Regulation of Na(v)1.2 channels by brain-derived neurotrophic factor, TrkB, and associated Fyn kinase

Abstract

Voltage-gated sodium channels are responsible for action potential initiation and propagation in neurons, and modulation of their function has an important impact on neuronal excitability. Sodium channels are regulated by a Src-family tyrosine kinase pathway, and this modulation can be reversed by specifically bound receptor phosphoprotein tyrosine phosphatase-beta. However, the specific tyrosine kinase and signaling pathway are unknown. We found that the sodium channels in rat brain interact with Fyn, one of four Src-family tyrosine kinases expressed in the brain. Na(V)1.2 channels and Fyn are localized together in the axons of cultured hippocampal neurons, the mossy fibers of the hippocampus, and cell bodies, dendrites, and axons of neurons in many other brain areas, and they coimmunoprecipitate with Fyn from cotransfected tsA-201 cells. Coexpression of Fyn with Na(V)1.2 channels decreases sodium currents by increasing the rate of inactivation and causing a negative shift in the voltage dependence of inactivation. Reconstitution of a signaling pathway from brain-derived neurotrophic factor (BDNF) to sodium channels via the tyrosine receptor kinase B (TrkB)/p75 neurotrophin receptor and Fyn kinase in transfected cells resulted in an increased rate of inactivation of sodium channels and a negative shift in the voltage dependence of inactivation after treatment with BDNF. These results indicate that Fyn kinase is associated with sodium channels in brain neurons and can modulate Na(V)1.2 channels by tyrosine phosphorylation after activation of TrkB/p75 signaling by BDNF.

Figure 1.

Interaction of Na_V1.2 channels with Fyn kinase. A, Rat brain membrane preparations were isolated by sucrose density centrifugation as described in Materials and Methods. Sodium channels were solubilized, and antibodies against the sodium channel α subunit (anti-SP20), the β1 subunit (anti-β1CT), or nonimmune rabbit IgG (Rb IgG) were used to immunoprecipitate sodium channel complexes. Antibody against Src-family kinases (SFKs; anti-SRC2) was used to immunoblot the immunoprecipitated (IP) complex. B, Sodium channels from rat brain membranes were solubilized and immunoprecipitated with antibodies against the sodium channel α subunit (anti-SP20) or Rb IgG. The IP complex was immunoblotted with antibodies against individual Src-family kinases (Src, Yes, Fyn, and Lyn, respectively). Each IP sample was from the same membrane lysate to ensure that the same level of each kinase was present in each sample. C, Membrane preparations were isolated from adult rat and postnatal day 2 rat brain. The α and β1 subunits were immunoprecipitated with anti-SP20 or anti-β1CT, respectively, and the IP complex was immunoblotted with anti-Fyn antibody. Each experiment was replicated at least three times with consistent results, using the same protocol illustrated in A–C.

Figure 2.

Role of Fyn catalytic activity in association and phosphorylation of Na_V1.2 channels. A, Na_V1.2 α subunit was expressed in absence or presence of the β1 subunit cotransfected in a 1:1 molar ratio of cDNA in tsA-201 cells. Membrane lysates were isolated, and anti-SP20 or nonimmune rabbit IgG (Rb IgG) was used to immunoprecipitate sodium channel complexes. The immunoprecipitated (IP) samples were immunoblotted with anti-Fyn. B, Na_V1.2 α subunit, β1 subunit, and either wild-type or catalytically inactive K299M Fyn were coexpressed in tsA-201 cells, and sodium channels were immunoprecipitated with anti-SP20 or nonimmune Rb IgG. Wild-type Fyn (top) and K299M Fyn (bottom) were detected by immunoblotting with anti-Fyn antibodies. C, Na_V1.2 α and β1 subunits were coexpressed with either wild-type (wt) Fyn or K299M Fyn in tsA-201 cells, membranes were isolated, and sodium channel complexes were immunoprecipitated with anti-SP19 or nonimmune Rb IgG. The resulting samples were immunoblotted with anti-phosphotyrosine antibody, 4G10, and then the blot was stripped and reblotted with anti-SP20. Each experiment was replicated at least three times with consistent results, using the same protocol illustrated in A–C.

Figure 3.

Localization of Na_V1.2 and Fyn in transfected tsA-201 cells. A, Na_V1.2 and Fyn were coexpressed in tsA-201 cells. The cells were fixed and double immunostained using anti-SP20 and anti-Fyn3G antibodies, as described in Materials and Methods. Optical sections near the top and center of the cell are shown. Regions of overlap are shown in yellow. B, Na_V1.2 and Fyn were expressed separately in tsA-201 cells. The cells were fixed and single immunostained using anti-SP20 or anti-Fyn3G antibodies, as described in Materials and Methods. Optical sections near the top and the center of the cell are shown. C, Fyn was expressed alone in tsA-201 cells, and the cells were double immunostained for Fyn and caveolin. Regions of overlap are shown in yellow. Scale bars, 10 μm. D, Cells expressing Na_V1.2 only and cells coexpressing Na_V1.2 and Fyn were examined in the confocal microscope, and the number of Na_V1.2 puncta per cell were counted. The average number of puncta per cell was 1.35 ± 0.55 (n = 23) and 33.9 ± 6.5 (n = 14) in the absence and presence of Fyn, respectively. Each experiment was replicated at least three times with consistent results, using the same protocol illustrated in A–C. Error bars represent SEM.

Figure 4.

Localization of Na_V1.2 and Fyn in cultured rat hippocampal neurons. A, Cultured hippocampal neurons were fixed and triple labeled with anti-Fyn (left, green), anti-Na_V1.2 (center left, red), and anti-neurofilament (center right, white). The merged image of Na_V1.2 and Fyn is shown on the right. Regions of overlap are shown in yellow. B, C, Magnified images of the boxed areas (b and c, respectively) in A (right). Scale bars: A, 20 μm; B, 10 μm; C, 6 μm.

Figure 5.

Localization of Na_V1.2 channels and Fyn in hippocampus and cerebellum. Sections of rat brain were cut, fixed, and double labeled for Na_V1.2 and Fyn as described in Materials and Methods. Shown are Na_V1.2 (red, left), Fyn (green, center), and merged images (right). Regions of overlap are shown in yellow. A, CA3 region of the hippocampus. B, Magnified images of the mossy fibers in the dentate gyrus. DG, Dentate gyrus; G, granule cells; MF, mossy fibers. C, High magnification of mossy fibers in the CA3 region. D, Purkinje neurons (P) and molecular layer (M) of the cerebellum. Scale bars: A, 100 μm; B, 50 μm; C, 10 μm; D, 50 μm.

Figure 6.

Effects of Fyn expression on functional properties of sodium channels. Sodium currents were recorded from cells transfected with Na_V1.2 alone (filled circles) or Na_V1.2 plus Fyn (open squares). A, Left, Sodium currents evoked by depolarizations to potentials from −110 to 10 mV in 10 mV steps from a holding potential of −110 mV from cells transfected with Na_V1.2 alone (top) or Na_V1.2 plus Fyn (bottom). Right, Time course of inactivation during 100-ms-long depolarizations. The decay time course after the peak was fit with a single exponential. Time constants (Tau) are plotted versus test potential. Inset, Normalized sodium currents in response to a depolarization to −15 mV in cells transfected with Na_V1.2 alone (slower) and with (faster) Fyn. B, Mean current–voltage relationships. Peak sodium currents were measured during depolarizations to the indicated potentials from cells transfected with Na_V1.2 alone (filled circles; n = 63) and Na_V1.2 plus Fyn (open squares; n = 46). C, Mean normalized conductance (G)–voltage relationships derived from these current–voltage curves according to G = I/(V − V_Rev), where I is the measured current at voltage V, and V_Rev is the extrapolated reversal potential. D, Mean normalized inactivation curves. Cells were depolarized for 100 ms with prepulses to the indicated potentials (−110 to −15 mV in 5 mV steps) followed by a 5 ms test pulse to 0 mV. Peak test pulse current was measured and plotted as a function of prepulse potential. Normalized mean peak test pulse current is plotted as a function of prepulse potential. E, Mean normalized slow inactivation curves. Cells were depolarized for 1 s with prepulses to the indicated potentials followed by repolarization for 20 ms to −110 mV to allow recovery from fast inactivation and depolarization to −10 mV for a 10 ms test pulse. Peak test pulse current was measured at each prepulse potential and plotted as a function of prepulse potential. Error bars represent SEM.

Figure 7.

Time course of the effect of BDNF treatment on sodium currents in tsA-201 cells expressing Na_V1.2 channels with TrkB, p75, and Fyn. From a holding potential of −70 mV, cells were hyperpolarized to −110 mV for 110 ms, and sodium currents were evoked by 10 ms test pulses to −10 mV before and after addition of 7.1 nm BDNF. A, Example of test pulse sodium currents in control (CON) and after addition of BDNF. Calibration: 500 pA, 2 ms. B, Mean time course of peak sodium current reduction in response to Fyn addition using the same pulse protocol as in A. Linear rundown was subtracted from the individual current records for each cell. The mean linear rundown was 3.4%/min. The BDNF-induced decline in sodium current (B, fit curve) had an initial rate of 21.2%/min, 6.1-fold faster than mean rundown. Error bars represent SEM.

Figure 8.

Functional effects of BDNF treatment on cells expressing Na_V1.2 channels with TrkB, p75, RPTPβ, and Fyn. A, Mean current–voltage curves from cells expressing Na_V1.2, Fyn, TrkB, p75, and RPTPβ in the absence of BDNF (filled circles; n = 8) or after exposure to 7.1 nm BDNF for 60 min (open squares; n = 8). B, Conductance–voltage relationships, constructed as described in Figure 4C, from the data in A. C, Mean normalized inactivation curves as described in Figure 4D for cells without (filled circles; n = 6) and with BDNF (open squares; n = 7). D, Time course of inactivation during depolarizations measured as described in Figure 6A. Time constants (Tau) are plotted versus test pulse potential. Error bars represent SEM.