Multiple molecular interactions determine the clustering of Caspr2 and Kv1 channels in myelinated axons

Abstract

Clustering of Kv1 channels at the juxtaparanodal region (JXP) in myelinated axons depends on their association with the Caspr2/TAG-1 adhesion complex. The interaction between these channels and Caspr2 was suggested to depend on PDZ (PSD-95/Discs large/zona occludens-1) scaffolding proteins. Here, we show that at a subset of the JXP, PSD-93 colocalizes with Caspr2, K(+) channels and its related protein postsynaptic density protein-95 (PSD-95). The localization of PSD-93 and PSD-95 depends on the presence of Caspr2, as both scaffolding proteins failed to accumulate at the JXP in mice lacking either Caspr2 or TAG-1. In contrast, Caspr2 and K(+) channels still colocalized and associated in PSD-93, PSD-95 or double PSD-93/PSD-95 null mice. To directly evaluate the role of PDZ domain proteins in the function of Caspr2, we examined the ability of transgenic Caspr2 molecules lacking either their cytoplasmic domain (Caspr2dCT), or their PDZ-binding sequence (Caspr2dPDZ), to restore Kv1 channel clustering in Caspr2 null mice. We found that while Kv1 channels were distributed throughout internodes in nerves expressing Caspr2dCT, they were clustered at the JXP in axons expressing a full-length Caspr2 (Caspr2FL) or the Caspr2dPDZ transgene. Further proteomic analysis revealed that Caspr2 interacts with a distinct set of scaffolding proteins through its PDZ- and protein 4.1-binding sequences. These results demonstrate that while the molecular assembly of the JXP requires the cytoplasmic domain of Caspr2, its carboxy-terminal PDZ-binding motif is dispensable for Kv1 channel clustering. This mechanism is clearly distinct from the one operating at the axon initial segment, which requires PSD-93 for Kv1 channel clustering.

Figure 1.

PSD-93 is found at the JXP of myelinated axons. A–D, Immunofluorescence labeling of teased adult rat sciatic nerves using antibodies to PSD-93 (A), PSD-95 (B), SAP97 (C), and SAP102 (D). The nodes of Ranvier (A, B) and paranodal junction (C, D) were labeled with antibodies to Na⁺ channels (NaCh) and Caspr, respectively. E, Immunolabeling of dorsal and ventral roots with antibodies to PSD-93, Kv1.2 and βIV spectrin. Labeling for βIV spectrin and PSD-93 is shown in the top, while the labeling for βIV spectrin and Kv1.2 is shown in the bottom as indicated. Note that PSD-93 immunoreactivity is detected in all Kv1.2-positive JXP in the dorsal but not the ventral roots. F–H, At the juxtaparanodal region, PSD-93 colocalizes with Kv1.2 (F), Caspr2 (G) and PSD-95 (H). I–K, Teased sciatic nerves isolated from wild-type (WT), PSD-93 null (PSD-93^−/−), or Caspr null mice (Caspr^−/−), were immunolabeled for PSD-93 (red), Na⁺ channels (green) and Caspr (blue). The organization of the nodal environ is schematically shown on the right. JX, Juxtaparanodes; PN, paranodes; ND, nodes of Ranvier; IN, internodes. Scale bars, 10 μm.

Figure 2.

Juxtaparanodal accumulation of PSD-93 depends on Caspr2 and TAG-1. Immunofluorescence labeling of teased sciatic nerves isolated from wild-type (WT; A, D, G, J), Caspr2 null (Caspr2^−/−; B, E, H, K), or TAG-1 null mice (TAG1^−/−; C, F, I, L) using the following antibody combinations: A--C, Caspr2 (red), Na⁺ channels (green) and Caspr (blue); D--F, TAG-1 (red) and Caspr (blue); G--I, PSD-93 (red), Na⁺ channels (green) and Caspr (blue); J–L, PSD-95 (red), Na⁺ channels (green) and Caspr (blue). Scale bar, 10 μm.

Figure 3.

K⁺ channels localized at the JXP and are associated with Caspr2 in mice lacking PSD-93 and PSD-95. Immunofluorescence labeling of teased sciatic nerves and optic nerve sections obtained from wild-type (WT; A, E), PSD-93 null (PSD-93^−/−; B, F), PSD-95 mutant (PSD-95^−/−; C, G), or double PSD-93/PSD-95 mutant mice (PSD-93^−/−/PSD-95^−/−; D, H), using antibodies to Kv1.2 (red), Na⁺ channels (green) and Caspr (blue). Insets in A--D show staining for Caspr2 (red), Na⁺ channels (green) and Caspr (blue). Insets in E--H show high magnification staining of Kv1.2 (red) and Na⁺ channels (green). Scale bar, 10 μm. I, Caspr2, Kv1.2, and Kv2.1 were immunoprecipitated from brain membrane lysates prepared from wild-type (WT), Caspr2 null (Caspr2^−/−), PSD-93 null (PSD-93^−/−), PSD-95 mutant (PSD-95^−/−), or double PSD-93/PSD-95 mutant mice (PSD-93^−/−/PSD-95^−/−) as indicated. From one brain that was used for each set of immunoprecipitation, 45% of the lysate was used for Kv1.2 and Kv2.1, whereas 10% was used for Caspr2. Protein complexes were resolved on SDS-gel and blotted with an antibody to Caspr2. The location of molecular mass markers is shown on the left in KDa.

Figure 4.

Characterization of Caspr2 transgenes. A, Schematic presentation of the different Caspr2 constructs used. The cytoplasmic domain of Caspr2 contains a 4.1 (red square) and a PDZ (blue circle)-binding sequences. C2FL encodes for a full-length HA-tagged (yellow square) Caspr2, whereas the C2dPDZ and C2dCT constructs encode for a Caspr2 lacking the PDZ-binding and the entire cytoplasmic region, respectively. B, Caspr2 transgenes reach the cell surface. Cell surface biotinylation was performed using HEK-293 cells (none), cells expressing an HA-tagged Caspr (C1HA), or HEK-293 cells expressing the indicated Caspr2 transgenes. Cell lysates were subjected to immunoprecipitation with an antibody to HA-tag followed by immunoblotting with the same antibody (bottom) or with streptavidin-HRP (top). The location of molecular mass markers is shown on the left in KDa. C1HA served as a control for a protein that does not reach the cell surface. C, COS-7 cells expressing the indicated transgenes, were labeled with an antibody to the extracellular domain of Caspr2 in either the absence (−) or the presence (+) of Triton X-100 (Tx100) as indicated. Cell nuclei are labeled with Dapi (Blue). Scale bar, 30 μm.

Figure 5.

Caspr2FL and Caspr2dPDZ colocalize and associate with K⁺ channels. A, Genomic PCR analysis. Tail DNA isolated from the animals indicated was used as a template for PCR using primer sets that recognize the transgene (tg), ceramide galactosyltransferase (Cgt), Caspr2 or neomycin (Neo). B, Expression of transgenic transcripts in DRG neurons. mRNA isolated from DRGs from the animals indicated were used as a template for RT-PCR using primer sets that recognize the transgene (tg), Caspr2 or GAPDH as a control. C, Expression of Caspr2 transgenes. Brain lysates were prepared from wild-type mice (WT), mice lacking Caspr2 (KO), or the indicated Caspr2 transgenic expressed on the Caspr2 null background. Immunoprecipitation (IP) was performed using an antibody to HA-tag (HA) or to the cytoplasmic domain of Caspr2 (CT), followed by immunoblotting with antibodies to HA-tag, the cytoplasmic domain (Caspr2-CT) or the extracellular domain (Caspr2-ECD) of Caspr2 as indicated below each panel. D, Association of Caspr2 transgenes with K⁺ channels. Brain membrane lysates prepared from the mice lines indicated were subjected to IP with antibodies to HA-tag (HA), Kv1.2 or Kv2.1, followed by immunoblotting with an antibody to HA-tag. E, Localization of Caspr2 transgenes in sciatic nerve. Teased sciatic nerve fibers isolated from the indicated mouse lines were immunolabeled using an antibody to gliomedin (Gldn; red) to label the nodes, Caspr (blue) to label the paranodal junction, and an antibody that recognizes the extracellular domain of Caspr2 (ECD; green). Scale bar, 10 μm.

Figure 6.

Rescue of the juxtaparanodal protein complex by Caspr2 lacking the PDZ-binding motif. Triple immunofluorescence labeling of teased sciatic nerves isolated from wild-type mice (WT), Caspr2 nulls (Caspr2^−/−), or Caspr2 nulls expressing the FL (Caspr2^−/−/FL), dCT (Caspr2^−/−/dCT), and dPDZ (Caspr2^−/−/dPDZ) transgenes, using antibodies to Caspr (blue), Caspr2-ECD (green), and Kv1.2 (reg; left column), TAG-1 (red; middle column), or PSD93 (red; right column). The red and blue channels are shown in the upper part, while the green and the blue channels are shown in the lower part of each panel. Note that Caspr2, Kv1.2 and TAG-1 are present at the JXP region in WT, Caspr2^−/−/FL and Caspr2^−/−/dPDZ nerves, but not in nerves isolated from Caspr2^−/− or Caspr2^−/−/CT. Scale bar, 10 μm.

Figure 7.

Association of Caspr2 cytoplasmic domain with MPPs requires its protein 4.1-binding sequence. A, Proteomic identification of scaffolding proteins associated with the cytoplasmic domain of Caspr2. ID, GenBank identification; Coverage, percentage of the protein sequence covered by the peptides identified; CT and GST, number of times each protein was identified using the cytoplasmic domain of Caspr2 (CT) or with GST control. B, Expression of MPPs in DRG neurons and Schwann cells. RT-PCR analysis of mRNA isolated from cultured DRG neurons and Schwann cells using specific primers for the different MPPs and β-actin as control. C, Caspr2 fusion proteins used for pull-down experiments. A schematic representation of the different constructs along with coomassie brilliant blue staining of an SDS gel containing the isolated proteins. D, Association of Caspr2 with MPP2, MPP6 and Kv1.2 require its PDZ- and protein 4.1 binding motif. GST-fusion proteins containing the cytoplasmic domain of Caspr2 (GST-C2CT), a cytoplasmic domain lacking the PDZ-binding sequence (GST-C2dPDZ), or a cytoplasmic domain lacking its juxtamembrane protein-4.1-binding motif (GST-C2d4.1) were used to pulldown myc-tagged proteins from transfected HEK-293 cells, or Kv1.2 from rat brain as indicated. Protein complexes were separated on SDS-gels and immunoblotted with an antibody to Myc-tag or to Kv1.2.