The ataxia3 Mutation in the N-Terminal Cytoplasmic Domain of Sodium Channel Nav1.6 Disrupts Intracellular Trafficking (original) (raw)
Articles, Neurobiology of Disease
, Xiaoyang Cheng, Valerie Drews, David A. Buchner, Julie M. Jones, Monica J. Justice, Stephen G. Waxman, Sulayman D. Dib-Hajj and Miriam H. Meisler
Journal of Neuroscience 4 March 2009, 29 (9) 2733-2741; https://doi.org/10.1523/JNEUROSCI.6026-08.2009
Abstract
The ENU-induced neurological mutant ataxia3 was mapped to distal mouse chromosome 15. Sequencing of the positional candidate gene Scn8a encoding the sodium channel Nav1.6 identified a T>C transition in exon 1 resulting in the amino acid substitution p.S21P near the N terminus of the channel. The cytoplasmic N-terminal region is evolutionarily conserved but its function has not been well characterized. ataxia3 homozygotes exhibit a severe disorder that includes ataxia, tremor, and juvenile lethality. Unlike Scn8a null mice, they retain partial hindlimb function. The mutant transcript is stable but protein abundance is reduced and the mutant channel is not detected in its usual site of concentration at nodes of Ranvier. In whole-cell patch-clamp studies of transfected ND7/23 cells that were maintained at 37°C, the mutant channel did not produce sodium current, and function was not restored by coexpression of β1 and β2 subunits. However, when transfected cells were maintained at 30°C, the mutant channel generated voltage-dependent inward sodium currents with an average peak current density comparable with wild type, demonstrating recovery of channel activity. Immunohistochemistry of primary cerebellar granule cells from ataxia3 mice demonstrated that the mutant protein is retained in the _cis_-Golgi. This trafficking defect can account for the low level of Nav1.6-S21P at nodes of Ranvier in vivo and at the surface of transfected cells. The data demonstrate that the cytoplasmic N-terminal domain of the sodium channel is required for anterograde transport from the Golgi complex to the plasma membrane.
Introduction
Scn8a encodes the sodium channel Nav1.6, which is expressed in neurons of the CNS and PNS. In addition to its localization on cell bodies, dendrites, and unmyelinated axons, Nav1.6 is the predominant sodium channel at mature nodes of Ranvier in central and peripheral myelinated axons (Catterall et al., 2005; Meisler and Kearney, 2005). Nav1.6 contributes to persistent current, resurgent current and repetitive firing in cerebellar Purkinje cells and DRG neurons (Raman et al., 1997; Cummins et al., 2005; Rush et al., 2005; Levin et al., 2006; Chen et al., 2008). Mutations of Scn8a in the mouse can result in tremor, ataxia, and other movement disorders (Meisler et al., 2004). Mice with null mutations in Scn8a exhibit paralysis and lethality by P21. A mutation of human SCN8A was identified in a family with ataxia and cognitive impairment (Trudeau et al., 2006).
The pore-forming α subunit of voltage-gated sodium channels is comprised of four homologous protein domains, each with six transmembrane segments, whose sequences are highly conserved in vertebrate and invertebrate channels. The 24 transmembrane segments contain residues affecting ion specificity, voltage dependence, and rates of activation and inactivation (Catterall et al., 2005). Most pathogenic missense mutations responsible for human sodium channelopathies are located in the transmembrane segments and adjacent cytoplasmic linkers (George, 2005; Kearney et al., 2006; Dib-Hajj et al., 2007). The ∼130 residues of the sodium channel cytoplasmic N-terminal domain are also evolutionarily conserved, but few functions have been associated with this region of the protein. Five missense mutations in the N terminus of Nav1.1, between residues 78 to 102, have been identified in patients with epilepsy (Fujiwara et al., 2003; Nabbout et al., 2003; Fukuma et al., 2004; Harkin et al., 2007). These patient mutations alter residues of the N terminus that are conserved in Nav1.6, but the functional effects of the mutations have not been determined.
Screening of ENU-mutagenized mice for abnormal phenotypes and positional cloning of the mutated genes is an effective method for probing the in vivo functions of mammalian genes. We now report the positional cloning of an ENU-induced mutation that causes juvenile lethality in the ataxia3 mouse. Identification of a missense mutation in the cytoplasmic N terminus of Nav1.6 provided an opportunity to investigate the cellular role of this domain of the sodium channel protein.
Materials and Methods
Animals.
The ENU-induced neurological mutant ataxia3 was generated in the Developmental Mutagenesis Program at the Baylor College of Medicine (Kile et al., 2003). The mutant is formally designated MGI:2671787, nurm14Jus. Mutagenesis was performed in strain C57BL/6J background, and the mutant stock is maintained by crossing heterozygotes to C57BL/6J mice to preserve the homogeneous genetic background.
Genetic mapping.
Mapping was performed by crossing the mutant strain to the 129S6/SvEv inbred strain to the N2 generation. N2 siblings were intercrossed and DNA from 22 offspring were analyzed with microsatellite markers that map to all mouse autosomes and the X chromosome (Research Genetics). Significant linkage was detected to D15Mit42, and a total of 12 markers on Chromosome 15 were analyzed. Intercross statistics were evaluated using Map Manager QTL at p = 0.01 (Ken Manley, Roswell Park Cancer Institute, Buffalo, NY).
cDNA constructs.
The plasmid pcDNA3-Nav1.6R encodes the full-length mouse Nav1.6 with the amino acid substitution Y371S that renders the channel resistant to micromolar concentrations of tetrodotoxin (TTX-R) (Herzog et al., 2003; Cummins et al., 2005). The S21P mutation (c.61T>C) was introduced into NaV1.6R using the QuickChange XL II site-directed mutagenesis kit (Stratagene). The human β1 and β2 constructs (Lossin et al., 2002) were provided by Dr. A. L. George (Vanderbilt University, Nashville, TN).
Western blots.
The membrane fraction was prepared from brain homogenates by centrifugation at 100,000 × g as previously described (West et al., 1992). Western blots loaded with 50 μg of membrane protein were stained with anti-Nav1.6 antibody (1:200) from Sigma (S0438, lot 076K1101) as described (Levin and Meisler, 2004).
Northern blots.
Northern blots contained 2 μg of polyA+ RNA prepared from brain homogenates as previously described (Burgess et al., 1995). The probe was a 32P-labeled RT-PCR product corresponding to exons 1–6 of mouse Scn8a.
Immunohistochemistry of sciatic nerve.
Staining was performed as previously described (Kearney et al., 2002). Rabbit polyclonal anti-Nav1.6 (Sigma, #S0438, antiserum lot 076K1101) was diluted 1:100. Anti-Caspr (University of California, Davis/ National Institute of Neurological Disorders and Stroke/National Institute of Mental Health NeuroMab Facility, clone K65/35) was diluted 1:1000. The secondary antibodies were biotinylated goat anti-rabbit IgG (1:250; Jackson Laboratories) and anti-mouse-Alexa-488 (1:500; Invitrogen). Polyclonal antibody labeling was visualized with strepavidin–Alexa-568 (1:400; Invitrogen). Images were captured with an Olympus FluoView 500 Laser Scanning Confocal Microscope in the University of Michigan Microscopy and Image Analysis Laboratory.
Cell transfections.
The cell line ND7/23 was generated by fusion of DRG neurons with neuroblastoma cell line N18Tg2 (Wood et al., 1990). Wild type NaV1.6R and NaV1.6R-S21P mutant constructs were transfected into ND7/23 cells using Lipofectamine 2000 (Invitrogen) as described previously (Wittmack et al., 2004). Briefly, ND7/23 cells were plated on 10 mm coverslips in 24-well plates, flooded with 0.5 ml of DMEM plus10% FBS and incubated at 37°C for 40 h. Cells were cotransfected with wild-type NaV1.6R or S21P mutant constructs (0.8 μg/well) together with GFP (0.2 μg/well) (pEGFP, Clontech). Lipofectamine 2000 reagent (1.5 μl) and DNA (1 μg total) were each mixed with 50 μl of DMEM media (serum free, antibiotics free) and incubated at room temperature for 5 min. The two solutions were then combined and incubated at room temperature for 20 min. The mixture (100 μl per well) was added to the ND7/23 cell culture medium (0.5 ml of DMEM plus10% FBS per well) and incubated at 37°C for 3 h. After replacement with fresh DMEM supplemented by 10% FBS, the cells were incubated in 95% air-5% CO2 for 18 h at 37°C or 30°C (low temperature) before electrophysiological measurements.
Electrophysiology.
Whole-cell voltage-clamp recordings were performed at room temperature (20−22°C) using an Axopatch 200B amplifier (Axon Instruments). ND7/23 cells with robust green fluorescence signal were selected for patch-clamp. Fire-polished electrodes (0.6–1.3 MΩ) were fabricated from 1.6 mm outer diameter borosilicate glass micropipettes (World Precision Instruments). The pipette potential was adjusted to zero before seal formation, and liquid junction potential was not corrected. Capacity transients were canceled and voltage errors were minimized with 80% series resistance compensation. Currents were acquired with Clampex 9.2 at 6 min after establishing whole-cell configuration, sampled at a rate of 50 or 100 kHz, and filtered at 5 kHz.
For current-voltage relationships, cells were held at −120 mV and stepped to a range of potentials (−65 to + 60 mV in 5 mV increments) for 100 ms each. Peak inward currents (I) were plotted as a function of depolarizing potential to generate I–V curves. Activation curves were obtained by converting current (I) to conductance (G) at each voltage (V) using the equation G = I/(V −_V_rev), where _V_rev is the reversal potential which was determined for each cell individually. Activation curves were then fit with Boltzmann function in the form of G = G_max/(1 + exp[(V_1/2,act− V)/k]), where _G_max is the maximal sodium conductance, _V_1/2,act is the potential at which activation is half-maximal, V is the test potential, and k is the slope factor.
Steady-state fast inactivation was achieved with a series of 500 ms prepulses (−140 to −10 mV in 10 mV increments), and the remaining available channels were activated by a 20 ms test pulse to −10 mV. Peak inward currents obtained from steady-state fast-inactivation protocols were normalized to the maximal peak current (_I_max) and fit with Boltzmann functions: I/_I_max = 1/(1 + exp[(V − _V_1/2,inact)/_k_]), where V represents the inactivating prepulse potential, _V_1/2,inact represents the midpoint of inactivation curve.
The pipette solution contained (in mm): 140 CsF, 10 NaCl, 1 EGTA, 10 dextrose, and 10 HEPES, pH 7.35 (adjusted with CsOH), and the osmolarity was adjusted to 315 mOsmol/L with sucrose. The extracellular bath solution contained (in mm): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 20 TEACl, 5 CsCl, 0.1 CdCl2, 10 HEPES, pH 7.35 (adjusted with NaOH), and the osmolarity was 328 mOsmol/L. Tetrodotoxin (TTX; 300 nm) was added to the bath solution to block endogenous voltage-gated sodium currents in ND7/23 cells (Zhou et al., 2003; John et al., 2004; Wittmack et al., 2004; Rush et al., 2005; Wittmack et al., 2005), permitting currents from wild type Nav1.6 or S21P mutant channels to be recorded in isolation.
Data analysis.
Data were analyzed using Clampfit 9.2 (Molecular Devices) and Origin 7.5 (Microcal Software), and presented as means ± SE. The Kruskal–Wallis nonparametric test was used to analyze current density data. One way ANOVA was used to assess the statistical significance of changes in characteristics of channel activation and inactivation.
Gradient isolation and culture of cerebellar granule cells.
Cerebellar granule cells were prepared and cultured from 14- to 18-d-old mice (P14–18) by modification of previously described methods (Brewer, 1997). Each brain was removed and placed in 2 ml of ice-cold Hibernate A (BrainBits) supplemented with B27 (Invitrogen) and 0.5 mm l-glutamine. The cerebellum was dissected and diced into smaller pieces. The tissue was incubated in a warm trypsin/Hibernate solution for 30–40 min with agitation every 5–10 min, and then transferred to 2 ml of fresh HibernateA/B27/l-glutamine and triturated 10 times with a P1000 pipette tip. After addition of 2 ml of HibernateA/B27/l-glutamine, the tissue was allowed to settle. Aliquots (2 ml) of supernatant were removed to a 15 ml conical tube. Trituration was repeated 3 times. 8 ml of the triturated cell suspension was placed on a 4 ml step gradient prepared from 1 ml aliquots of 35%, 25%, 20% and 15% (v/v) Optiprep (Sigma):HepesNaCl, 1:1, in HibernateA/B27/l-glutamine. The cell suspension was centrifuged at 800 × g for 15 min. Cell debris floating above the 4 ml gradient was discarded. The bottom two 2 ml, excluding the pellet, were resuspended in 12 ml of NeurobasalA Full media (Neurobasal A/0.5 mm l-glutamine). To 50 ml of suspension was added 1 ml of B27 supplement, 10 μl 50 mg/ml gentamicin and 2.5 μl of b-FGF (Invitrogen) prepared by resuspending 10 μg of b-FGF in 100 μl 10 mm Tris pH 1.7 containing 0.1% BSA. The suspension was centrifuged at 1500 × g and the cell pellet was resuspended in 4 ml of Neurobasal A Full media. The cells were plated on glass coverslips coated with poly-l-lysine (Sigma) and laminin (Sigma) and incubated at 37°C, 5% CO2. Seventy-five percent of the medium was changed 24 h after isolation and this was repeated every 3–4 d.
Immunohistochemistry of cerebellar granule cells.
Neurons were fixed in 4% PFA in PBS for 15 min with rocking at room temperature, washed, and permeabilized in 0.1% Triton, 6% BSA for 30 min at room temperature. The cells were blocked in 10% normal goat serum in PBS for 30 min at room temperature. All antibodies were diluted in Wash Buffer (1% BSA, 0.1% Triton X-100 in PBS). Primary antibodies were incubated overnight with rocking at 4°C. Secondary antibodies were incubated at room temperature for 1 h. Although the rabbit polyclonal anti-Nav1.6 (Sigma #SO438) generates specific staining of nodes of Ranvier (see above and Kearney et al., 2002), this antibody produced high background staining of null cerebellar granule cells that lack Nav1.6 (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Therefore, mouse Nav1.6 in cultured cells was detected with anti-Nav1.6 Na+ channel, clone K87A/10 mouse monoclonal IgG (Neuromab Facility, lot number: 440-8HK-09) used at 1:100 dilution. The secondary antibody was anti-mouse-Alexa-488 (1:500; Invitrogen). The ER marker, protein disulfide isomerase (PDI), was detected with rabbit PDI Polyclonal Antibody (Assay Designs) used at 1:500 dilution. The secondary antibody was goat-anti-rabbit Alexa 594 (1:800, Invitrogen). The _cis_-Golgi marker, GM130, was detected with mouse monoclonal anti-GM130 used at 1:20 dilution (BD Transduction Laboratories). The secondary antibody was goat-anti-mouse Alexa 594 (1:800, Invitrogen). For double-labeling with monoclonal anti-Nav1.6 and monoclonal GM130, the anti-Nav1.6/Alexa 488 staining was completed first and followed by anti-GM130/Alexa 594 staining. Neurofilament-200 was detected with polyclonal anti-Neurofilament-200 (Sigma, product no. N 4142) used at 1:80 dilution. The secondary antibody was goat-anti-rabbit Alexa 594 (1:800, Invitrogen). Cell nuclei were stained with 1 μg/ml DAPI (Invitrogen) in PBS for 15 min. Coverslips were mounted on microscope slides with Prolong Gold (Invitrogen) and dried overnight. Images were captured with an Olympus FluoView 500 Laser Scanning Confocal Microscope in the University of Michigan Microscopy and Image Analysis Laboratory.
Results
Phenotype of the ataxia3 mutant
The ataxia3 mutant was identified in a large-scale mutagenesis program after administration of the mutagen ENU to mice of strain C57BL/6J. A G3 breeding scheme was used to detect recessive mutations with visible phenotypes (Kile et al., 2003). The ataxia3 mutant was recognized by its locomotor abnormalities that include pronounced tremor, ataxic gait, and partial loss of hindlimb function. Affected animals have impaired use of their hindlimbs by 2 weeks of age (Fig. 1A), and do not survive beyond 1 month (Fig. 1B). In the initial crosses between heterozygous carriers, we observed 27 affected animals among a total of 106 offspring, consistent with autosomal recessive inheritance and complete penetrance of the phenotype (p = 0.99).
Figure 1.
Phenotype and mapping of the ataxia3 mutant. A, Impaired hindlimb function in ataxia3 homozygote. B, Juvenile lethality of the ataxia3 homozygote. C, the ataxia3 mutation maps to the distal region of mouse chromosome 15. The haplotypes for 22 chromosomes from 11 F2 mice are indicated. Solid symbols, B6 alleles; open symbols, 129 alleles. The ataxia3 mutation did not recombine with the markers D15Mit193, D15Mit42 and D15Mit14.
Genetic mapping of ataxia3 to mouse chromosome 15
To map the mutation, C57BL/6J.ataxia3/+ heterozygotes were crossed to strain 129S6/SvEvTac for two generations. Heterozygous mice from the N2 generation were intercrossed. Twenty- two offspring from the intercross were genotyped with microsatellite markers. The ataxia3 locus was nonrecombinant with three markers on distal chromosome 15: D15Mit193, D15Mit42 and D15Mit14 (Fig. 1C). The observed gene order and recombination distances (cM ± SE) were as follows: D15Mit228 - 14 ± 5 - D15Mit33,D15Mit96 - 4.6 ± 3 - ataxia3,D15Mit42,D15Mit14,D15Mit193.
Identification of a mutation in Scn8a encoding sodium channel Nav1.6
The nonrecombinant markers for the ataxia3 locus are located between 55 and 58 cM on mouse chromosome 15, while the Scn8a gene encoding Nav1.6 is located at 60 cM. The tremor, ataxia, and hindlimb impairment in ataxia3 mice strongly resemble the phenotypes of known mutants of Scn8a (Meisler et al., 2004). We therefore evaluated Scn8a as a positional candidate gene for ataxia3. The Nav1.6 cDNA was amplified by RT-PCR as previously described (Buchner et al., 2004) and the products were gel-purified and sequenced. The nucleotide substitution c.61T>C (GenBank NM_001077499) was identified in the ataxia3 cDNA (Fig. 2A). The destruction of a _Hinf_1 site by the T>C mutation can be used for genotyping the mutant mice (Fig. 2C). The resulting substitution of serine residue 21 by proline (p.S21P) is located close to the N terminus of the channel (Fig. 2B). Serine residue 21 is evolutionarily conserved in vertebrate sodium channels including the muscle channel Nav1.4 and the cardiac channel Nav1.5, and also in invertebrate sodium channels, suggesting a conserved function common to these channels (Fig. 2D). The retention of some hindlimb function in the ataxia3 mice (see above) indicates that Nav1.6-S21P is not a null allele, since the null mice develop complete paralysis of the hindlimbs (Meisler et al., 2004).
Figure 2.
The ataxia3 mutation causes a Ser –>Pro substitution at a conserved residue of Nav1.6. A, Sequence chromatogram demonstrating the T –> C mutation in ataxia3. B, Location of S21P in the cytoplasmic N-terminal domain of Nav1.6. C, Genotyping assay based on loss of a _Hinf_1 restriction site due to the T>C mutation in ataxia3. D, Evolutionary conservation of serine 21 in the human sodium channel gene family (SCN1A-SCN11A) and invertebrate sodium channels.
Noncomplementation of ataxia3 and a null allele of Scn8a
To demonstrate that the Nav1.6-S21P mutation is responsible for the mutant phenotype of the mice, we tested the ability of the mutant allele to complement the recessive lethality of the null allele Scn8atg1 (Burgess et al., 1995). Heterozygous ataxia3/+ mice were crossed with null heterozygotes. The Scn8aS21P/tg1 compound heterozygotes were born in the predicted Mendelian ratio of 24% (5/22) but none of the compound heterozygotes survived beyond weaning. All of the compound heterozygotes exhibited the tremor and ataxia that are characteristic of each homozygous mutant. The failure of the ataxia3 mutant to rescue the motor abnormalities and lethality of a null allele of Scn8a, together with the phenotypic similarity of ataxia3 to Scn8a mutants and the genetic mapping of ataxia3 near Scn8a, strongly support the identification of Nav1.6-S21P as the causal mutation in ataxia3 mice.
Abundance and localization of Nav1.6-S21P in mutant mice
To determine whether the c.61T>C mutation destabilizes the Scn8a transcript, we prepared a Northern blot with polyA+ RNA from brain and hybridized it with a Nav1.6 cDNA probe. The size and abundance of the 10 kb mRNA were comparable in the wild-type and homozygous ataxia3 RNA (Fig. 3A). Both samples also contain the minor Scn8a transcript of 12 kb that terminates at an alternative polyadenylation site (Drews et al., 2005)
Figure 3.
Nav1.6 transcript and protein in ataxia3 mice. A, A Northern blot containing 3 μg of polyA+ RNA from wild-type and homozygous ataxia3 brain was hybridized with a radiolabeled 1 kb cDNA fragment from Nav1.6. B, Western blot containing 50 μg of membrane protein from wild-type and homozygous ataxia3 brain homogenates was stained with polyclonal anti-Nav1.6 antiserum as described (Levin and Meisler, 2004). The blot was stained with Ponceau S to visualize total protein (bottom). C, Nav1.6 at nodes of Ranvier. Teased sciatic nerve was stained with polyclonal anti-Nav1.6 antiserum as described (Kearney et al., 2002). Red, anti-Nav1.6; green, anti-Caspr. Images were captured with an Olympus FluoView 500 Laser Scanning Confocal Microscope. As a quantitative positive control, nodal Nav1.6 is visible in the homozygous medJ mutant in which channel protein is reduced to 10% of wild-type levels (Kearney et al., 2002; Buchner et al., 2003).
To examine the abundance of the Nav1.6 channel protein, membrane protein was purified from ataxia3 and wild-type mouse brain. Western blotting with anti-Nav1.6 antibody detected a low level of full-length Nav1.6 protein in homozygous ataxia3 brain (Fig. 3B). The protein is not detected in the null control (Fig. 3B). The abundance of Nav1.6 protein in the homozygous ataxia3 mutant is greatly reduced in comparison with heterozygote and wild-type brain (Fig. 3B).
To determine whether Nav1.6-S21P is present at the nodes of Ranvier, where the normal channel is highly concentrated, sciatic nerves were dissected, teased onto poly-l-lysine coated coverslips and immunostained for Nav1.6 and the paranodal marker Caspr as previously described (Kearney et al., 2002). Nodal Nav1.6 was readily detected in nerves from ataxia3/+ heterozygotes that produce 50% of the normal level of wild-type channel (Fig. 3C). In contrast, Nav1.6 protein could not be detected at any nodes in sciatic nerve from ataxia3 mice (Fig. 3C). As a control, we were able to detect Nav1.6 in nodes from sciatic nerve of Scn8amedJ/medJ mice which produce 10% of the wild-type level of channel protein (Fig. 3C) (Kearney et al., 2002). The absence of Nav1.6 at the nodes of Ranvier in ataxia3 mice is consistent with a defect in trafficking of the S21P protein.
Channel activity in transfected ND7/23 cells
Electrophysiological measurement of channel activity is a sensitive method for detection of functional sodium channels at the cell surface. To determine whether functional Nav1.6-S21P channels are present at the cell membrane, we used an assay in which ND7/23 cells, a DRG-derived cell line, are transfected with a TTX-resistant version of wild-type Nav1.6 cDNA (NaV1.6R) with the amino acid substitution Y371S (Herzog et al., 2003). In the presence of 300 nm TTX, endogenous sodium currents in ND7/23 cells are blocked and currents derived from the transfected wild-type NaV1.6R and its derivatives can be studied in isolation (see Materials and Methods).
ND7/23 cells were cotransfected with EGFP and NaV1.6R or the derivative NaV1.6R-S21P and analyzed by whole-cell voltage clamp electrophysiology. Transfected cells with robust green fluorescence were recorded. All cells that formed whole-cell configuration were included in the calculation of current density, with peak current measured at −5 mV. Activation and inactivation properties were analyzed for the subset of cells with large currents (_I_Na > 1 nA).
Most (26/30) cells transiently transfected with wild type Nav1.6R and incubated at 37°C produced a TTX-R current >200 pA, the smallest Na current that could be unequivocally attributed to the recombinant sodium channels. The Nav1.6R channels exhibited the expected current-voltage relationship (Fig. 4A). The average peak current density of wild-type NaV1.6R channels at −5 mV was 84.9 ± 17.5 pA/pF (n = 30) (Fig. 4B), with _V_1/2 of activation −18.3 ± 1.2 mV and _V_1/2 of steady-state fast inactivation of −67.7 ± 1.0 mV (n = 12) (Fig. 4C), in agreement with our previous reports (Wittmack et al., 2004, 2005; Rush et al., 2005).
Figure 4.
Electrophysiological characterization of NaV1.6 in ND7/23 cells. A, Representative family traces of Na+ currents from ND7/23 cells transfected with either wild-type NaV1.6R or NaV1.6R-S21P mutant channels. WT37, wild-type channel and incubation at 37°C; S21P37, mutant channel and incubation at 37°C; WT30, wild-type channel and cells incubated at 30°C; S21P30, mutant channel and cells incubated at 30°C. Cells were held at −120 mV and Na+ currents were elicited by step depolarizations from −65 to + 60 mV in 5 mV increments every 5 s. B, Peak current densities of wild-type NaV1.6R and S21P channels in cells that were incubated at 37°C or 30°C. WT37, n = 30; S21P37, n = 18; WT30, n = 21; S21P30, n = 28). C, Boltzmann's fit for voltage-dependent activation of Na+ currents from ND7/23 cells transfected with wild-type NaV1.6R channels at 37°C (n = 12) and 30°C (n = 13); and S21P mutant channels at 30°C (n = 17). D, Boltzmann's fit for voltage-dependent steady-state fast inactivation of Na+ currents from ND7/23 cells transfected with wild-type NaV1.6R channels and incubated at 37°C (n = 12) or 30°C (n = 13), and S21P mutant channels from cells incubated at 30°C (n = 17). Cells were held at −120 mV and a series of 500 ms prepulses from −140 mV to −10 mV were evoked followed by 40 ms test pulses to −10 mV.
In contrast to the observations with the wild-type construct, 17 of 18 cells transfected with S21P mutant channels failed to produce currents >200 pA (Fig. 4A,B). One cell produced a 550 pA peak current, resulting in an average peak current density for S21P mutant channels of 5.8 ± 1.5 pA/pF (mean ± SE) (n = 18).
We tested the ability of cotransfected β1 and β2 subunits to rescue the channel activity of the S21P construct. The majority of ND7/23 cells transfected with wild-type Nav1.6, β1 and β2 subunits (7 of 8) produced sodium currents with average current density = 86.7 ± 21.8 pA/pF (n = 8). In contrast, only 2 of 14 cells expressing NaV1.6R-S21P produced sodium currents, and the average current density was only 6.9 ± 0.8 pA/pF (n = 14). Thus overexpression of β1 and β2 subunits did not rescue the activity of NaV1.6-S21P mutant channels. These experiments indicate that under physiological conditions, Nav1.6-S21P does not exhibit channel activity, possibly due to failure to reach the cell surface.
Rescue of NaV1.6-S21P channel activity at low temperature (30°C)
Incubation of cells at lower temperature can permit the production of functional protein from mutants that are defective in protein folding or transport (Ulloa-Aguirre et al., 2004; Rusconi et al., 2007). To test the hypothesis that impaired trafficking is responsible for the lack of functional Nav1.6-S21P channels at the cell surface at 37°C, we incubated ND7/23 cells at 30°C for 18 h after transfection with the mutant cDNA. The reduction in incubation temperature did not alter the level of functional wild-type NaV1.6R, and 19/21 transfected cells produced voltage-dependent inward currents with an average peak current density of 173.1 ± 28 pA/pF (n = 21) (Fig. 4A,B). Under these culture conditions, the S21P mutant channel also exhibited robust activity: 26 of 28 transfected cells generated voltage-dependent inward currents with an average peak current density of 123.8 ± 14.3 pA/pF (n = 28) (Fig. 4; Table 1). The restoration of channel activity at 30°C is consistent with the model that Nav1.6-S21P retains normal biophysical properties, but the channel is not transported to the cell surface at 37°C.
Table 1.
Summary of electrophysiological characterization of wild-type Nav1.6 and S21P mutant channels
Analysis of Boltzmann fits for channel activation and steady-state fast inactivation of WT channels did not show a temperature-dependent effect on these properties. No significant difference was observed on the voltage dependence of activation and steady-state fast inactivation of WT channels at 30°C (_V_1/2,activation = −18.6 ± 1.2 mV, _V_1/2,inactivation = −69.0 ± 1.1 mV, n = 13) (Fig. 4C,D), compared with the values at 37°C (Table 1). The S21P mutation did not change the voltage dependence of channel activation (_V_1/2,activation = −20.5 ± 1.1 mV, n = 17) (Fig. 4C) compared with WT channels. However the S21P mutation caused a small but statistically significant −3.7 mV hyperpolarizing shift in steady-state fast inactivation (Fig. 4D) (_V_1/2,inactivation = −72.7 ± 1.1 mV, n = 17, p < 0.05 vs wild-type NaV1.6R at 30°C).
Subcellular localization of mutant Nav1.6-S21P protein
To determine the intracellular location of Nav1.6-S21P protein that fails to reach the cell surface we isolated cerebellar granule cells from ataxia3 and wild-type mice at P14–18 and maintained them in culture at 37°C for 16 d. During this period, isolated neurons redifferentiate and regenerate neuronal processes and their components (Osorio et al., 2005; Van Wart and Matthews, 2006).
After 1 week in culture, both wild-type and mutant Nav1.6 protein colocalized with the _cis_-Golgi marker GM130 (Fig. 5A). This localization is consistent with the high carbohydrate content of voltage-gated sodium channels (Waechter et al., 1983; Schmidt and Catterall, 1987). Neither wild-type nor mutant Nav1.6 was colocalized with the endoplasmic reticulum marker α-protein disulfide isomerase (PDI) (Fig. 5B). These data indicate that Nav1.6-S21P escapes the ER quality control system and is transported to the Golgi apparatus during biosynthesis.
Figure 5.
Subcellular localization of Nav1.6 in cerebellar granule cells after 1 week of culture. A, Costaining of Nav1.6 (green) and GM130, a marker for the _cis_-Golgi (red). B, Costaining of Nav1.6 (green) and α-protein disulfide isomerase (PDI), a marker of the endoplasmic reticulum (ER) (red). Cell nuclei are stained with DAPI (blue). Images were captured with an Olympus FluoView 500 Laser Scanning Confocal Microscope. Scale bars, 20 μm. a3, ataxia3.
After 16 d in culture, abundant neuronal processes were detected in both wild-type and mutant cultures by staining for neurofilament-200 (Fig. 6A, left panel). In the wild-type neurons (401 of 717), most of the Nav1.6 protein was located in the cell soma and processes, while in the ataxia3 neurons (263 of 279), the Nav1.6-S21P retained a perinuclear localization (Fig. 6A, middle and right panels). Costaining with the _cis_-Golgi marker GM130 confirmed the complete retention of Nav1.6-S21P in the Golgi apparatus (Fig. 6B). The data demonstrate failure of anterograde transport of Nav1.6-S21P out of the Golgi complex. This biosynthetic defect can account for the failure Nav1.6-S21P to reach the cell surface in transfected cells and its absence from nodes of Ranvier in vivo.
Figure 6.
Subcellular localization of Nav1.6 in cerebellar granule cells after 16 d of culture. A, left panel, abundant neuronal processes in wild-type and ataxia3 mutant cells visualized by staining for neurofilament. middle and right, representative images of wild-type and mutant cells. B, Costaining of Nav1.6 (green) and the _cis_-Golgi marker (red). In the mutant ataxia3 cells, Nav1.6 remains localized in the Golgi. Cell nuclei are stained with DAPI (blue). Images were captured with an Olympus FluoView 500 Laser Scanning Confocal Microscope. Scale bars, 20 μm. a3, ataxia3.
Discussion
We have identified and characterized a novel missense mutation of mouse Scn8a, p.Ser21Pro, that results in juvenile lethality in vivo. S21P is located in an evolutionarily conserved portion of the cytoplasmic N-terminal domain that is present in neuronal, cardiac and muscle channels and in invertebrate sodium channels, suggesting that this domain participates in a cellular function that is common to all of these sodium channels. Our evidence for a role in intracellular trafficking is consistent with this expectation. The mutated serine residue 21 is located at one end of a predicted α helix (www.predictprotein.org), and substitution of proline for serine at this position could destabilize the helix. Alternatively this mutation may eliminate a site of protein phosphorylation, although the surrounding sequence is not a good match for any consensus motif for protein phosphorylation, with a predicted phosphorylation potential of only 0.14 (http://ca.expasy.org). Full-length Nav1.6-S21P protein was detected at reduced abundance on Western blots, but it could not be detected at nodes of Ranvier by immunostaining. In transfected cells, Nav1.6-S21P did not exhibit channel activity at 37°C, but activity was rescued when transfected cells were maintained at 30°C. The data are consistent with the view that the mutant protein is trapped within an intracellular compartment at 37°C and does not reach the cell membrane.
Pathogenic mutations that impair intracellular trafficking have been identified of the cardiac channel SCN5A (Nav1.5) in patients with Brugada syndrome (Valdivia et al., 2004; Poelzing et al., 2006; Tan et al., 2006) and the neuronal channel SCN1A (Nav1.1) in a patient with Severe Myoclonic Epilepsy of Infancy (Rusconi et al., 2007). These mutations were not located in the N-terminal domain, and were shown to result in reduced or absent inward sodium currents in heterologous expression systems such as HEK 293 cells. The channel activity of these mutants has been rescued by incubation of transfected cells at reduced temperatures, treatment with channel blockers, or over-expression of β-subunits (Valdivia et al., 2004; Poelzing et al., 2006; Tan et al., 2006; Pfahnl et al., 2007; Rusconi et al., 2007). Lowering the incubation temperature is thought to slow the kinetics of protein folding, allowing more time for the mutant proteins to acquire a functional conformation before sorting to the cell membrane (Ulloa-Aguirre et al., 2004). In the case of the Nav1.6-S21P mutation, lower temperature appeared to rescued protein trafficking while overexpression of β-subunits did not.
To determine the fate of the mutant Nav1.6-S21P protein, we examined primary cultures of cerebellar granule cells from ataxia3 and wild-type mice. In wild-type neurons after 1 week in culture, most of the Nav1.6 protein was located in the Golgi complex. This is the first demonstration that Nav1.6, which is thought to be a glycoprotein, accumulates in the Golgi during redifferentiation of immature neurons in culture. After an additional 9 d in culture, wild-type Nav1.6 was distributed throughout the soma and neuronal processes, while Nav1.6-S21P was completely retained in the Golgi apparatus. These observations lead to the specific hypothesis that mutation of the cytoplasmic N-terminal domain prevents anterograde trafficking of channel protein out of the _cis_-Golgi, perhaps by preventing association with protein chaperones. Chaperones involved in the processing of sodium channels in the Golgi have not been identified, and the Nav1.6-S21P mutant will be a useful reagent for identifying the interacting protein(s) required for anterograde transport of Nav1.6 from the Golgi complex to the cell membrane.
Mutations in the N terminus of two other ion channels have been associated with retention in the Golgi. Deletion of residues 1–76 from the N terminus of Kir2.1 results in Golgi accumulation (Stockklausner and Klocker, 2003). Amino acid substitutions at residues 12 and 13 of the α-adrenergic receptor protein also prevented delivery to the cell surface and resulted in accumulation in the Golgi (Dong and Wu, 2006). Comparison of the primary sequences of these three channels did not identify a shared N-terminal sequence.
Accessory proteins that bind to the cytoplasmic domains of sodium channels can influence channel density at the cell surface (Cusdin et al., 2008). Binding of annexin II light chain (p11) between residues 74 and 103 of the N terminus of Nav1.8 (Poon et al., 2004) increases the channel density at the surface of DRG neurons (Okuse et al., 2002). Binding of FGF13, a member of the fibroblast growth factor family, to the C terminus of Nav1.6 also results in increased current density (Wittmack et al., 2004). β subunit binding sites are present in the extracellular portion of domain 4 (Catterall et al., 2005) as well as the cytoplasmic C terminus (Spampanato et al., 2004), and interaction with β subunits can enhance targeting and trafficking of sodium channel α subunits to the cell surface (Chen et al., 2002, 2004; Lopez-Santiago et al., 2006). A trafficking-defective mutation in the C terminus of Nav1.1 was rescued by overexpression of β subunits (Rusconi et al., 2007). The lack of rescue of Nav1.6-S21P by β subunits indicates that the N terminus interacts with a different class of proteins.
This work has demonstrated that the neuromuscular disorder in ataxia3 mice is a consequence of deficiency of Nav1.6 at the cell membrane, resulting from retention in the Golgi complex. In contrast to Nav1.6 null mice, which exhibit complete hindlimb paralysis, the ataxia3 mutants retain a small amount of hindlimb function, suggesting that there is a low level of functional Nav1.6-S21P in vivo. This is consistent with the observation of channel activity in a small proportion of cells transfected with the mutant channel at 37°C, and the detection of a small amount of channel protein on Western blots of membrane protein from mutant brain. The amount of functional protein in vivo can be estimated by comparison with other Scn8a mutants. Mice with the genotype Scn8amedJ/medJ produce 10% of normal Nav1.6 and survive to adulthood (Kearney et al., 2002; Meisler et al., 2004). Mice with the genotype Scn8a medJ/medJ, Scnm1R187X/R187X produce 5% of normal channel protein and do not survive beyond 3 weeks, but they do retain partial hindlimb function like the ataxia3 mice (Kearney et al., 2002). Mice with 1.5% of normal levels of Nav1.6 exhibit complete hindlimb paralysis and lethality at 3 weeks (Howell et al., 2008) (Howell et al., 2008). The phenotype of the ataxia3 mice suggests that ∼5% of Nav1.6-S21P reaches the cell surface in vivo.
The identification of a mutation that impairs transport of Nav1.6 out of the Golgi complex opens several new avenues of investigation. It will be feasible to screen for proteins whose interaction with the N terminus is prevented by the S21P mutation, and to determine whether trafficking of the mutant channel from the Golgi is rescued by sodium channel blockers such as phenytoin, lidocaine or mexiletine that can function as pharmacological chaperones in cultured cells (Valdivia et al., 2004; Rusconi et al., 2007; Zhao et al., 2007). The ataxia3 mutation defines a new function of the cytoplasmic N terminus of Nav1.6 and provides an in vivo model for testing pharmacological rescue of ion channelopathies caused by trafficking defects.
Footnotes
- This work was supported by National Institutes of Health Grants R01 NS34509 (M.H.M.), U01 HD39372, and R01 CA115503 (M.J.J.), and a postdoctoral fellowship from the University of Michigan Center for Genetics in Health and Disease (L.M.S.). S.D.D.-H. and S.G.W. were supported by the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs and by grants from the National Multiple Sclerosis Society. We thank Chris Dinh and Andrew Salinger for technical assistance with genetic mapping, and the Isom laboratory for advice regarding neuronal cell culture.
- Correspondence should be addressed to Miriam H. Meisler, 4909 Buhl Box 5618, University of Michigan, Ann Arbor, MI 48109-5618. meislerm{at}umich.edu