Fibroblast growth factor homologous factor 2B: association with Nav1.6 and selective colocalization at nodes of Ranvier of dorsal root axons

Abstract

Voltage-gated sodium channels interact with cytosolic proteins that regulate channel trafficking and/or modulate the biophysical properties of the channels. Na(v)1.6 is heavily expressed at the nodes of Ranvier along adult CNS and PNS axons and along unmyelinated fibers in the PNS. In an initial yeast two-hybrid screen using the C terminus of Na(v)1.6 as a bait, we identified FHF2B, a member of the FGF homologous factor (FHF) subfamily, as an interacting partner of Na(v)1.6. Members of the FHF subfamily share approximately 70% sequence identity, and individual members demonstrate a cell- and tissue-specific expression pattern. FHF2 is abundantly expressed in the hippocampus and DRG neurons and colocalizes with Na(v)1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. However, retinal ganglion cells and spinal ventral horn motor neurons show very low levels of FHF2 expression, and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. Thus, FHF2 is selectively localized at nodes of dorsal root sensory but not ventral root motor axons. The coexpression of FHF2B and Na(v)1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The preferential expression of FHF2B in sensory neurons may provide a basis for physiological differences in sodium currents that have been reported at the nodes of Ranvier in sensory versus motor axons.

Figure 1.

Na_v1.6 binds to several FHF2 isoforms in a Y2H assay. FHF members were expressed as fusion proteins with the VP16 activation domain, and the C terminus of Na_v1.6 was expressed as a fusion protein with the Gal4 DNA-binding domain. Pairwise binding of Na_v1.6C and members of the FHF family, as indicated, was tested by the filter-lift β-gal assay. A blue signal indicates a positive interaction between the two proteins. The interacting partners c-jun/c-fos served as a positive controls, and the noninteracting pair Rb/lamin served as a negative control.

Figure 2.

FHF2 and Na_v1.6 are colocalized in the brain. A, Pan sodium channel and FHF2 antibodies were used to immunoprecipitate (IP) the voltage-gated sodium channels from a rat brain lysate. Anti-mouse IgG was used as a negative control to rule out nonspecific binding. Western blotting analysis of the IP complex was performed by the pan sodium channel antibody. Lane 1 shows a robust immunoreactive signal from the cell lysate that was used for the IP assay, consistent with the presence of intact sodium channel proteins in this sample. Nonspecific antibodies do not immunoprecipitate a channel complex (lane 2). As predicted, pan sodium channel antibody immunoprecipitated a channel complex (lane 3). The reduced signal in lane 3 compared with lane 1 is likely attributable to the avidity of the antibody in the two different assays. FHF2 coimmunoprecipitated voltage-gated sodium channels from the brain lysate (lane 4). Comparison of the immunoreactive bands in lanes 1 and 4 might be explained by the interaction of FHF2 with only a subset of the cellular pool of channels. The molecular weight marker (in kilodaltons) is shown on the left. B, Colocalization of Na_v1.6 and FHF2 in the hippocampus. Na_v1.6- and FHF2-specific antibodies were used to immunolabel sections of rat hippocampus. FHF2 (green) and Na_v1.6 (red) proteins were detected in the pyramidal cells of Ammon's horn, and the merged images (yellow) show significant colocalization of the two proteins. The inset shows the pyramidal cells at 40× magnification. C, GFP antibody was used to IP Na_v1.6 from lysates of HEK 293 cells transfected with either Na_v1.6 plus GFP (control; lane 3) or Na_v1.6 plus FHF2B-GFP (lane 4). The IP complex was probed with the pan sodium channel antibody and detected no association between Na_v1.6 with GFP (lane 3), but an association of Na_v1.6 with FHF2B (lane 4). Lanes 1 and 2 show Western blotting analysis of the cell lysates probed with pan sodium channel antibody (top) and GFP (bottom) to show comparable levels of Na_v1.6/GFP (lane 1) and Na_v1.6/FHF2B-GFP (lane 2) in the samples used for the immunoprecipitation assay.

Figure 3.

FHF2 is expressed in neurons of dorsal root ganglion. In situ hybridization using digoxigenin-labeled antisense (A) and sense (B) probes is shown. Significant staining of DRG neurons of all size classes was observed using the antisense probe (A). Sense riboprobes yielded no signals (B). Immunohistochemistry was performed using Na_v1.6- and FHF2-specific antibodies. Slides were viewed under a Nikon Eclipse E800 microscope. FHF2 (C) and Na_v1.6 (D) proteins were detected in small and large DRG neurons. A merge of the two images (E) shows significant colocalization of the two proteins. Scale bars, 50 μm.

Figure 4.

FHF2 is expressed at the nodes of Ranvier in the dorsal roots, where it colocalizes with Na_v1.6. Images of a section of the dorsal root immunolabeled with antibodies against Na_v1.6 (red), FHF2 (green), and Caspr (blue) show the colocalization of FHF2 and Na_v1.6, with Na_v1.6 present at every node of Ranvier, which shows FHF2 expression (A). Nodal regions were delineated by staining for Caspr, which is a marker of paranodes (D). As expected, Na_v1.6 staining was limited to the narrow gaps representing the nodes (B). FHF2 staining is restricted to a narrow band (C). The colocalization of Na_v1.6 and FHF2 at the nodes of Ranvier is demonstrated by the merger of the three images (E). Scale bar, 10 μm. The inset in A shows FHF2 in unmyelinated axons of the teased sciatic nerve. The picture is a merged image (yellow) of FHF2 (green) and peripherin (red), a marker of unmyelinated axons.

Figure 5.

FHF2 is not expressed at the nodes of Ranvier in ventral roots or optic nerve. Images of a section of the ventral root (A) and optic nerve (B) immunolabeled with antibodies against Na_v1.6 (red), FHF2 (green), and Caspr (blue) are shown. Nodal regions were delineated by Caspr staining. As expected, Na_v1.6 staining was limited to the narrow gaps at nodes (A, B, insets). No FHF2 staining is detectable in either tissue. The staining of the nodes by Na_v1.6 argues against the possibility that the FHF2 antibody could not access the nodal region. Scale bar, 10 μm.

Figure 6.

Expression of FHF2 mRNA in DRG neurons, ventral horn of spinal cord, Purkinje neurons, and retinal ganglion neurons. In situ hybridization using FHF2 digoxigenin-labeled riboprobe shows that FHF2 is abundantly expressed in DRG neurons (A) compared with motor neurons in the ventral horn (B), Purkinje cells (C), or retinal ganglion neurons (D). All tissues were processed together, and the colorimetric reaction was stopped before it reached saturation to allow a comparison of the levels of FHF2 expression.

Figure 7.

FHF2B increases functional Na_v1.6 sodium current density in transiently transfected ND7/23 cells. Representative examples of sodium current traces are shown. ND7/23 cells were transiently transfected with Na_v1.6_R and either GFP (A) or FHF2B-GFP (B). Cells were held at a potential of -120 mV and depolarized to a range of potentials (-65 to +60 mV) for 40 msec. C, Maximum peak current densities were measured and plotted (pA/pF). Cells transfected with GFP alone showed essentially zero sodium current (6.0 ± 0.5 pA/pF; n = 11) compared with those transfected with Na_v1.6_R and GFP, which displayed robust transient sodium currents (85.4 ± 21.2 pA/pF; n = 11; ^*p < 0.005). Cells transfected with Na_v1.6_R and FHF2B-GFP (202.5 ± 17.5 pA/pF; n = 22) produced a more than twofold increase in current density when compared with cells transfected with Na_v1.6 and GFP control (85.4 ± 21.2 pA/pF; n = 11; ^**p < 0.001).

Figure 8.

FHF2B causes a depolarizing shift in steady-state inactivation of Na_v1.6 sodium currents. A, Steady-state activation and inactivation curves are shown for ND7/23 cells transiently transfected with Na_v1.6 and either GFP (closed circles) or FHF2B-GFP (open circles). For activation, cells were held at -120 mV and depolarized to arange of potentials (-65 to +20), and the normalized conductance was plotted as a function of voltage. For inactivation, cells were held at -120 mV, and the peak current amplitude was measured by a 40 msec test pulse to -10 mV, after 500 msec prepulses to potentials over the range -120 to +20 mV. Boltzmann fits to the data are shown. FHF2B caused a significant (p < 0.02) depolarizing shift in inactivation but no shift inactivation parameters. B, Development of closed state inactivation at -70 mV was measured and showed greater inactivation with GFP transfected (closed circles) than FHF2B transfected cells (open circles). Lines are drawn to guide the eye. Cells were held at -120 mV, prepulsed to -70 mV for increasing amounts of time (1-3000 msec) and then stepped to -10 mV to determine the fraction of current inactivated during the prepulse. These data are consistent with the depolarizing shift in steady-state inactivation shown in A. C, Recovery from inactivation at -80 mV was measured and showed no differences between GFP-transfected (closed circles) and FHF2B-transfected (open circles) cells. Lines are drawn to guide the eye. Cells were held at -120 mV, and the peak current amplitude was measured by a test pulse to -10 mV, after a 40 msec inactivating pulse to -10 mV and a recovery period between 0.1 and 200 msec at -80 mV.