Article |
Address correspondence to Bénédicte Dargent, INSERM UMR 641, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P. Dramard, 13916 Marseille cedex 20, France. Tel.: 33-491 698 859. Fax: 33-491 090 506. email: dargent.b{at}jean-roche.univ-mrs.fr
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Abstract |
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Key Words: axonal initial segment; ankyrin G tethering; sorting; neuronal polarity; sodium channels
J. J. Garrido's present address is Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, 28049 Cantoblanco, Spain.
Abbreviations used in this paper: AIS, axonal initial segment; [AIS], AIS concentration; BFA, brefeldin A; CAM, cell adhesion molecules; DA, distal axon; MAP2, microtubule associated protein 2; PB, phosphate buffer; SD, somatodendritic.
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Introduction |
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Little is known about the biogenesis of the AIS, but information has recently emerged indicating the pivotal role of the cytoskeletal adaptor complex ankyrin G/ß IV spectrin (Bennett and Baines, 2001; Salzer, 2003). The expression of either ankyrin G (Zhou et al., 1998; Jenkins and Bennett, 2001) or ß IV spectrin (Komada and Soriano, 2002) is mandatory for the assembly of molecules of the L1 CAM family and sodium channels. At the molecular level, sodium channels from rat brain are composed of an subunit, the pore-forming protein Nav1, and the auxiliary subunits, ß2 or ß4 and ß1 or ß3 (Catterall, 2000; Isom, 2001; Salzer, 2003; Yu et al., 2003). This complex molecular organization has hampered dissection of targeting and/or clustering motifs of sodium channels. Therefore, we developed an approach based on CD4 chimera expression in cultured hippocampal neurons to assess whether any of the large intracellular regions of Nav1.2 contained sufficient information for sorting and specific membrane organization. Using this approach, we have recently shown that sodium channel targeting and clustering at the AIS is specified by a motif within the cytoplasmic loop linking homologous domains IIIII (the IIIII linker) of the Nav1 proteins (Garrido et al., 2003). This signal was sufficient to relocalize the somatodendritic potassium channel Kv2.1 at the AIS of hippocampal neurons (Garrido et al., 2003). When the ankyrin binding motif of neurofascin (Davis and Bennett, 1994), a member of L1 CAMs, was replaced by the IIIII linker of sodium channel Nav1.2, the resulting neurofascinsodium channel chimera was concentrated at the AIS of hippocampal neurons (Lemaillet et al., 2003). This process involves a direct association with the ankyrin repeat domain of ankyrin G (Lemaillet et al., 2003).
The upstream events leading to membrane protein segregation within the AIS have yet to be dissected (Winckler and Mellman, 1999). Taking into account very recent studies on axonal sorting, several models are conceivable, that may not be mutually exclusive. One scenario is that an AIS protein is nonselectively inserted in the plasma membrane of both somatodendritic and axonal domains and subsequently eliminated in the dendrites, the soma, and in the distal part of the axon by endocytosis where it is tethered by ankyrin G at the AIS. Such a multistep process would be coordinated by independent molecular determinants, i.e., internalization and tethering motifs. It is significant that somatodendritic endocytosis has been shown to be involved in the axonal compartmentalization of a CD4 chimera bearing the COOH terminus of Nav1.2 (Garrido et al., 2001) and more recently, of the synaptic protein VAMP2 (Sampo et al., 2003). In each case, the abrogation of the internalization signal impaired axonal polarization (Garrido et al., 2001; Sampo et al., 2003). A second possibility is that an AIS protein is selectively sorted and inserted in the axonal domain, as observed in the case of NgCAM, the avian homologue of L1 (Sampo et al., 2003). After lateral diffusion, a fraction is tethered at the AIS whereas the distal population is eliminated by endocytosis. Alternatively, membrane proteins preassembled with ankyrin G can be selectively sorted to the AIS, as a consequence of polarized transport along microtubules involving KIF5, a member of the kinesin super family (Nakata and Hirokawa, 2003). Finally, the possibility that segregation of a given protein at the AIS involves both direct routing and transcytosis cannot be excluded. For example, Sampo et al. (2003) have shown that NgCAM is selectively routed to axons via a targeting motif localized in its extracellular domain. Wisco et al. (2003) have shown that NgCAM is preferentially inserted in the somatodendritic domain and subsequently sorted to the axons by transcytosis, a process mediated by an internalization motif located in the cytoplasmic COOH terminus of NgCAM.
The present study was aimed at analyzing the trafficking of a CD4 chimera bearing the IIIII linker of Nav1.2 (CD4-Nav1.2 IIIII) in hippocampal neurons, to obtain new insights into the mechanisms that determine protein segregation in the AIS. At the steady-state, the surface distribution of CD4-Nav1.2 IIIII is restricted to the AIS of transfected hippocampal neurons (Garrido et al., 2003). We show here that CD4-Nav1.2 IIIII is preferentially inserted in the somatodendritic domain but is subsequently eliminated by endocytosis, whereas it is accumulated at the AIS by a diffusion trap due to the high concentration of ankyrin G. Ankyrin G tethering involves a conserved glutamate residue within the sodium channel clustering motif whereas endocytosis is governed by a segment located in the NH2 terminus of linker IIIII of Nav1.2.
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Results |
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Kinetics of insertion of CD4-Nav1.2 IIIII in the plasma membrane of hippocampal neurons
We next examined whether the insertion of newly synthesized CD4-Nav1.2 IIIII occurs preferentially in the somatodendritic domain or in the axonal domain. With this aim, we looked at the time course of cell surface insertion of CD4-Nav1.2 IIIII in hippocampal neurons by using brefeldin A (BFA) to block Golgi apparatus trafficking (Cid-Arregui et al., 1995; Jareb and Banker, 1997). After a 4-h posttransfection period, hippocampal neurons were treated overnight with BFA, resulting in protein accumulation in the Golgi apparatus. After removal of BFA, the cell surface distribution of CD4-Nav1.2 IIIII was visualized by immunostaining at different time intervals (Fig. 6 A). At t = 0, upon BFA removal and after cell permeabilization, an intracellular accumulation was observed in the perinuclear region (unpublished data) and staining that was consistent with BFA block. After 30 min of recovery, CD4 surface staining was visualized on the somatodendritic membrane with an equivalent signal at the AIS in the majority of cells (53%). This cell population was designated somatodendritic (SD)-AIS. A second population SD-[AIS], with brighter CD4 staining at the AIS was also observed in 24% of the cells. At this stage of recovery, no signal was visualized on the distal part of axons (DA; Fig. 6 A). These two cell populations were also predominant 60 min after BFA removal but decreased markedly afterwards. Concomitantly, CD4-Nav1.2 IIIII appeared in the distal region of axons. A surface distribution in both domains with an enrichment at the AIS (SD-[AIS]-DA) was predominant after 4 and 8 h of recovery (82 and 88%, respectively). Restriction at the AIS, as observed in the absence of BFA treatment, began to be preponderant 24 h after removal (Fig. 6 A). In parallel, we determined the kinetics of the insertion of CD4-Nav1.2 IIIII E1111A (Fig. 6 B), a mutant that is no longer segregated at the AIS (Fig. 1). After 30 min of recovery, the E1111A mutant was visualized on the somatodendritic membrane with an equivalent signal at the AIS in 81% of cells (cell population designated SD-AIS). At this stage of recovery, no signal was visualized on the distal part of axons, similar to that observed with CD4-Nav1.2 IIIII (Fig. 6 A). A surface distribution in both domains (SD-A), but with no enrichment at the AIS was largely predominant after 2 h of recovery (77%). These observations indicated that CD4-Nav1.2 IIIII is preferentially inserted in the somatodendritic membrane and in the AIS, where it is enriched, and subsequently in the distal part of the axons.
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Discussion |
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The time course of the appearance of CD4-Nav1.2 IIIII at the cell surface revealed that the neo-synthesized protein is preferentially inserted in the somatodendritic domain rather than in distal regions of axons. This finding is unlikely to result from a side effect of BFA block because it has been shown recently that the initial insertion of membrane proteins in axonal tips is not perturbed by this type of treatment (Wisco et al., 2003). We also observed that an uniform somatodendritic distribution preceded enrichment at the AIS. Previous studies have indicated that the accumulation of membrane proteins like sodium channels and neurofascin at the AIS is coordinated by ankyrin G (Jenkins and Bennett, 2001) and is impaired by inactivation of either ankyrin G or ß IV spectrin gene expression (Zhou et al., 1998; Komada and Soriano, 2002). When the IIIII linker of Nav1.2 was fused to a cytosolic protein like the green fluorescent protein, the resulting chimera was trapped within the AIS (Garrido et al., 2003). Moreover, the AIS motif of Nav1.2 contains a 9-amino acid ankyrin binding site, highly conserved within the sodium channel Nav1 family (Lemaillet et al., 2003). A single glutamate residue (Nav1.2 E 1111) within this motif plays a critical role in CD4-Nav1.2 IIIII tethering to cytoskeleton at the AIS. Substitution of alanine or glutamine for this residue resulted in a loss of chimera segregation at the AIS, a finding underlining the functional impact of removing the charge. Consistently, CD4 chimeras bearing the IIIII linkers of Nav 1.6 (Garrido et al., 2003) and 1.8 were concentrated at the AIS and resistant to Triton X-100 extraction. Their accumulation at the AIS was abolished by mutation of the equivalent glutamate residue to alanine (unpublished data). Besides, in cultured hippocampal neurons, ankyrin B, which contains the conserved ankyrin repeat domain that binds to sodium channels (Bennett and Lambert, 1999; Lemaillet et al., 2003), is distributed in distal regions of axons, a localization distinct from that of ankyrin G. However, at the steady-state, CD4-Nav1.2 IIIII was restricted at the AIS. For all these reasons, it is tempting to propose that CD4-Nav1.2 IIIII diffuses laterally in the somatic plasma membrane and is subsequently trapped by the cystokeleton adaptor complex ankyrin G-ß IV spectrin, at the AIS. A similar mechanism could be involved in NgCAM accumulation. This axonal protein, that possesses the highly conserved ankyrin binding site of the L1 family (Garver et al., 1997), is initially incorporated in the somatodendritic domain of hippocampal neurons (Wisco et al., 2003). This interpretation is consistent with the fact that perturbation of the diffusion barrier formed by the AIS results in a somatic localization of NgCAM and sodium channels (Winckler et al., 1999; Nakada et al., 2003).
Despite the importance of anchoring, we show here that cytoskeleton tethering, presumably by ankyrin G-ß IV spectrin, is not sufficient for CD4-Nav1.2 IIIII segregation at the AIS, endocytosis is also required. The internalized population of CD4-Nav1.2 IIIII was observed throughout the soma and dendrites and to a lesser extent, in the distal part of axons. It was never seen within the AIS, in all observed cells. The presence of endocytotic vesicles in these regions is consistent with the steady-state distribution of CD4-Nav1.2 IIIII. The requirement for endocytosis was further demonstrated by fact that a mutation (1010-1030) that abolished internalization of CD4-Nav1.2 IIIII altered its steady-state distribution without inhibiting accumulation at the AIS. Consistent with this conclusion is the observation that CD4-Nav1.8 IIIII, which was not internalized either in COS-7 cells or in hippocampal neurons, was distributed at the steady-state in soma, dendrites, and axons, and was enriched at the AIS. These findings also indicate that the internalization of CD4-Nav1.2 IIIII is not involved in its accumulation at the AIS but rather in its elimination in the somatodendritic domain and in the distal part of axons. They imply that transcytosis is unlikely to be involved in the accumulation of CD4-Nav1.2 IIIII at the AIS. However, we cannot exclude the possibility that the presence of internalized CD4-Nav1.2 IIIII in the distal part of axons may reflect transcytosis from the somatodendritic domain to the axonal tips, as described for NgCAM (Wisco et al., 2003). The sequence that governs endocytosis of CD4-Nav1.2 IIIII does not encompass a canonic internalization signal such as the di-leucine or tyrosine-based motifs (Yxx
) recognized by the clathrin-mediated endocytotic pathway (Bonifacino and Traub, 2003). Hence, further investigation will be required to analyze the endocytotic pathway recognized by the IIIII linker of Nav1.2. A differential regulation of endocytosis in the somatodendritic versus the axonal domain cannot be excluded.
In conclusion, our present study shows that endocytosis and domain-selective tethering confers CD4-Nav1.2 IIIII segregation at the AIS. However, whether the Nav1.2 sodium channel follows a similar trafficking pathway remains to be explored. Multiple mechanisms probably play a role in establishing polarized sorting to the AIS. For instance, it is conceivable that ankyrin G-ß IV spectrin may also act during sorting, involving preassembly with sodium channels and the CAM family. Consistent with this hypothesis is the recent observation that when RNAi was used to eliminate expression of Nav1.2 in cultured neurons, it resulted not only in a loss of sodium channels but also perturbed ankyrin G compartmentalization at the AIS (unpublished data). Hence further investigations are required for a better understanding of the molecular and cellular processes involved in the construction of the AIS.
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Materials and methods |
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Cell culture and transfection
Primary hippocampal neurons were prepared from embryonic day 18 rats according to Goslin and Banker (1989) with slight modifications (Garrido et al., 2001). Neurons were transfected after 7 to 9 d in vitro using Lipofectamine 2000 (Invitrogen) (Garrido et al., 2003). COS-7 cells were cultured as described previously (Garrido et al., 2001) and transfected at a 80% confluence with Lipofectamine 2000, according to the manufacturer's instructions. Transfected cells were processed for immunofluorescence 24 h after transfection.
BFA treatment
Neurons (9 d in vitro) were transfected as described above. After 4 h, brefeldin A (Sigma-Aldrich) was added to yield 0.75 µg/ml final concentration in conditioned medium. After 14 h, coverslips were washed three times in conditioned medium and returned to the air/CO2 incubator at 37°C. Coverslips were removed, fixed at the indicated times, and processed for immunostaining. At t = 0, before washing, 10% of the transfected neurons displayed CD4 staining at the cell surface (n = 1,600, four independent experiments).
Immunofluorescence
To immunodetect the steady-state surface distribution of the chimeras in neurons, cells were fixed in 4% paraformaldehyde for 20 min. Nonspecific binding was blocked with 0.22% gelatin in 0.1 M phosphate buffer (PB). Cells were incubated for 1 h with anti-CD4 antibodies (monoclonal, 1:1,000; a gift from Jean Mérot, INSERM U533, Nantes, France); polyclonal T4-4, 1:1,500, National Institutes of Health AIDS and Reference Reagent Program). After a permeabilization step (0.066% saponin, 0.22% gelatin in PB), endogenous proteins were immunodetected either with anti-ankyrin G antibodies (monoclonal, 1:30; Zymed Laboratories; polyclonal 1:60, a gift from Gisèle Alcaraz, INSERM UMR 641, Marseille, France) or with an anti-MAP2 monoclonal, (1:400; Sigma-Aldrich) The secondary antibodies were goat antimouse or goat antirabbit conjugated to Alexa 563 or Alexa 488 (1:800; Molecular Probes). Coverslips were mounted in Mowiol.
Detergent extraction
Neurons were preincubated with anti-CD4 antibody at 16°C for 30 min, washed, and incubated in extraction buffer (30 mM Pipes, 1 mM MgCl2, 5 mM EDTA, 0.5% Triton X-100) for 10 min at 37°C as described previously (Garrido et al., 2003). Then, neurons were rinsed in PB, fixed, and processed by immunofluorescence.
Immunoendocytosis assay
COS-7 cells were exposed to the primary antibody diluted in PB with 0.22% gelatin for 45 min at 4°C, extensively washed, and then transferred to preheated DME medium and returned to the air/CO2 incubator at 37°C for 20 min. Cells were then fixed, permeabilized, and subjected to secondary antibody binding. For double immunostaining, a monoclonal anti-EEA1 antibody (1:400; Transduction Laboratories) was added to permeabilized cells followed by secondary antibodies. To dissociate membrane bound antibody before fixation, cells were incubated 10 min at 4°C in 0.2 M acetic acid and 0.5 M NaCl.
Neurons were incubated with primary antibody in glial-conditioned medium supplemented with 0.1% BSA for 30 min at 37°C, washed, and fixed. After the permeabilization step, cells were processed as described above. For differential staining of cell surface and internalized populations, endocytosis was stopped by transferring neurons to DME-Hepes at 16°C. Living cells were then exposed to Alexa 488conjugated secondary antibody for 40 min at 16°C, washed, fixed, and further incubated 30 min with an excess of unlabeled secondary antibody (0.2 mg/ml diluted in PB with 0.22% gelatin). After a 15-min postfixation step, neurons were permeabilized and incubated with Alexa 563conjugated secondary antibody.
Confocal microscopy
Labeling was viewed with a confocal laser scanning microscope (model TCS; Leica). Optical sections taken in the xy plane from consecutive z positions every 0.450.5 µm, using the standard microscope software (Scanware; Leica). Images were processed with Adobe Photoshop software.
Online supplemental material
Fig. S1 shows the alignment of the NH2-terminal region of the linker IIIII sequences of voltage-dependent sodium channels. Fig. S2 shows that the cytoplasmic IIIII linker of Nav1.8 is not recognized by an endocytotic pathway in transfected COS-7 cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200312155/DC1.
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Acknowledgments |
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This work was supported by INSERM and the "Association Francaise contre les Myopathies" grants and the "Fondation pour la Recherche Médicale" (M.-P. Fache and J.J. Garrido).
Submitted: 23 December 2003
Accepted: 6 July 2004
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References |
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Bennett, V., and A.J. Baines. 2001. Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 81:13531392.
Bennett, V., and S. Lambert. 1999. Physiological roles of axonal ankyrins in survival of premyelinated axons and localization of voltage-gated sodium channels. J. Neurocytol. 28:303318.[CrossRef][Medline]
Boiko, T., A. Van Wart, J.H. Caldwell, S.R. Levinson, J.S. Trimmer, and G. Matthews. 2003. Functional specialization of the axon initial segment by isoform-specific sodium channel targeting. J. Neurosci. 23:23062313.
Bonifacino, J.S., and L.M. Traub. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395447.[CrossRef]
Catterall, W.A. 2000. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron. 26:1325.[Medline]
Cid-Arregui, A., R.G. Parton, K. Simons, and C.G. Dotti. 1995. Nocodazole-dependent transport, and brefeldin Asensitive processing and sorting, of newly synthesized membrane proteins in cultured neurons. J. Neurosci. 15:42594269.[Abstract]
Davis, J.Q., and V. Bennett. 1994. Ankyrin binding activity shared by the neurofascin/L1/NrCAM family of nervous system cell adhesion molecules. J. Biol. Chem. 269:2716327166.
Garrido, J.J., F. Fernandes, P. Giraud, I. Mouret, E. Pasqualini, M.P. Fache, F. Jullien, and B. Dargent. 2001. Identification of an axonal determinant in the C-terminus of the sodium channel Na(v)1.2. EMBO J. 20:59505961.
Garrido, J.J., P. Giraud, E. Carlier, F. Fernandes, A. Moussif, M.P. Fache, D. Debanne, and B. Dargent. 2003. A targeting motif involved in sodium channel clustering at the axonal initial segment. Science. 300:20912094.
Garver, T.D., Q. Ren, S. Tuvia, and V. Bennett. 1997. Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin. J. Cell Biol. 137:703714.
Goslin, K., and G. Banker. 1989. Experimental observations on the development of polarity by hippocampal neurons in culture. J. Cell Biol. 108:15071516.[Abstract]
Isom, L.L. 2001. Sodium channel beta subunits: anything but auxiliary. Neuroscientist. 7:4254.
Jareb, M., and G. Banker. 1997. Inhibition of axonal growth by brefeldin A in hippocampal neurons in culture. J. Neurosci. 17:89558963.
Jenkins, S.M., and V. Bennett. 2001. Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments. J. Cell Biol. 155:739746.
Kobayashi, T., B. Storrie, K. Simons, and C.G. Dotti. 1992. A functional barrier to movement of lipids in polarized neurons. Nature. 359:647650.[CrossRef][Medline]
Komada, M., and P. Soriano. 2002. ßIV-spectrin regulates sodium channel clustering through ankyrin-G at axon initial segments and nodes of Ranvier. J. Cell Biol. 156:337348.
Lemaillet, G., B. Walker, and S. Lambert. 2003. Identification of a conserved ankyrin-binding motif in the family of sodium channel alpha subunits. J. Biol. Chem. 278:2733327339.
Nakada, C., K. Ritchie, Y. Oba, M. Nakamura, Y. Hotta, R. Iino, R.S. Kasai, K. Yamaguchi, T. Fujiwara, and A. Kusumi. 2003. Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization. Nat. Cell Biol. 5:626632.[CrossRef][Medline]
Nakata, T., and N. Hirokawa. 2003. Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head. J. Cell Biol. 162:10451055.
Peles, E., and J.L. Salzer. 2000. Molecular domains of myelinated axons. Curr. Opin. Neurobiol. 10:558565.[CrossRef][Medline]
Salzer, J.L. 2003. Polarized domains of myelinated axons. Neuron. 40:297318.[Medline]
Sampo, B., S. Kaech, S. Kunz, and G. Banker. 2003. Two distinct mechanisms target membrane proteins to the axonal surface. Neuron. 37:611624.[Medline]
Wilson, J.M., M. de Hoop, N. Zorzi, B.H. Toh, C.G. Dotti, and R.G. Parton. 2000. EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts. Mol. Biol. Cell. 11:26572671.
Winckler, B., and I. Mellman. 1999. Neuronal polarity: controlling the sorting and diffusion of membrane components. Neuron. 23:637640.[CrossRef][Medline]
Winckler, B., P. Forscher, and I. Mellman. 1999. A diffusion barrier maintains distribution of membrane proteins in polarized neurons. Nature. 397:698701.[CrossRef][Medline]
Wisco, D., E.D. Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch, and B. Winckler. 2003. Uncovering multiple axonal targeting pathways in hippocampal neurons. J. Cell Biol. 162:13171328.
Yu, F.H., R.E. Westenbroek, I. Silos-Santiago, K.A. McCormick, D. Lawson, P. Ge, H. Ferriera, J. Lilly, P.S. DiStefano, W.A. Catterall, et al. 2003. Sodium channel beta4, a new disulfide-linked auxiliary subunit with similarity to beta2. J. Neurosci. 23:75777585.
Zhou, D., S. Lambert, P.L. Malen, S. Carpenter, L.M. Boland, and V. Bennett. 1998. AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing. J. Cell Biol. 143:12951304.