the outer leaflet of the dentate gyrus compared with controls (C). E, β1 (+/+) mice stained with anti-Nav1.3. F, β1 (-/-) mice stained with anti-Nav1.3 illustrating increased staining in interneurons of the hippocampus. G, Higher magnification of the dentate gyrus labeled with anti-Nav 1.3 antibodies illustrating increased staining in neurons located in this region of β1 (-/-) mice. H, Tissue section incubated with no primary antibodies to illustrate the specificity of antibody staining. Scale bars, 200 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Berglund EO, Murai KK, Fredette B, Sekerkova G, Marturano B, Weber L, Mugnaini E, Ranscht B (1999) Ataxia and abnormal cerebellar microorganization in mice with ablated contactin gene expression. Neuron 24: 739-750. - PubMed
    1. Bhat MA, Rios JC, Lu Y, Garcia-Fresco GP, Ching W, St Martin M, Li J, Einheber S, Chesler M, Rosenbluth J, Salzer JL, Bellen HJ (2001) Axonglia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin. Neuron 30: 369-383. - PubMed
    1. Boiko T, Rasband M, Levinson S, Caldwell J, Mandel G, Trimmer J, Matthews G (2001) Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon. Neuron 30: 91-104. - PubMed
    1. Boyle MET, Berglund EO, Murai KK, Weber L, Peles E, Ranscht B (2001) Contactin orchestrates assembly of the septate-like junctions at the paranode in myelinated peripheral nerve. Neuron 30: 385-397. - PubMed
    1. Burgess DL, Kohrman DC, Galt J, Plummer NW, Jones JM, Spear B, Meisler MH (1995) Mutation of a new sodium channel gene, Scn8a, in the mouse mutant “motor endplate disease.” Nat Genet 10: 461-465. - PubMed

Publication types

MeSH terms