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. 2004 Aug 18;24(33):7387-99.
doi: 10.1523/JNEUROSCI.0322-04.2004.

Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface

Bhaval S Shah  1 , Anthony M Rush, Shujun Liu, Lynda Tyrrell, Joel A Black, Sulayman D Dib-Hajj, Stephen G Waxman
Affiliations

Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface

Bhaval S Shah et al. J Neurosci. .

Abstract

The upregulation of voltage-gated sodium channel Na(v)1.3 has been linked to hyperexcitability of axotomized dorsal root ganglion (DRG) neurons, which underlies neuropathic pain. However, factors that regulate delivery of Na(v)1.3 to the cell surface are not known. Contactin/F3, a cell adhesion molecule, has been shown to interact with and enhance surface expression of sodium channels Na(v)1.2 and Na(v)1.9. In this study we show that contactin coimmunoprecipitates with Na(v)1.3 from postnatal day 0 rat brain where this channel is abundant, and from human embryonic kidney (HEK) 293 cells stably transfected with Na(v)1.3 (HEK-Na(v)1.3). Purified GST fusion proteins of the N and C termini of Na(v)1.3 pull down contactin from lysates of transfected HEK 293 cells. Transfection of HEK-Na(v)1.3 cells with contactin increases the amplitude of the current threefold without changing the biophysical properties of the channel. Enzymatic removal of contactin from the cell surface of cotransfected cells does not reduce the elevated levels of the Na(v)1.3 current. Finally, we show that, similar to Na(v)1.3, contactin is upregulated in axotomized DRG neurons and accumulates within the neuroma of transected sciatic nerve. We propose that the upregulation of contactin and its colocalization with Na(v)1.3 in axotomized DRG neurons may contribute to the hyper-excitablity of the injured neurons.

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Figures

Figure 1.
Figure 1.
GPI-anchored contactin and contactin-GFP proteins are present at the surface of HEK-Nav1.3 cells. Live nonpermeabilized HEK-Nav1.3 cells were probed with anti-contactin antibody (A) and nuclei labeled with DAPI (B). No contactin-immunofluorescent signal is detected. Contactin (C) or contactin-GFP (D) constructs were transfected into HEK-Nav1.3 cells, and the live cells were reacted with anti-contactin antibody. Surface labeling of contactin (red) is apparent for both contactin and contactin-GFP constructs. HEK-Nav1.3 cells transfected with contactin-GFP also show GFP signal (green).
Figure 2.
Figure 2.
Contactin binds to Nav1.3N and Nav1.3C in vitro. Recombinant proteins GST, GST-Nav1.3N, GST-Nav1.3L1, GST-Nav1.3L2, GST-Nav1.3L3, or GST-Nav1.3C (0.5 μg) immobilized on glutathione-Sepharose beads were incubated with extracts prepared from HEK 293 cells transfected with either Ctn-GFP or GFP. Immunoblot analysis of the bound proteins and cell extracts were probed with anti-GFP antibody. A, Schematic diagram of the sodium channel showing the intracellular regions used for the GST pull down assay. B, Coomassie blue-stained gel showing the GST-fusion proteins separated by SDS-PAGE. C, GST pull down assay using cell extracts from HEK 293 cells transfected with Ctn-GFP. Only GST-Nav1.3N and GST-Nav1.3C captured Ctn-GFP. D, GST-Nav1.3N and GST-Nav1.3C were used in a pull down assay using cell extracts from HEK 293 cells transfected with GFP. Neither GST alone nor the GST-Nav1.3 derivatives bind to GFP.
Figure 3.
Figure 3.
Contactin forms a complex with Nav1.3 in vivo and in HEK 293 cells. A, Interaction of contactin and Nav1.3 in HEK-Nav1.3 cells transfected with the Ctn-GFP construct. Immunoblot analysis (lanes 1-3) of cell extracts prepared from control HEK 293 cells transfected with Ctn-GFP, HEK-Nav1.3 cells, or HEK-Nav1.3 cells transfected with Ctn-GFP, using pan sodium channel (top panel) or GFP (bottom panel) antibodies show that Nav1.3 is detected only in HEK-Nav1.3 cell lysates (top panel, lanes 2, 3), whereas Ctn-GFP is detected only in lysates of cells transfected with Ctn-GFP construct (bottom panel, lanes 1, 3). Endogenous sodium channels (top panel, lane 1) or contactin (bottom panel, lane 2) are not detected in the cell lysates. Reciprocal immunoprecipitation analysis using Nav1.3-specific and GFP-specific antibodies (lanes 4-6). Anti-GFP antibodies coimmunoprecipitated Nav1.3 channels from HEK-Nav1.3-Ctn-GFP cell lysates (top panel, lane 6), whereas Nav1.3-specific antibodies coimmunoprecipitated Ctn-GFP (bottom panel, lane 6). B, Interaction of contactin and Nav1.3 in HEK-Nav1.3 cells is not dependent on the GFP tag or exogenous β1 subunit. HEK-Nav1.3 cell line was transfected with wild-type contactin alone or together with human β1 subunit, as indicated. Western blot analysis of HEK-Nav1.3 cells expressing Nav1.3 and Ctn was performed using pan sodium channel (top panel) and contactin (bottom panel) antibodies. This analysis shows robust expression of Nav1.3 channel (top panel, lane 1) and wild-type contactin (bottom panel, lane 1). Channel complexes were immunoprecipitated using Nav1.3-specific antibodies (lanes 3, 5) or control, nonspecific IgG (lanes 2, 4). Nav1.3 is detected only in the immunoprecipitation analysis using Nav1.3-specific antibody (lanes 3, 5). Control IgG did not immunoprecipitate proteins that reacted with the pan sodium channel or the contactin antibodies (lanes 2, 4). The coexpression of β1 subunit was not necessary for the interaction of Nav1.3 and wild-type contactin (compare lanes 3, 5). C, Contactin interacts with Nav1.3 in P0 rat brain. Western blot analysis using pan sodium channel antibodies and contactin-specific antibodies detect a sodium channel (top panel, lane 1) and contactin-immunoreactive (bottom panel, lane 1) bands of the expected sizes in P0 rat brain homogenate. The immunoprecipitated protein complexes were probed by pan sodium channel antibody (top panel, lane 3) and contactin antibody (bottom panel, lane 3). The volume of homogenate loaded on the gel in lane 1 is 5% of the volume that was used for the immunoprecipitation assay. Anti-Nav1.3 antibodies coimmunoprecipitated contactin. Control IgG antibodies failed to immunoprecipitate the channel or contactin (lane 2). IP, Immunoprecipitate; WB, Western blot.
Figure 4.
Figure 4.
Contactin enhances Nav1.3 at the cell surface in transfected HEK 293 cells. HEK-Nav1.3 cells or HEK-Nav1.3 cells transfected with contactin were stained with anti-pan sodium channel and anti-contactin antibodies. HEK-Nav1.3 cells show prominent cytoplasmic Nav1.3 staining. HEK-Nav1.3 transfected with contactin show enhanced peripheral Nav1.3 labeling. B-D show HEK-Nav1.3 cells stained with anti-pan sodium channel antibody (B), contactin (C), and overlay (D). HEK-Nav1.3 cells transfected with contactin show Nav1.3 staining (F), contactin staining (G), and overlay (H). Confocal microscopic images were quantified using Scion Image software. A, E, Typical intensity measurements of Nav1.3 immunoreactivity in HEK-Nav1.3 cells (A) or HEK-Nav1.3 cells transfected with contactin (E) are shown. Densitometric measurements of Nav1.3 signal were taken through the cell axis, as indicated by the white line (B, F). The fluorescence density in arbitrary units was plotted against the sectional length in pixels. Scale bar, 10 μm.
Figure 5.
Figure 5.
Contactin increases functional cell surface expression of Nav1.3 in transfected HEK-Nav1.3 cells. A, Representative families of traces from control HEK 293 cells transfected with Ctn-GFP that do not express recombinant Nav1.3 (top); HEK-Nav1.3 cells transfected with GFP (middle); and HEK-Nav1.3 cells transfected with Ctn-GFP (bottom). B, Current densities (in picoamperes per picofarad) recorded from control HEK 293 cells transfected with Ctn-GFP showed only very low levels of current. HEK-Nav1.3 cells transfected with GFP displayed a sodium current of 287.9 ± 36.8 pA/pF. HEK-Nav1.3 cells stably expressing Nav1.3 transfected with Ctn-GFP showed a significant (*p < 0.01) and nearly threefold increase in current density (873 ± 87 pA/pF). C, Representative families of traces from HEK-Nav1.3 cells transfected with Ctn-GFP (top), β1-red (middle), or Ctn-GFP and β1-red (bottom). D, Current densities recorded from HEK-Nav1.3 cells transfected with Ctn-GFP, β1-red, or Ctn-GFP plus β1-red. HEK 293 cells stably expressing Nav1.3 cotransfected with Ctn-GFP and β1-red showed no significant difference in current density in comparison to HEK-Nav1.3 cells transfected with Ctn-GFP alone. β1-red transfection alone showed no effect on current density.
Figure 6.
Figure 6.
Contactin does not change steady-state characteristics of Nav1.3 in transfected HEK-Nav1.3 cells. Normalized current-voltage relationships are shown (A). Steady-state, voltage-dependent inactivation (500 msec prepulses) (B), and recovery from inactivation (C) were determined as described in Materials and Methods. The time course for recovery from inactivation of peak current at -120 mV is shown. Squares, HEK-Nav1.3 cells transfected with GFP. Circles, HEK-Nav1.3 cells transfected with Ctn-GFP. No differences in these properties were seen in HEK-Nav1.3 cells transfected with either Ctn-GFP or GFP. C, Inset, Averaged normalized currents were elicited by a depolarizing pulse from -120 to -10 mV and are overlaid. The currents are indistinguishable from one another.
Figure 7.
Figure 7.
Removal of surface contactin does not reduce the elevated Nav1.3 density at the cell surface in HEK-Nav1.3-Ctn cells. HEK-Nav1.3 cells were transfected with contactin construct (HEK-Nav1.3-Ctn) and treated with PI-PLC or were untreated (see Materials and Methods). Live cells (nonpermeabilized) were immunostained for contactin using a polyclonal anti-contactin antibody in untreated (A), and 16 hr (B), 19 hr (C), and 21 hr (D) after initial treatment with PI-PLC to ascertain level of cell surface expression of contactin over time. Scale bar, 10 μm. E, Peak current density for HEK-Nav1.3 treated with PI-PLC and HEK-Nav1.3-Ctn treated with PI-PLC or untreated was plotted in picoamperes per picofarad. HEK-Nav1.3-Ctn treated with PI-PLC do not show significant difference in the Nav1.3 current density compared with untreated cells.
Figure 8.
Figure 8.
Contactin expression is increased in axotomized DRG neurons. A, In control DRG, contactin is present in most neurons and is peripherally localized. B, In axotomized DRG neurons, there is a substantial increase in contactin immunostaining. C, Western blot analysis shows that transection of the sciatic nerve causes an increase in the contactin protein levels in the DRG. The Western blot was probed with anti-contactin antibody (lanes 1, 2). The increase in contactin protein signal can be seen in ipsilateral (I) DRGs (lane 2) in comparison to contralateral (C) DRGs (lane 1). The blot was then stripped and reprobed with anti-Nav1.9 antibodies (lanes 3, 4). The expected decrease in Nav1.9 protein signal can be seen in ipsilateral DRGs (lane 4) in comparison to contralateral DRGs (lane 3). D, Nav1.3 is not detectable in control DRG neurons. E, There is a substantial increase in Nav1.3 immunofluorescence in DRG neurons (same section as shown in B) after transection of the sciatic nerve. F, Merged images of B and E. Scale bar, 50 μm.
Figure 9.
Figure 9.
Nav1.3 and contactin colocalize in sciatic nerve neuroma. Images of uninjured control (contralateral) and transected (ipsilateral) sciatic nerve show that contactin (left) and Nav1.3 (middle) accumulate within axon-like profiles within the neuroma (right, short arrows), with less immunostaining in proximal regions of the transected nerve. Contactin and Nav1.3 exhibit extensive colocalization (right, yellow) within the neuroma. Black arrow “ligature” indicates site of ligation. Scale bar, 500 μm.
Figure 10.
Figure 10.
Nav1.3 and contactin colocalize in axons within neuroma. This section shows axons, at the level indicated by white short arrows in Figure 9, at higher magnification. Contactin (A) and Nav1.3 (B) exhibit substantial colocalization (C, yellow) in most axons within sciatic nerve neuroma. Scale bar, 25 μm.
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