Sites and molecular mechanisms of modulation of Na(v)1.2 channels by Fyn tyrosine kinase

Abstract

Voltage-gated sodium channels are important targets for modulation of electrical excitability by neurotransmitters and neurotrophins acting through protein phosphorylation. Fast inactivation of Na(V)1.2 channels is regulated via tyrosine phosphorylation by Fyn kinase and dephosphorylation by receptor phosphoprotein tyrosine phosphatase-beta, which are associated in a signaling complex. Here we have identified the amino acid residues on Na(V)1.2 channels that coordinate binding of Fyn kinase and mediate inhibition of sodium currents by enhancing fast inactivation. Fyn kinase binds to a Src homology 3 (SH3)-binding motif in the second half of the intracellular loop connecting domains I and II (L(I-II)) of Na(V)1.2, and mutation of that SH3-binding motif prevents Fyn binding and Fyn enhancement of fast inactivation of sodium currents. Analysis of tyrosine phosphorylation sites by mutagenesis and functional expression revealed a multisite regulatory mechanism. Y66 and Y1893, which are in consensus sequences appropriate for binding to the Fyn SH2 domain after phosphorylation, are both required for optimal binding and regulation by Fyn. Y730, which is located near the SH3-binding motif in L(I-II), and Y1497 and Y1498 in the inactivation gate in L(III-IV), are also required for optimal regulation. Phosphorylation of these sites likely promotes fast inactivation. Fast inactivation of the closely related Na(V)1.1 channels is not modulated by Fyn, and these channels do not contain an SH3-binding motif in L(I-II). Subtype-selective modulation by tyrosine phosphorylation/dephosphorylation provides a mechanism for differential regulation of sodium channels by neurotrophins and tyrosine phosphorylation in unmyelinated axons and dendrites, where Na(V)1.2 channels are expressed in brain neurons.

Figure 1.

Effect of deletion of the SH3 domain of Fyn on association with Na_V1.2 channels. A, Na_V1.2 and wild-type Fyn with a C-terminal Myc epitope tag were coexpressed in tsA-201 cells. B, Na_V1.2 and ΔSH3,4Fyn with a C-terminal Myc tag were coexpressed in tsA-201 cells. C, Na_V1.2 and lipΔSH3,4Fyn with a C-terminal Myc tag were coexpressed in tsA-201 cells. D, Na_V1.2 and ΔSH3Fyn with a C-terminal Myc tag were coexpressed in tsA-201 cells. A–D, Anti-rabbit IgG was used as a negative control (lane 1), and anti-SP19, an antibody which recognizes the Na_V1.2 α subunit, was used to immunoprecipitate sodium channel complexes (lane 2). Immunoprecipitation (IP) samples were probed with either 4G10 (middle) or anti-Myc (B, C) or anti-Fyn (A, D) (bottom). Then the blot was stripped and reprobed with SP20 recognizing the Na_V1.2 α subunit (top). Similar results were obtained in at least three experiments like those illustrated in A–D and in additional experiments with similar, but not identical, experimental design.

Figure 2.

Binding of Fyn by L_I–II of the Na_V1.2 α subunit. A, L_I–II, L_II–III, L_III–IV, and C-terminal (C-term) domains of Na_V1.2 with N-terminal lipid anchors and C-terminal Myc epitope tags were expressed in tsA-201 cells, solubilized, and immunoblotted with anti-Myc as described in Materials and Methods. B, Coimmunoprecipitation of Na_V1.2 intracellular domains with Fyn. The indicated Na_V1.2 constructs and Fyn were coexpressed in tsA-201 cells and solubilized. Lysate samples for each construct were resolved by SDS-PAGE and immunoblotted with anti-Fyn (lane 1). The indicated Na_V1.2 domain constructs were immunoprecipitated with nonimmune rabbit IgG (lane 2) or anti-Myc (lane 3) and analyzed by SDS-PAGE and immunoblotting with anti-Fyn. C, Immunoprecipitation of L_I–IIa and L_I–IIb with Fyn. L_I–IIa and L_I–IIb with N-terminal lipid anchor and C-terminal Myc epitope tag were analyzed as in B. Similar results were obtained in at least three experiments like those illustrated in A–C and in additional experiments with similar, but not identical, experimental design. IP, Immunoprecipitation; WT, wild-type.

Figure 3.

Effect of mutation of the SH3-binding motif and phosphorylated tyrosine residues in Na_V1.2 channels. A, Na_V1.2/638G and Fyn were coexpressed in tsA-201 cells and solubilized. Lysate samples (lane 1) were analyzed by SDS-PAGE and immunoblotting with anti-SP20 (top) or anti-Fyn (bottom). Additional samples were immunoprecipitated with nonimmune rabbit IgG (lane 2) or anti-SP19 (lane 3) and analyzed by SDS-PAGE and immunoblotting with anti-SP20 (top) or anti-Fyn (bottom). B, Na_V1.2/638G, β1 subunit, and Fyn were coexpressed in tsA-201 cells and analyzed as in A. C, Left, Na_V1.2 or Na_V1.2/638G mutant channels were coexpressed with Fyn in tsA-201 cells, solubilized, immunoprecipitated with nonimmune rabbit IgG (lane 1) or anti-SP19 (lane 2), analyzed by SDS-PAGE, and immunoblotted with anti-Fyn (bottom). C, Right, Na_V1.2/Y66F or Na_V1.2/Y730F mutant channels were coexpressed with Fyn in tsA-201 cells, solubilized, immunoprecipitated with nonimmune rabbit IgG (lane 1) or anti-SP19 (lane 2), analyzed by SDS-PAGE, and immunoblotted with anti-SP20 (Na_V1.2; top) or anti-Fyn (bottom). D, Na_V1.2, Na_V1.2/Y1497F,Y1498F (YYFF), or Na_V1.2/Y1893F channels were coexpressed with Fyn in tsA-201 cells, solubilized, immunoprecipitated with nonimmune rabbit IgG (lane 1) or anti-SP19 (lane 2), analyzed by SDS-PAGE, and immunoblotted with anti-Fyn. Similar results were obtained in at least three experiments like those illustrated in A–D and in additional experiments with similar, but not identical, experimental design.

Figure 4.

Effects of mutations in the SH3 domain of Na_V1.2 on modulation by Fyn. A, Examples of effects of Fyn coexpression on the time course of sodium currents during depolarizations to −15 mV from a holding potential of −110 mV. Normalized currents recorded in the absence [slower trace] and presence (faster trace) of coexpressed Fyn from WT channels (top) or Na_V1.2/638G (bottom) are superimposed. B, Mean time constants for inactivation of currents conducted by Na_V1.2/638G channels during depolarizations to the indicated potentials in the absence (filled circles) or presence (open squares) of coexpressed Fyn. The dotted and dashed curves represent the Na_V1.2 WT data reported by Ahn et al. (2007) (accompanying study) for the channel expressed alone and in the presence of Fyn, respectively. The effect of Fyn on the time constants of fast inactivation of Na_v1.2/638G is significantly less than wild type at all potentials (p < 0.05). C, Normalized mean conductance–voltage relationships for cells expressing Na_V1.2/638G in the absence (filled circles) and presence (open squares) of coexpressed Fyn derived from depolarizations to the indicated potentials. D, Mean normalized inactivation curves of Na_V1.2/638G channels in the absence (filled circles) and presence (open squares) of coexpressed Fyn. The voltage dependence of inactivation of WT channels as determined by Ahn et al. (2007) (accompanying study) in the absence (dotted line) and presence (dashed line) of coexpressed Fyn are superimposed. 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. Mean normalized peak test pulse current is plotted as a function of prepulse potential. E, Mean normalized inactivation curves of Na_V1.2/P636A channels in the absence (filled circles) and presence (open squares) of coexpressed Fyn. The protocol was identical to that of D. F, Mean time constants for inactivation of Na_V1.2/P636A channels during depolarizations to the indicated potentials in the absence (filled circles) or presence (open squares) of coexpressed Fyn. Time constants were derived from monoexponential fits to the decaying phase of the sodium current recorded in response to depolarizations to the indicated potentials. Error bars represent SEM.

Figure 5.

Effect of mutation Na_V1.2/Y730F on modulation by Fyn. A, Mean voltage dependence of activation of Na_v1.2/Y730F channels in the absence (filled circles) and presence (open squares) of coexpressed Fyn kinase. Protocol was as described in Figure 4C. B, Mean normalized inactivation curves of Na_V1.2/Y730F channels in the absence (filled circles) and presence (open squares) of coexpressed Fyn, determined as described in Figure 4D. C, Mean time constants for inactivation of Na_V1.2/Y730F channels during depolarizations to the indicated potentials in the absence (filled circles) or presence (open squares) of coexpressed Fyn, determined as described in Figure 4B. Error bars represent SEM.

Figure 6.

Effect of mutations Na_V1.2/Y1497F and Na_V1.2/Y1498F on modulation by Fyn. A, C, Mean normalized inactivation curves from cells expressing Na_V1.2/Y1497F (A) and Na_V1.2/Y1498F (C) channels in the absence (filled circles) and presence (open squares) of coexpressed Fyn determined as described in Figure 4D. B, D, Mean time constants for inactivation of Na_V1.2/Y1497F (B) and Na_V1.2/Y1498F (D) channels during depolarizations to the indicated potentials in the absence (filled circles) or presence (open squares) of coexpressed Fyn, determined as described in Figure 4B. Error bars represent SEM.

Figure 7.

Effects of mutations in predicted SH2-binding domains on modulation by Fyn. A, C, D, Mean normalized inactivation curves from cells expressing Na_V1.2/Y66F (A), Na_V1.2/Y1893F (C), and Na_V1.2/Y1975F (D) channels in the absence (filled circles) and presence (open squares) of coexpressed Fyn, determined as described in Figure 4D. B, Mean time constants for inactivation of Na_V1.2/Y66F channels during depolarizations to the indicated potentials in the absence (filled circles) or presence (open squares) of coexpressed Fyn determined as described in Figure 4B. The dotted and dashed curves represent the Na_V1.2 wild-type (WT) data reported by Ahn et al. (2007) for the channel expressed alone and in the presence of Fyn, respectively. Inactivation of Y66F channels in control was significantly slower than WT channels at potentials between −40 and −15 mV (p < 0.05 by ANOVA with Tukey's posttest). Error bars represent SEM.

Figure 8.

Effect of Fyn coexpression on the inactivation properties of Na_V1.1 channels. A, Mean normalized inactivation curves from cells expressing Na_V1.1 channels in the absence (filled circles) and presence (open squares) of coexpressed Fyn, determined as described in Figure 4D. B, Mean time constants for inactivation of Na_V1.1 channels during depolarizations to the indicated potentials in the absence (filled circles) or presence (open squares) of coexpressed Fyn, determined as described in Figure 4B. Error bars represent SEM.