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. 2005 Nov 15;569(Pt 1):179-93.
doi: 10.1113/jphysiol.2005.097220. Epub 2005 Sep 15.

Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels

Jun-Yang Lou  1 , Fernanda Laezza, Benjamin R Gerber, Maolei Xiao, Kathryn A Yamada, Hali Hartmann, Ann Marie Craig, Jeanne M Nerbonne, David M Ornitz
Affiliations

Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels

Jun-Yang Lou et al. J Physiol. .

Abstract

Genetic ablation of the fibroblast growth factor (Fgf) 14 gene in mice or a missense mutation in Fgf14 in humans causes ataxia and cognitive deficits. These phenotypes suggest that the neuronally expressed Fgf14 gene is essential for regulating normal neuronal activity. Here, we demonstrate that FGF14 interacts directly with multiple voltage-gated Na(+) (Nav) channel alpha subunits heterologously expressed in non-neuronal cells or natively expressed in a murine neuroblastoma cell line. Functional studies reveal that these interactions result in the potent inhibition of Nav channel currents (I(Na)) and in changes in the voltage dependence of channel activation and inactivation. Deletion of the unique amino terminus of the splice variant of Fgf14, Fgf14-1b, or expression of the splice variant Fgf14-1a modifies the modulatory effects on I(Na), suggesting an important role for the amino terminus domain of FGF14 in the regulation of Na(v) channels. To investigate the function of FGF14 in neurones, we directly expressed Fgf14 in freshly isolated primary rat hippocampal neurones. In these cells, the addition of FGF14-1a-GFP or FGF14-1b-GFP increased I(Na) density and shifted the voltage dependence of channel activation and inactivation. In fully differentiated neurones, FGF14-1a-GFP or FGF14-1b-GFP preferentially colocalized with endogenous Nav channels at the axonal initial segment, a critical region for action potential generation. Together, these findings implicate FGF14 as a unique modulator of Nav channel activity in the CNS and provide a possible mechanism to explain the neurological phenotypes observed in mice and humans with mutations in Fgf14.

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Figures

Figure 1
Figure 1. FGF14 interacts with Nav α subunits
A, sequence alignment of FGF14-1a, FGF14-1b, FGF12-1b, FGF13-1b and the conserved FGF core domain (derived from sequence alignment of all mouse FGFs). The N-terminal region of FGF14 is encoded by alternative, divergent exons (‘Exon1/NT’). The critical 41 amino acid Nav channel-binding domain of FGF12-1b is underlined. Two human dominant mutations linked to spinocerebellar ataxia are marked by an arrowhead (F149S; Van Swieten et al. 2003) and a filled circle (frameshift mutation; Dalski et al. 2005). Identical and conserved amino acid residues are shaded. B, FGF14-1a–Myc, FGF14-1b–Myc, FGF14ΔNT–Myc or hSpry–Myc was expressed with human Nav1.5 or Nav1.1 in HEK293 cells. Whole cell lysates were immunoprecipitated with anti-Myc-agarose (IP:Myc) and immunoblots (IB) were performed with either anti-Myc or anti-pan-Nav channel antibodies. Arrowheads indicate Nav channels (∼250 kDa).
Figure 2
Figure 2. Functional modulation of Nav1.5 and Nav1.1 by FGF14
A, representative whole-cell voltage-gated inward Na+ currents (INa) recorded from HEK-hNav1.5 cells transiently expressing GFP, FGF14-1a–GFP, or FGF14-1b–GFP in response to voltage steps from −90 to +60 mV from a holding potential of −130 mV (inset). B, peak current densities measured in individual HEK-hNav1.5 cells expressing GFP, FGF14-1a–GFP, FGF14-1b–GFP or GFP-FGF14ΔNT; horizontal bars represent mean values (**P < 0.005, ***P < 0.0005 for FGF14–GFP-expressing, compared to GFP-expressing cells). C, voltage dependences of INa activation and steady-state inactivation. For activation, conductances in individual cells at each test potential were calculated and normalized to the conductance measured at −20 mV in the same cell; mean ±s.e.m. normalized values are plotted. For inactivation, currents measured during the test pulse to −10 mV (see Methods) from each prepulse potential were normalized to the value measured on depolarization from −130 mV in the same cell. Mean ±s.e.m. normalized values are plotted as a function of prepulse potential. The fitted parameters are provided in Table 1. D, representative whole-cell INa recordings from HEK293 cells coexpressing Nav1.1 and GFP, FGF14-1a–GFP or FGF14-1b–GFP; the voltage protocol is illustrated in the inset. E, peak current densities for all HEK-Nav1.1 cells are shown, and horizontal bars represent mean values (***P < 0.0005 for FGF14-1b–GFP expressing cells compared to GFP expressing cells). F, voltage dependences of channel activation and steady-state inactivation are determined as described above; fitted parameters are provided in Table 1.
Figure 3
Figure 3. FGF14-1b binds to Nav α subunits and modulates INa in Neuro2A cells
A, RT-PCR analysis of Nav subunit expression in Neuro2A cells. Control cDNA was from adult rat brain (lane B), E15 rat heart (lane H) and adult rat dorsal root ganglia (lane D). Adult brain cDNA is positive for Nav1.1, 1.2, 1.3, 1.6, for β1-β4 and Gapdh; E15 heart cDNA is positive for Nav1.4 and 1.5 and for β1–β4; and adult DRG cDNA is positive for Nav1.7, 1.8, 1.9 and β1–β4. In Neuro2A cells, Nav1.7 is robustly expressed; Nav1.2, Nav1.3 and Nav1.4 are also detected. Note selective expression of β1 and β3 in Neuro2A cells. B, expression of endogenous Nav α subunits and coimmunoprecipitation with FGF14-1b–Myc. Cells were transfected with Fgf14-1b–Myc or hSpry–Myc(−). Robust expression of both proteins is evident in immunoblots (IB) of fractionated proteins prepared from transfected cells. Endogenous Nav α subunit expression was detected using an anti-pan-Nav α subunit specific antibody. Whole cell lysates were immunoprecipitated (IP) with anti-Myc-agarose and probed (IB) with either an anti-Myc or an anti-pan-Nav channel antibody. Arrowheads indicate Nav α subunits (∼250 kDa). C, representative whole-cell INa recorded from Neuro2A cells transiently expressing GFP, FGF14-1a–GFP or FGF14-1b–GFP; the voltage protocol is illustrated in the inset. D, peak current densities measured in individual Neuro2A cells expressing GFP, FGF14-1a–GFP or FGF14-1b–GFP; horizontal bars represent mean values (**P < 0.005). E, voltage dependences of Nav channel activation and steady-state inactivation were determined as described in the legend to Fig. 2; fitted parameters are provided in Table 1.
Figure 4
Figure 4. FGF14 expression and function in hippocampal neurones
A, expression of Fgf14 in the CA1 and CA3 regions in the dentate gyrus of postnatal day 11 mouse hippocampus. B, expression of Fgf14 is also readily detected in rat hippocampal neurones maintained in vitro for 10 days (DIV10). C and D, representative whole-cell INa recorded from postnatal rat hippocampal neurones expressing GFP (C), FGF14-1a–GFP (D), FGF14-1b–GFP (E); the voltage protocol is illustrated in the inset. E, peak current densities measured in individual hippocampal neurones expressing GFP, FGF14-1a–GFP or FGF14-1b–GFP are illustrated; horizontal bars represent mean values (*P < 0.05). F, voltage dependences of channel activation and steady-state inactivation were determined as described in the legend to Fig. 2; fitted parameters are presented in Table 1.
Figure 5
Figure 5. Co-localization of native Nav channels and FGF14 at the axonal initial segment of hippocampal neurones
Rat hippocampal neurones were transfected with Fgf14-1a–GFP (A–E), Fgf14-1b–GFP (F–J) or GFP (K–O). Fluorescence images revealed a distinct distribution pattern for FGF14-1a–GFP (A) and FGF14-1b–GFP (F) compared to GFP (K). Fluorescence signals from anti-Nav1.2 and anti-MAP2 were visualized in the red (B, G, L) and blue (C, H, M) channels, respectively. Overlays of colour channels are shown (D, E, I, J, N, O). FGF14-1a–GFP and FGF14-1b–GFP were preferentially targeted to MAP2-negative processes (E, J) and colocalized with Nav1.2 (D, I), while GFP was distributed homogeneously (N, O). Arrows indicate the axon initial segment region in the three sets of images. Confocal images of another neurone transfected with Fgf14-1b–GFP (P) showing GFP fluorescence localized in the AIS. Nav channels, detected with an anti-pan-Nav channel antibody (Q), appear colocalized with FGF14-1b–GFP in the merged image (R). S, AIS enrichment Index (fluorescence intensity ratio in the AIS versus dendrites) was measured in GFP- (0.96 ± 0.06, n = 4), in FGF14-1a–GFP- (3.46 ± 0.22, n = 5, P < 0.05 compared to GFP) and in FGF14-1b–GFP-expressing neurones (3.39 ± 0.4, n = 5, P < 0.05 compared to GFP). Scale bars = 10 μm.
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