Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain - PubMed (original) (raw)

Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain

C F Ratcliffe et al. J Cell Biol. 2001.

Abstract

Sequence homology predicts that the extracellular domain of the sodium channel beta1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. We show here that beta1 subunits associate with neurofascin, a neuronal cell adhesion molecule that plays a key role in the assembly of nodes of Ranvier. The first Ig-like domain and second fibronectin type III-like domain of neurofascin mediate the interaction with the extracellular Ig-like domain of beta1, confirming the proposed function of this domain as a cell adhesion molecule. beta1 subunits localize to nodes of Ranvier with neurofascin in sciatic nerve axons, and beta1 and neurofascin are associated as early as postnatal day 5, during the period that nodes of Ranvier are forming. This association of beta1 subunit extracellular domains with neurofascin in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier.

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Figures

Figure 1.

Figure 1.

Sodium channel β1 and β3 subunits coimmunoprecipitate with neurofascin in cotransfected tsA-201 cells. (A) Representation of HA.11-tagged neurofascin 186. (B) TsA-201 cells were cotransfected with neurofascin 186 (NF186) and sodium channel subunits as indicated. Cell lysates were immunoprecipitated with the indicated antibodies and the blot was probed with monoclonal antibody anti-HA.11. Each cell lysate was probed with anti-HA.11 or specific antisodium channel antibodies as shown to confirm that all proteins were expressed (bottom).

Figure 2.

Figure 2.

Neurofascin and β1 localize to nodes of Ranvier and associate in rat brain. Rat sciatic nerve was labeled with neurofascin antibody anti-FNIII (A) or anti-β1CT (B). Expression of both β1 and neurofascin is concentrated at nodes of Ranvier. (C) P5 or adult rat brain lysates were immunoprecipitated with the indicated antibodies. Blots were probed with neurofascin antimucin antibody. (D) Expression of β1 in both P5 and adult rat brain lysates. (E and F) Teased sciatic nerve stained with anti-β1extra from 3-d-old (E) and 10-d-old (F) rats, demonstrating the localization of β1 at the nodes of Ranvier in development. NF, neurofascin. Bars: 2 μm (A and B) and 10 μm (E and F).

Figure 3.

Figure 3.

β1 and neurofascin associate in cis but not in trans. (A) TsA-201 cells cotransfected with β1 and neurofascin (NF) were lysed and immunoprecipitated (IP) as described previously. (B) TsA-201 cells were transfected separately and cocultured. Lysed cells were immunoprecipitated with the indicated antibodies and the blot was probed with anti-HA.11. The bottom panel shows cell lysates probed with anti-β1CT or anti-HA.11 to demonstrate that proteins were well expressed. (C) Cells transfected with β1 were stained with anti-β1CT, and (D) cells transfected with HA-tagged neurofascin 186 were stained with anti-HA.11.

Figure 4.

Figure 4.

The extracellular domain of β1 interacts with neurofascin. (A) TsA-201 cells cotransfected with NF186Δic and β1 were lysed and probed with the indicated antibodies. Blots were probed with anti-HA.11. Cell lysates were also probed with anti-HA.11 and anti-β1CT to show that both proteins were expressed. (B) Cells cotransfected with neurofascin and β1β2β2 were lysed and immunoprecipitated (IP) with the indicated antibody. Blots were probed with anti-HA.11. Cell lysates were probed with anti-HA.11 and anti-β2CT to show expression of both proteins. (C) Cells cotransfected with β1ec–GPI and neurofascin were lysed and immunoprecipitated with antibodies as indicated. Cell lysates were probed with anti-HA.11 or anti-β1extra to show both proteins were expressed.

Figure 5.

Figure 5.

Determination of the neurofascin binding site. (A) TsA-201 cells were cotransfected with α and β1 subunits and GPI-tagged constructs Ig1-GPI, Ig2-GPI, Ig3-GPI, Ig4-GPI, Ig5-GPI, Ig6-GPI, FN1-GPI, FN2-GPI, FN4-GPI, and mucin-GPI, respectively. Cell lysates were immunoprecipitated with anti-SP20 and blots were probed with anti-HA.11 antibody. (B) Lysates were probed with anti-SP20, anti-β1CT, and anti-HA.11 to demonstrate that all proteins were expressed. (C) TsA-201 cells were cotransfected with (lane 1) α, β1, and neurofascin 186; (lane 2) α and neurofascin 186; (lane 3) α, β1, and Ig1-GPI; (lane 4) α and Ig1-GPI; (lane 5) α, β1, and FN2-GPI; and (lane 6) α and FN2-GPI. Cell lysates were immunoprecipitated with anti-SP20 and blots were probed with anti-HA.11 antibody.

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