Identification of an axonal determinant in the C-terminus of the sodium channel Nav1.2
2001; Springer Nature; Volume: 20; Issue: 21 Linguagem: Inglês
10.1093/emboj/20.21.5950
ISSN1460-2075
Autores Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle1 November 2001free access Identification of an axonal determinant in the C-terminus of the sodium channel Nav1.2 Juan José Garrido Juan José Garrido INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Fanny Fernandes Fanny Fernandes INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Pierre Giraud Pierre Giraud INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Isabelle Mouret Isabelle Mouret INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Eric Pasqualini Eric Pasqualini INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Marie-Pierre Fache Marie-Pierre Fache INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Florence Jullien Florence Jullien INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Bénédicte Dargent Corresponding Author Bénédicte Dargent INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Juan José Garrido Juan José Garrido INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Fanny Fernandes Fanny Fernandes INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Pierre Giraud Pierre Giraud INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Isabelle Mouret Isabelle Mouret INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Eric Pasqualini Eric Pasqualini INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Marie-Pierre Fache Marie-Pierre Fache INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Florence Jullien Florence Jullien INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Bénédicte Dargent Corresponding Author Bénédicte Dargent INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France Search for more papers by this author Author Information Juan José Garrido1, Fanny Fernandes1, Pierre Giraud1, Isabelle Mouret1, Eric Pasqualini1, Marie-Pierre Fache1, Florence Jullien1 and Bénédicte Dargent 1 1INSERM U464, Institut Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur-Nord, Boulevard P.Dramard, 13916 Marseille cedex 20, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5950-5961https://doi.org/10.1093/emboj/20.21.5950 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To obtain a better understanding of how hippocampal neurons selectively target proteins to axons, we assessed whether any of the large cytoplasmic regions of neuronal sodium channel Nav1.2 contain sufficient information for axonal compartmentalization. We show that addition of the cytoplasmic C-terminal region of Nav1.2 restricted the distribution of a dendritic–axonal reporter protein to axons. The analysis of mutants revealed that a critical segment of nine amino acids encompassing a di-leucine-based motif mediates axonal compartmentalization of chimera. In addition, the Nav1.2 C-terminus is recognized by the clathrin endocytic pathway both in non-neuronal cells and the somatodendritic domain of hippocampal neurons. The mutation of the di-leucine motif located within the nine amino acid sequence to alanines resulted in the loss of chimera compartmentalization in axons and of internalization. These data suggest that selective elimination by endocytosis in dendrites may account for the compartmentalized distribution of some proteins in axons. Introduction Neurons possess the remarkable ability to selectively target proteins to distinct axonal or somatodendritic domains. This leads to the asymmetric organization of the neuronal plasma membrane which is essential for vectorial communication. The mechanisms by which hippocampal neurons target proteins to dendrites are starting to be unraveled. Carrier vesicles containing dendritic proteins are sorted at the level of the trans-Golgi network (for review see Winckler and Mellman, 1999) and are excluded from axons (Burack et al., 2000). At present, three major groups of sorting signals that account for the vectorial trafficking of dendritic proteins have been identified. The first group includes tyrosine-based signals (Jareb and Banker, 1998), whereas the second group comprises di-leucine-based motifs (Poyatos et al., 2000). It is noteworthy that both of these sorting signals are also involved in protein targeting to the basolateral membrane of epithelial cells, such as the MDCK line (for review see Mellman, 1996). Thus, neurons can recognize the same motifs for somatodendritic targeting that epithelial cells use for basolateral targeting. In addition, novel somatodendritic sorting sequences falling outside these two defined groups have recently been characterized (West et al., 1997a; Stowell and Craig, 1999; Lim et al., 2000; Ruberti and Dotti, 2000). Whereas information on dendritic targeting signals is emerging, much less is known about the mechanisms that lead to the polarized distribution of axonal proteins in hippocampal neurons (Winckler and Mellman, 1999; Burack et al., 2000). Most of the proteins that are directed to apical membranes in epithelial cells are distributed both on dendrites and axons when expressed in hippocampal neurons, indicating that apical sorting signals are not recognized as axonal determinants (Jareb and Banker, 1998). It is thought that the sorting of proteins to axons is under the control of specific sequence motifs distinct from those ensuring dendritic targeting, but little information is available about their structure (Winckler and Mellman, 1999). In addition, the possibility remains open that mechanisms downstream of carrier vesicle transport operate to target proteins to axons. A recent study (Burack et al., 2000) involving the visualization of carrier vesicles containing green fluorescent protein-tagged NgCAM (neuron–glial cell adhesion molecules) has shown that this axonal membrane protein follows a non-vectorial trafficking pathway and is transported in vesicles within both axons and dendrites. This suggests that vesicles containing axonal proteins are not able to fuse with the dendritic membrane and are only inserted in axonal membranes. Alternatively, it has been proposed (Stowell and Craig, 1999; Winckler and Mellman, 1999; Burack et al., 2000) that insertion of axonal proteins into the somatodendritic domain occurs, but is followed by selective elimination; however, this has never been demonstrated. One of the major physiological roles of neuronal voltage-gated sodium channels is to generate action potentials at the axon hillock/initial segment, and to ensure propagation along myelinated or unmyelinated fibers to nerve terminals (Stuart et al., 1997; Catterall, 2000). Localization studies have shown that a member of the voltage-gated sodium channel family, Nav1.2, is preferentially distributed on fibers in adult rat brain (Westenbroek et al., 1989; Gong et al., 1999). Biochemical, developmental and genetic studies suggest that the cytoskeletal linker protein ankyrin G could play an important role in sodium channel clustering at the initial segment (for review see Bennett and Lambert, 1999). However, the upstream mechanisms that initially direct sodium channels to the axonal membrane remain poorly understood. During the early stages of the establishment of neuronal polarity, Nav1.2 was found to be preferentially distributed on axons of developing hippocampal neurons, and to display a high density at the initial axonal segment (Dargent et al., 1998). Molecular characterization in developing hippocampal neurons indicated that Nav1.2, the pore-forming α subunit, is associated with the auxiliary β2 subunit, but not the β1 subunit, in good agreement with previous studies (Scheinman et al., 1989; Gong et al., 1999). The observation that a myc-tagged β2 subunit expressed by transfection in hippocampal neurons was uniformly distributed on dendrites and axons (B.Sampo, J.A.Boudier, E.Carlier, F.Jullien, J.L.Boudier, A.Le Bivic, R.A.Maue and B.Dargent, submitted) eliminated the possibility that the sodium channel β2 subunit carries a dominant axonal sorting signal and suggested that Nav1.2 contains intrinsic information for axonal compartmentalization. Therefore, the goal of the present study was to examine this hypothesis. A strategy based on the expression of chimeras in hippocampal neurons demonstrated that the cytoplasmic C-terminal domain of Nav1.2 restricted the distribution of a dendritic–axonal reporter protein to axons. The analysis of mutants revealed a region of nine amino acids encompassing a di-leucine-based motif within the proximal region of the Nav1.2 C-terminus that determines axonal compartmentalization. Results The C-terminal cytoplasmic domain of Nav1.2 contains an axonal determinant In preliminary experiments, we expressed Nav1.2 tagged with a myc epitope in COS-7 cells. After cell permeabilization, significant immunostaining was observed intracellularly, but no labeling was revealed on non-permeabilized cells using an antibody directed against an extracellular epitope (not shown), indicating that the level of surface expression was too low for detection by immunofluorescence. Similar results were obtained when Nav1.2 was transfected in hippocampal neurons. To circumvent this problem, and taking into account the fact that targeting/clustering motifs have been identified within cytoplasmic regions of potassium channels (Zito et al., 1997; Lim et al., 2000) and of the AMPA receptor (Ruberti and Dotti, 2000), we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We therefore constructed chimeric proteins (Figure 1B) in which the cytoplasmic tail of the human CD4 receptor, a type I membrane protein, was replaced by loop I–II, loop II–III or the C-terminal cytoplasmic tail of Nav1.2. In parallel, we replaced the cytoplasmic N-terminus of the human transferrin receptor (hTfR), a type II membrane protein, with that of Nav1.2. The CD4–Nav1.2 loop III–IV chimera was not generated. This region is involved in fast channel inactivation, is relatively highly conserved (Catterall, 2000) and is thus unlikely to contain subtype-specific sorting signals. Figure 1.Expression of chimeric proteins containing the cytoplasmic regions of Nav1.2 in hippocampal neurons. (A) Schematic representation of the neuronal sodium channel Nav1.2. The N- and C-termini are located intracellularly. Four homologous domains (I–IV), each containing six transmembrane segments, are connected by large cytoplasmic loops (I–II, II–III and III–IV). (B) Schematic representation of the chimeric proteins. Because of the topology of the intracellular regions of Nav1.2, two types of chimera were generated. The N-terminus of the human transferrin receptor, a type II membrane protein, was replaced by the N-terminus of Nav1.2 (hTfR–Nav1.2Nt). The other chimeras were composed of the extracellular and transmembrane regions of human CD4 receptor and loop I–II (CD4–I–II), loop II–III (CD4–II–III) and the C-terminal region of Nav1.2 (CD4–Nav1.2Ct). (C) Expression of the chimeras in hippocampal neurons. The constructs indicated were transfected into seven DIV hippocampal neurons. Two days later, neurons were fixed, permeabilized, and double stained for MAP2 and either hTfR or CD4. The N-terminal chimera is localized only in the somatodendritic domain, while the loop I–II chimera is mostly concentrated in soma, and to a lower extent throughout the dendrites. Note that the chimera containing loop II–III was concentrated within the axon initial segment (arrow), whereas only the CD4–Nav1.2Ct chimera was immunodetected all along the axon (arrow). Bar, 50 μm. Download figure Download PowerPoint We first verified using either an anti-hTfR antibody or an anti-CD4 antibody that the chimeric proteins were efficiently transported and inserted into the plasma membrane of non-permeabilized COS-7 cells (not shown, but see Figure 4). Chimeras were next transiently expressed in hippocampal neurons and their expression was analyzed by immunostaining either before or after cell permeabilization to detect surface and total distribution. Discrimination between axonal and somatodendritic domains was based on staining for MAP2, a somatodendritic marker, and on morphological criteria. Axons were identified as MAP2-negative processes. Figure 1C shows chimera distribution in permeabilized hippocampal neurons. The TfR–Nav1.2Nt chimera was visualized only in the somatodendritic domain. CD4–I–II was mainly localized within the soma and some labeling was detected in dendrites but not in axons. CD4–II–III was also distributed in the soma and within the axonal initial segment. A lower staining was visualized in dendrites and in the distal region of axons. CD4–Nav1.2Ct was detected within the soma and all along axons. Vesicular staining in dendrites was also observed. We next examined the surface expression of the chimeras at the steady state on non-permeabilized hippocampal neurons. hTfRΔNt shows a non-polarized distribution when expressed in hippocampal neurons (West et al., 1997a). TfR–Nav1.2Nt was visualized on the somatodendritic domain, but not in axons (100% of hTfR-positive cells, n = 30, three independent experiments; not shown), indicating that this chimera is not able to enter axons. CD4ΔCt was detected both in the somatodendritic and axonal membranes (Figure 2A), whereas CD4–I–II and CD4–II–III chimeras were not immunodetected at the plasma membrane (not shown), in contrast to what was observed in COS-7 cells. As shown in Figure 2C, the addition of the C-terminal region of Nav1.2 to CD4ΔCt restricted the surface distribution of the chimeric protein to axons. CD4-labeled axons were thinner and much longer than dendrites, and extended far beyond the cell body of transfected cells, forming numerous branched processes. The polarized surface expression of CD4–Nav1.2Ct was very reproducible (Table I; Figure 3B), and its distribution was more pronounced distally than proximally (Figure 2E). Endogenous Nav1.2 subunits are preferentially distributed on axons of developing hippocampal neurons, and in particular at the initial segment where they co-localize with ankyrin G (Bennett and Lambert, 1999). A direct comparison of the immunostaining of endogenous Nav1.2 with that of CD4–Nav1.2Ct could not be carried out because the only anti-Nav1.2 antibody that provides satisfactory staining in our hands is directed against the C-terminus (Gong et al., 1999). Therefore, we used an antibody against ankyrin G, a marker of the initial axonal segment. Double immunostaining showed that CD4–Nav1.2Ct was not concentrated at the initial segment, but was generally located in more distal regions of the axon than ankyrin G (Figure 2E–G). Finally, when the cytoplasmic tail of CD4 was replaced by the cytoplasmic C-terminus of the auxiliary β2 subunit, the corresponding chimera CD4–β2Ct was uniformly distributed on dendrites and axons (Figure 2H). Figure 2.Addition of the C-terminus of Nav1.2 restricts the surface distribution of CD4 to axons. Seven DIV hippocampal neurons were transfected with CD4ΔCt (A), CD4–Nav1.2Ct (C and E) and CD4–β2Ct (H) constructs. After 48 h, neurons were fixed and incubated with an anti-CD4 antibody prior to permeabilization, then exposed either to an antibody against MAP2, a somatodendritic marker (B, D and I) or to an anti-ankyrin G antibody (F). CD4ΔCt (A) was distributed both in dendrites and axons (arrows). In contrast, addition of the Nav1.2 C-terminus (C) restricts chimera expression to axons (arrows), whereas the addition of β2 C-terminus did not (H). The expression of CD4–Nav1.2Ct (green; E) was detected in axonal domains distal to ankyrin G (red; G), which is localized in the initial segment. Bar, 50 μm. Download figure Download PowerPoint Figure 3.The proximal region of the Nav1.2 C-terminus governs the axonal compartmentalization of the CD4 chimera. (A) Amino acid sequence of the Nav1.2 C-terminus (1777–2005) showing the major deletions (Δ1828, Δ1851 and Δ1871). Internal deletion Δ1839–1859 is underlined and Δ1853–1862 is in bold. Di-leucine motifs are represented in italics. (B) Schematic representation of mutations generated in the C-terminus of Nav1.2. The percentage of transfected CD4-positive hippocampal neurons with axonal polarity is indicated, taking as 100% the total population of transfected neurons, i.e. CD4-positive neurons. Hippocampal neurons were considered as showing restricted axonal expression when a cell process displayed immunoreactivity to anti-CD4 antibodies, but staining was absent at the surface of dendrites and soma. Data are represented as the mean ± SD from six different experiments and n represents the total number of neurons analyzed for each chimera. (C) Surface expression of CD4–Nav1.2Ct mutants. Seven DIV hippocampal neurons were transfected with the indicated constructs. Two days later, they were double stained for CD4 (green) and MAP2 (red). The surface distribution of the truncated mutant Δ1871 was restricted to axons (arrows), indicating that the C-terminal extremity of Nav1.2 is not required for the polarized expression of the chimera. In contrast, the Δ1851 and Δ1828 mutants were distributed on soma and dendrites as well as in axons. The internal deletion of nine amino acids between 1853 and 1862 resulted in the loss of CD4–Nav1.2Ct restriction in axons. Download figure Download PowerPoint Figure 4.Internalization of CD4–Nav1.2Ct through the clathrin-dependent pathway in non-neuronal cell lines. COS-7 cells were transfected with CD4–Nav1.2Ct (A and B), CD4ΔCt (C) and CD4–I–II (L) constructs. Living cells were subjected to an immunoendocytosis assay for 20 min. CD4–Nav1.2Ct showed a diffuse membrane staining after the pre-labeling step with an anti-CD4 antibody at 4°C (A). Following 20 min of incubation at 37°C, antibody-prelabeled CD4–Nav1.2Ct (B) displayed a punctate intracellular distribution, whereas CD4ΔCt (C) and CD4–I–II (L) were confined to the cell surface. (D–I) Following an immunoendocytosis assay, COS-7 cells expressing CD4–Nav1.2Ct were exposed to either anti-AP2 (E) or anti-transferrin receptor (H) monoclonal antibodies. Vesicles containing CD4–Nav1.2Ct co-localized with AP2 (arrowheads, F) and with transferrin receptor (arrowheads, I). (J and K) HeLa cells expressing wild-type dynamin (J) or mutant K44A dynamin (K) were transfected with CD4–Nav1.2Ct and an immunoendocytosis assay similar to that described above. A representative confocal section of CD4–Nav1.2Ct staining for each condition is shown. In HeLa cells expressing wild-type dynamin, CD4–Nav1.2Ct is efficiently endocytosed (J, confocal section), whereas in cells expressing mutant dynamin the chimeric protein remains in the plasma membrane (K, confocal section). Bar, 25 μm. Download figure Download PowerPoint Table 1. Differential surface distribution of CD4 chimeras containing the C-terminal region of Nav1.1, Nav1.2 and Nav1.6 Axonal Somatodendritic Non-polarized CD4–Nav1.1 31 ± 8 69 ± 8 CD4–Nav1.2 89 ± 0.5 11 ± 0.5 CD4–Nav1.6 82 ± 18 27 ± 12 Data (mean ± SD) are expressed as percentages, taking as 100% the total population of transfected neurons, i.e. CD4-positive neurons from three (CD4–Nav1.1, n = 50; CD4–Nav1.6, n = 81) and six (CD4–Nav1.2, n = 50) different experiments. To ascertain further that the C-terminus of Nav1.2 contains specific information for axonal compartmentalization, we generated chimeras in which the C-terminus of CD4 was replaced by that of two other neuronal sodium channel types: Nav1.1 or Nav1.6 (Goldin, 1999). Table I shows that these chimeras displayed distinct steady-state distributions when expressed in hippocampal neurons. CD4–Nav1.1Ct was distributed on the somatodendritic and axonal membranes (69% of cells), whereas CD4–Nav1.6Ct was found to be located in the somatodendritic domain (82% of cells). Altogether, these results indicate that the C-terminus of Nav1.2 carries specific information that restricts the expression of an axonal–dendritic reporter protein to axons. Identification of the axonal determinant located within the C-terminal region of Nav1.2 The next step was to identify the molecular determinant(s) of the C-terminal of Nav1.2 implicated in the polarized expression of CD4–Nav1.2Ct. With this aim, we generated truncated mutants by PCR (Δ1974, Δ1915, Δ1892, Δ1871, Δ1851 and Δ1828; Figure 3B). The surface distribution of the mutant proteins was analyzed by immunostaining in transfected hippocampal neurons. Deletion of the C–terminal domain distal to residues 1974, 1915, 1892 or 1871 did not alter the compartmentalized expression of chimeric proteins. The results obtained with the Δ1871 chimera are illustrated (Figure 3); they demonstrate an absence of staining in dendrites, whereas an axonal localization was still observed. This indicates that the distal C-terminal region (residues 1871–2005) of the Nav1.2 subunit does not contain axonal determinants. In contrast, mutants Δ1851 and Δ1828 displayed a non-polarized distribution, as judged by their surface expression detected on both dendrites and axons (Figure 3B and C). Thus, a critical determinant located between amino acids 1851 and 1871 governs the polarized expression pattern of CD4–Nav1.2Ct. To define this determinant more precisely, we constructed two internal deletions (Δ1839–1859 and Δ1853–1862; Figure 3). Both mutants showed a non-compartmentalized expression in transfected seven DIV hippocampal neurons (Figure 3B and C). Thus, a motif of nine amino acids governs the axonal compartmentalization of CD4–Nav1.2Ct. CD4–Nav1.2Ct chimera undergoes internalization through the clathrin-dependent pathway At least two distinct pathways may underlie the polarized expression of CD4–Nav1.2Ct in hippocampal neurons. The protein could be delivered directly to axons. However, in permeabilized cells, dendrites were positive for CD4 staining (Figure 1C). Therefore, it is possible that CD4–Nav1.2Ct is initially transported and inserted into both domains, but is retrieved from the dendritic plasma membrane while it is retained in the axonal membrane. To examine whether the C-terminal tail of Nav1.2 is recognized by components of the endocytotic pathway, we first applied an immuno-endocytosis assay to COS-7 cells expressing the constructs. When the surface population of CD4–Nav1.2Ct was pre-labeled with an anti-CD4 antibody at 4°C, followed by a 20 min incubation at 37°C, it was found to be located exclusively in intracellular vesicles visualized by confocal microscopy (Figure 4A and B). The disappearance of diffuse membrane staining indicated that antibody-labeled CD4–Nav1.2Ct was efficiently retrieved from the plasma membrane. The typical endocytotic pattern was already detected after 10 min at 37°C and was resistant to an acid wash (not shown). Other chimeric proteins such as CD4ΔCt, CD4–I–II (Figure 4C and L) and CD4–β2Ct (not shown) were not internalized. We then examined whether the internalization of CD4–Nav1.2Ct was mediated by the clathrin-dependent pathway. Figure 4D–F shows that vesicles containing CD4–Nav1.2Ct co-localized with adaptor protein 2 (AP2), which is involved in clathrin-coated pit assembly (Marsh and McMahon, 1999). Co-staining with the transferrin receptor, a marker of recycling endosomes (Figure 4G–I), and with the early endosome antigen 1 (EEA1; not shown) was also observed. To confirm these findings, experiments were carried out in HeLa cell lines expressing either wild-type dynamin II or a defective dynamin mutant (K44A; Schmid, 1997). After 10 min of incubation at 37°C, CD4–Nav1.2Ct was efficiently internalized in cells expressing wild-type dynamin (Figure 4J). In contrast, the localization of the chimeric protein was restricted to the plasma membrane in the mutant cell line, due to the inability of dynamin K44A to retrieve clathrin-coated vesicles (Figure 4K). These data strongly indicate that at least one internalization motif located in the C-terminal region of Nav1.2 is efficiently recognized by the clathrin-dependent endocytotic machinery in non-neuronal cells. We next evaluated the possibility that CD4–Nav1.2Ct was also internalized when expressed in hippocampal neurons. A similar endocytosis assay was applied to transfected neurons, with the exception that the anti-CD4 antibody was added directly to the culture medium at 37°C. When visualized by confocal microscopy, antibody-labeled CD4–Nav1.2Ct was detected in vesicles distributed throughout dendrites and the cell body (Figure 5A). Some vesicles were also visualized in the proximal regions of axons, but not in more distal segments. In contrast, CD4ΔCt (Figure 5B) was not internalized, its localization being confined to the neuronal plasma membrane, like CD4–β2Ct (not shown). The presence of internalized CD4–Nav1.2Ct in endosomes throughout the somatodendritic domain was further confirmed by co-localization either with EEAI, a marker of somatodendritic early endosomes (Wilson et al., 2000) (Figure 5C–E), or with transferrin receptor and AP2 (not shown). Thus, these observations indicate that CD4–Nav1.2Ct undergoes internalization in the somatodendritic domain of hippocampal neurons. Figure 5.Internalization of CD4–Nav1.2Ct in the somatodendritic domain of hippocampal neurons. Seven DIV hippocampal neurons were transfected with CD4–Nav1.2Ct (A) or CD4ΔCt constructs (B). Following an immunoendocytosis assay, cells were fixed, permeabilized and incubated with anti-MAP2 antibody (red). CD4–Nav1.2Ct-containing vesicles (green) are distributed throughout dendrites (arrows) and the soma (box). Some punctate staining also appeared to be concentrated in the proximal portion of the axon (asterisk). In contrast, internalization of CD4ΔCt was not detected. Inserts represent 0.5 μm confocal sections of neuronal cell bodies showing the presence (A) or absence (B) of endocytotic vesicles. The axons of transfected cells are indicated by arrowheads. Hippocampal neurons expressing CD4–Nav1.2Ct (D) were subjected to an immunoendocytosis assay as described above. Following fixation and permeabilization, cells were exposed to an anti-EEA1 antibody (C). The overlay (E) shows that vesicles containing CD4–Nav1.2Ct (red) are also positive for EEA1 (green; arrows). Bar, 25 μm. Download figure Download PowerPoint A di-leucine motif acts as an axonal determinant and an internalization signal The nine amino acid sequence that is critical for CD4–Nav1.2Ct restriction to axons encompasses a potential di-leucine internalization motif (IL 1857–1858; Figure 3). Therefore, we next addressed a possible relationship between the internalization of CD4–Nav1.2Ct and its restriction to axonal membranes at steady state. We first analyzed the ability of truncated and internal mutants to undergo internalization in COS-7 cells (Figure 6). Confocal microscopy analysis showed that the Δ1871 mutant was still efficiently internalized in COS-7 cells (Figure 6A), and no significant cell surface signal was detectable 20 min after pre-labeling, as previously observed with wild-type CD4–Nav1.2Ct (Figure 4B). In contrast, expression of the mutant Δ1828 was confined to the cell surface (Figure 6C). In the case of the Δ1851 mutant, both surface and internalized populations were identified (Figure 6B), reflecting slower internalization kinetics compared with Δ1871 or the wild-type CD4–Nav1.2Ct sequence. The internal deletion of nine amino acids (Δ1853–1862) greatly reduced chimera internalization (Figure 6D). A chimera containing an internal deletion between amino acids 1839 and 1859 was also endocytosed less efficiently in
Referência(s)