Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels
1 Departments of Molecular Biology & Pharmacology Anatomy & Neurobiology Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA
2 Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA
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 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 (INa) 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 INa, suggesting an important role for the amino terminus domain of FGF14 in the regulation of Nav 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 INa 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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Introduction
Voltage-gated Na+ (Nav) channels are responsible for the rapidly rising phase of action potentials in neurones and myocytes. Nav channels are heteromeric, consisting of a pore-forming subunit (Nav1.x), associated with subunits (1 to 4) (Dib-Hajj et al. 1998; Smith et al. 1998; Qu et al. 1999; Catterall, 2000; Yu et al. 2003; Grieco et al. 2005). To date, 10 subunit genes have been identified: Nav1.1–Nav1.9 and NaX. Heterologous expression of any one of the Nav1.1–1.9 subunits alone results in functional channels with rapidly inactivating currents (INa), each with distinct biophysical properties and pharmacological sensitivities (Goldin, 1999, 2000). Nav1.x subunits are differentially expressed, with Nav1.1, 1.2, 1.3 and 1.6 being primarily expressed in the central and peripheral nervous systems (Schaller et al. 1995; Schaller & Caldwell, 2000), and Nav1.7, 1.8 and 1.9 being expressed preferentially in the peripheral nervous system (Toledo-Aral et al. 1997; Vijayaragavan et al. 2004). Nav1.4 is expressed in adult skeletal muscle and Nav1.5 is expressed in cardiac muscle (Goldin, 2001). In neurones, Nav channels are concentrated at the axon initial segments (AIS) and at the nodes of Ranvier, specialized domains formed by a complex matrix of proteins (Salzer, 2002). Interactions of the intracellular regions of Nav channels with other proteins are required to maintain their functional activity and proper subcellular localization (Peles & Salzer, 2000).
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Intracellular fibroblast growth factors (FGFs) 11–14 lack amino (N)-terminal secretory signal peptides yet they retain the conserved FGF domain primary sequence and the FGF -trefoil structure (Fig. 1) (Munoz-Sanjuan et al. 2000; Olsen et al. 2003). Two members of this FGF subfamily have been found to interact with, and to modify, the properties of heterologously expressed Nav channels (Liu et al. 2001, 2003; Wittmack et al. 2004). FGF12-1b/FHF1B binds Nav1.5 and induces a hyperpolarizing shift in the voltage dependence of Nav1.5 channel inactivation (Liu et al. 2003). FGF13-1b/FHF2B increases Nav1.6 current density and induces a depolarizing shift in voltage dependence of Nav1.6 channel inactivation (Wittmack et al. 2004).
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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, FGF14NT–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).
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Two alternatively spliced FGF14 isoforms, FGF14-1a and FGF14-1b, are highly homologous to both FGF12-1b and FGF13-1b but contain divergent amino termini (Fig. 1). FGF14-1b is abundantly expressed in the central nervous system and FGF14-1a is prominently expressed in the thymus and, at low levels, in the central nervous system (Smallwood et al. 1996; Wang et al. 2000). Mice deficient in FGF14 develop ataxia and dystonia early in postnatal development and, as adults, show reduced response to dopamine agonists and increased sensitivity to epileptogenic compounds (Wang et al. 2002). Humans harbouring mutations in FGF14 develop early onset spinocerebellar ataxia (SCA) and have cognitive impairments (Van Swieten et al. 2003; Dalski et al. 2005).
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Here, we demonstrate a direct interaction with, and a unique ability to inhibit, cardiac and neuronal Nav channel subunits by both FGF14 isoforms in heterologous expression systems. We show that removal of the FGF14-1b N-terminus or expression of the Fgf14 splice isoform, Fgf14–1a, modifies the ability of FGF14 to suppress INa. In addition, FGF14-1b directly interacts with native Nav channels in neuroblastoma cells, resulting in markedly reduced INa densities. Overexpression of either FGF14-1a–green fluorescent protein (GFP) or FGF14-1b–GFP in freshly isolated hippocampal neurones, however, results in increased Nav current densities, an effect that is expected to increase the excitability of these cells. In mature hippocampal neurones maintained for several days in vitro, FGF14-1a–GFP and FGF14-1b–GFP are both enriched at the axonal initial segment and colocalized with native neuronal Nav channels.
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Methods
Plasmids and antibodies
Fgf14–1a, Fgf14–1b and Fgf14NT were amplified by PCR from a human brain cDNA library and cloned into pCS2-MT (Roth et al. 1991). GFP fusion proteins were made by subcloning Fgf14 from pCS2-MT into pQBI25-fC or -fN vectors (QBiogene, Irvine, CA, USA). Fgf14–1b was also subcloned into pIRES2-EGFP (Clontech, Palo Alto, CA, USA) to generate the bicistronic construct Fgf14–1b-IRES-EGFP (Fig. 1B). To provide FGF14NT with a transcription initiation codon, the amino acid sequence ‘MESK’ was added using PCR. The MESK sequence corresponds to the amino-terminal sequence encoded by the first exon of FGF12-1b. This sequence was not included in the construction of GFP–FGF14NT. hSpry-Myc was made by cloning the human Sprouty 2 cDNA into the pCS2-MT vector. Full-length human Nav1.1 expression vector (pScn1a) was obtained from A. George (Vanderbilt University, Nashville, TN, USA).
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Monoclonal anti-Myc, anti-HA and goat anti-mouse–horseradish peroxidase (HRP) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A monoclonal anti-pan-Nav channel antibody was purchased from Sigma (St Louis, MO, USA). Rabbit anti-Nav1.2 was purchased from Upstate Signalling (Charlottesville, VA, USA). Monoclonal mouse IgG1 anti-MAP2 was purchased from Chemicon (Temecula, CA, USA). Alexa fluorescent-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) were used as needed.
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Cell culture and transient transfection
All reagents were purchased from Sigma unless otherwise noted. HEK293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 4 mML-glutamine and 10% fetal bovine serum, 100 U ml–1 penicillin and 100 μg ml–1 streptomycin and incubated at 37°C with 5% CO2. The stable cell line HEK-hNav1.5 was maintained similarly, but media contained 500 μg ml–1 G418 (Invitrogen). Cells were transfected at 90–100% confluency using Lipofectamine and PLUS reagent (Invitrogen) according to the manufacturer's instructions.
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Rat hippocampal cultures were prepared from 18-day-old rat embryos by previously described methods (Goslin et al. 1998). Time mated female rats were killed by carbon dioxide asphyxiation using a protocol approved by the Washington University Animal Studies Committee. Embryos were isolated, placed on ice, decapitated, and hippocampi were dissected and dissociated using trypsin and trituration through a Pasteur pipette. Neurones were plated at low density (1–5 x 105 cells/dish) on poly L-lysine-coated coverslips in 60 mm culture dishes in minimum essential medium (MEM) supplemented with 10% horse serum. After 2–4 h, coverslips containing neurones were inverted over a glial feeder layer in serum-free MEM with N2 supplements, 0.1% ovalbumin, and 1 mM pyruvate (N2.1 medium; components from Invitrogen). The neurones grew over the feeder layer but were kept separate from the glia by wax dots on the neuronal side of the coverslips. To prevent the overgrowth of the glia, neurone cultures were treated with cytosine arabinoside (5 μM; Calbiochem, La Jolla, CA, USA) for 3 days in vitro (DIV). Cultures were maintained in N2.1 medium for up to 10 days. Neurones were chronically treated with 100 μMD,L-2amino-5-phosphonovaleric acid (APV, Research Biochemicals, Natick, MA, USA). Transfections were performed at DIV9 for imaging and at DIV0 for electrophysiology using Lipofectamine 2000 (Invitrogen).
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Immunofluorescence
Rat hippocampal neurones (DIV10) were fixed in freshly made 4% paraformaldehyde and 10% sucrose in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.25% Triton X-100. After blocking with 10% BSA for 30 min at 37°C, neurones were incubated at room temperature for 12–16 h with the following primary antibodies: polyclonal rabbit anti-Nav1.2 (1 : 1000), monoclonal IgG1 anti-pan-Nav (1 : 100) and monoclonal mouse IgG1 anti-Map2 (1 : 2000), diluted in PBS containing 3% BSA. Neurones were then washed three times in PBS and incubated for 2 h at 37°C with appropriate secondary antibodies: Alexa 568 anti-rabbit or Alexa 647 anti-IgG1 (both at 1 : 500) together with aminomethylcoumarin (AMCA) anti-rabbit (1 : 100, Vector Laboratories, Burlingame, CA, USA). Neurones were then washed three times with PBS and coverslips were mounted in elvanol (Tris-HCl, glycerol, and polyvinyl alcohol with 2% 1,4-diazabicyclo[2,2,2]octane). Images were acquired using an Axoplan 2 epifluorescence microscope (Zeiss, Oberkochen, Germany) with a 63x (1.4 NA) objective or an Olympus FV500 confocal microscope with 488 nm and 633 nm laser lines with a 60x objective (1.4 NA objective). Images were acquired with a CCD camera and MetaVue Software for the epifluorescence microscope (Universal Imaging Corp., Downingtown, PA, USA) and with Fluoview Software for the confocal microscope. Optical sections of 0.2 μm were acquired and averaged in a stack of three (final thickness = 0.6 μm).
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Image quantification
For the analysis of the AIS enrichment of FGF14-1a–GFP and FGF14-1b–GFP, samples were colabelled with anti-Nav1.2 (AIS marker) and anti MAP2 (dendritic marker) antibodies. Images were acquired using an Axoplan 2 epifluorescence microscope (Zeiss) with a 63x (1.4 NA) objective. A mask of the axonal initial segment and a mask of the dendrites were first generated by thresholding, respectively, the Nav1.2 or MAP2 images. The masks of the corresponding regions were then applied to the unthresholded GFP images. The AIS enrichement index (AEI) was defined as the ratio of total fluorescence intensity/area(AIS) divided by the total fluorescence intensity/area(dendrites). GFP had an AEI of 1, as expected for a uniformly distributed non-polarized protein (Rivera et al. 2003). Analysis was done using MetaMorph software (Universal Imaging).
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Immunoprecipitations
Transfected HEK293 cells or Neuro2A cells were washed twice with PBS and lysed in modified RIPA buffer (mM): 10 Tris-HCl, 150 NaCl, 1 EDTA, 1% NP-40, 0.25% sodium deoxycholate. Protease inhibitor cocktail and phenylmethanesulphonyl fluoride (PMSF) were added immediately before cell lysis. Cell extracts were collected and sonicated for 20 s, incubated for 10 min and centrifuged at 4°C, 15 000 g for 15 min. Supernatants were collected and precleared with non-immune rabbit-IgG-agarose beads for 1 h at 4°C. Rabbit anti-Myc-agarose beads (Sigma) were incubated with the supernatants overnight at 4°C with agitation and then washed 5 times with lysis buffer before adding 6x sample buffer containing 5 mM tris(2-carboxyethyl)phosphine (TCEP). Mixtures were heated for 5 min at 95°C and resolved on 4–15% polyacrylamide gradient gels (Bio-Rad, Hercules, CA, USA). Resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) overnight at 4°C and blocked in Tris-buffered saline (TBS) with 5% skimmed milk and 0.1% Tween-20. Membranes were then incubated in blocking buffer containing monoclonal anti-Myc (1 : 1000) or anti-pan-Nav channel (1 : 5000) antibodies for 1 h. Washed membranes were incubated with goat anti-mouse-HRP (1 : 5000) detected with SuperSignal Pico chemiluminescent substrate (Pierce, Rockford, IL, USA).
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Nav subunit expression
RNA was prepared from Neuro2A cells, E13.5 heart, E16.5 whole embryo, adult dorsal root ganglion (DRG) and adult brain. cDNA was prepared using the ProMega Reverse Transcription System according to the manufacturer's protocol. PCR amplification used 40 ng cDNA in a 20 μl reaction volume with the primers listed below. PCR cycle conditions: 68°C, 5 min; 35 cycles (93°C, 40 s; 68°C, 30 s); 68°C, 10 min. For primers and amplicon sizes see Supplemental material. Note that amplicon sizes for each Nav subunit are unique and that primers were chosen to minimize the chance of non-specific amplification of an incorrect Nav subunit.
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In situ Hybridization
A 550 bp mouse Fgf14 probe (Wang et al. 2000) was labelled with digoxigenin following the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN, USA). Free-floating brain sections (30 μm) were washed twice in PBS and treated with freshly prepared 10 μg ml–1 proteinase K (Invitrogen) at 37°C. After acetylation, sections were incubated in hybridization buffer containing 0.2 μg ml–1 digoxigenin-labelled riboprobe at 43°C overnight. Hybridized sections were washed by successively immersing in 4x SSC (mM: 150 NaCl and 15 sodium citrate, pH 7.0, room temperature), 2x SSC containing 50% formamide (50°C, 30 min), 2x SSC (37°C, 10 min), 2x SSC containing 20 μg ml–1 RNase A (37°C, 30 min), 2x SSC (37°C, 20 min), and 0.1 x SSC (room temperature, 10 min). The hybridization signals were detected with the digoxigenin detection reagents (Roche Diagnostics) and photographed on a Zeiss Axioskop microscope.
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Electrophysiology
Transfected cells were dissociated and re-plated at low-density approximately 12 h post-transfection. Recordings were performed at room temperature (20–22°C) 12–18 h post-transfection (for both rat hippocampal neurones and mammalian cell lines) using an Axopatch 1D amplifier (Axon Instruments, Union City, CA, USA). Borosilicate glass pipettes with resistance of 1–2 M (HEK293 and Neuro2A) or 2.5–5 M (hippocampal neurones) were made using a P-87 Micropipette Puller (Sutter Instruments, Novato, CA, USA). The recording solutions were as follows: extracellular (mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 20 Hepes, pH 7.3; intracellular: 140 CsF, 1 EGTA, 10 NaCl, 10 Hepes, pH 7.3. After seal formation and cell break-in, membrane capacitance was calculated by integrating the area under the capacitative transients recorded in response to a 5 ms hyperpolarizing test pulse from –70 mV to –80 mV. Capacitative transients and series resistances were compensated electronically by 80–90%. Data were acquired at 50 kHz and filtered at 5 kHz prior to digitization and storage. All experimental parameters were controlled by Clampex 9.2 software (Axon Instruments) and interfaced to the electrophysiological equipment using a Digidata 1322A analog–digital interface (Axon Instruments).
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Voltage-dependent inward currents were evoked by depolarizations to test potentials between –100 mV and +60 mV from a holding potential (between –130 mV and –90 mV). Steady-state fast inactivation of Nav channels was measured with a paired-pulse protocol. From the holding potential, cells were stepped to varying test potentials between –130 mV and –10 mV (prepulse) prior to a test pulse to –10 mV or –20 mV. The extracellular solution used to record INa in neurones contained bicuculline (10 μM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 μM) and D,L-2amino-5-phosphonovaleric acid (APV, 100 μM) to block synaptic activity mediated, respectively, by GABA, AMPA and NMDA receptors.
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Data analysis
Current densities were obtained by dividing INa amplitude by membrane capacitance. Current–voltage relationships were generated by plotting current density as a function of the test potential. Conductance GNa was calculated by the following equation:
where INa is the current amplitude at voltage Vm, and Erev is the Na+ reversal potential. Steady-state activation curves were derived by plotting normalized GNa as a function of test potential and fitted using the Boltzmann equation:
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where GNa,Max is the maximum conductance, Va is the membrane potential of half-maximal activation, Vm is the membrane voltage and k is the slope factor.
For steady-state inactivation, normalized current amplitude (INa/INa,Max) at the test potential was plotted as a function of prepulse potential (Vm) and fitted using the Boltzmann equation:
where Vh is the potential of half-maximal inactivation and k is the slope factor.
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Data analysis was performed using Clampfit 9.2 software (Axon Instruments) and Prism 4 software (GraphPad, San Diego, CA, USA). Results are presented as means ±S.E.M. and the statistical significance of differences between groups was assessed using Student's t test and was set at P < 0.05.
Results
FGF14 interacts with multiple Nav channel subunits
Primary sequence comparison showed a high degree of sequence similarity between the core domain of FGF14 and two previously described intracellular FGFs, FGF12-1b and FGF13-1b (Fig. 1A) (Liu et al. 2001, 2003; Wittmack et al. 2004). The Fgf14 gene is alternatively spliced and encodes proteins with distinct amino-termini, FGF14-1a and FGF14-1b (65 and 69 amino acid residues, respectively) but are otherwise identical in the core FGF domain and carboxy-terminus (Wang et al. 2002). Two recently reported mutations in Fgf14, linked to autosomal dominant spinocerebellar ataxia (SCA), occur in the conserved FGF domain (Fig. 1A) (Van Swieten et al. 2003; Dalski et al. 2005).
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The Nav channel-interacting domain on FGF12-1b is localized to the amino terminal end of the FGF domain, amino acid residues 1–41 (Fig. 1A, underline), and shows 68% amino acid identity with the corresponding regions of FGF14-1a and FGF14-1b and 76% amino acid identity with FGF13-1b (Liu et al. 2001; Liu et al. 2003). Initial experiments were therefore focused on determining if FGF14 also interacts directly with Nav subunits using biochemical assays. For these experiments, FGF14-1a, FGF14-1b and an N-terminal deletion mutant (FGF14NT) were fused with six copies of the Myc epitope tag to generate FGF14-1a–Myc, FGF14-1b–Myc, and FGF14NT–Myc, respectively. We tested the ability of these tagged FGF14 molecules to interact with full-length cardiac and neuronal Nav subunits in HEK293 cells stably expressing human Nav1.5 and in wild-type HEK293 cells transiently expressing human Nav1.1 (Fig. 1B). Approximately 24 h after cotransfection with FGF14-1a–Myc, FGF14-1b–Myc, FGF14NT–Myc and hSpry–Myc, cell lysates were collected and immunoprecipitated with an anti-Myc antibody (Fig. 1B). Expression of Nav1.5 and Nav1.1 were readily detected using an anti-pan-Nav subunit antibody directed against the intracellular loop between domains III and IV. As illustrated in Fig. 1B, Nav1.5 and Nav1.1 coimmunoprecipitated with both FGF14-1a-Myc and FGF14-1b-Myc. Interestingly, deletion of the FGF14 N-terminus abolished binding to Nav1.5, but not to Nav1.1. These data demonstrate that FGF14 can interact with several Nav subunits and suggest that the amino terminal domain(s) of FGF14 may modulate these interactions.
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FGF14-1b inhibits INa in HEK293 cells expressing Nav1.5 or Nav1.1
To explore the functional significance of the biochemical interaction between FGF14 and Nav subunits, whole-cell voltage-clamp recordings were obtained from HEK293 cells coexpressing Nav subunits and FGF14. In initial experiments, GFP (control) or one of the GFP-tagged Fgf14 constructs (Fgf14-1a–GFP, Fgf14-1b–GFP or GFP-Fgf14NT) were transfected into HEK293 cells stably expressing human Nav1.5 (HEK-Nav1.5). Approximately 12–18 h after transfection, fluorescent cells were identified and whole-cell recordings were obtained (Fig. 2A and D). Robust, rapidly activating and inactivating inward currents (INa) were observed in GFP-expressing (control) cells, similar to non-fluorescent cells (not shown). INa amplitudes in HEK-Nav1.5 cells were markedly lower in cells coexpressing FGF14-1a–GFP or FGF14-1b–GFP compared with cells expressing GFP alone (Fig. 2A). In all experiments, INa was stable for the duration (5–10 min) of the recordings regardless of the coexpressed fluorescent protein. In addition, INa in all transfection groups exhibited typical current–voltage relationships, i.e. maximal INa was observed to occur at approximately –20 mV and the currents reversed at approximately 45 mV (not shown).
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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-FGF14NT; 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.
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Although there was considerable variability in peak INa densities recorded from individual Nav1.5 + GFP-expressing cells (Fig. 2B), coexpression of FGF14-1b–GFP or FGF14-1a–GFP led to a 91 ± 3% (P < 0.0005) and a 45 ± 8% (P < 0.005), respectively, decrease in mean current density. Co-expression of GFP–FGF14NT did not measurably affect peak INa densities in Nav1.5-expressing cells (Fig. 2B). To exclude the possibility that the C-terminal GFP tag on FGF14-1b contributed to the observed attenuation of INa, an FGF14-1b construct with an N-terminal GFP tag (GFP–FGF14-1b) and an untagged FGF14-1b (using a bicistronic vector, FGF14-1b-IRES–GFP) were also generated. Expression of either of these constructs in HEK-Nav1.5 cells resulted in mean ±S.E.M. peak INa densities not significantly different from those determined in cells expressing FGF14-1b–GFP. The mean ±S.E.M. peak INa densities were 103 ± 34 pA pF–1 for GFP–FGF14-1b (n= 10) and 103 ± 29 pA pF–1 for FGF14-1b-IRES–GFP (n= 12). Co-expression of FGF14-1b–GFP in cells expressing Nav1.1 also resulted in marked attenuation in peak INa (Fig. 2D). In contrast to the findings for Nav1.5, Nav1.1 cells coexpressing FGF14-1a–GFP were indistinguishable from controls (Fig. 2D). INa amplitudes in Nav1.1-expressing cells transfected with GFP-Fgf14NT were also not measurably different from control cells (Fig. 2E).
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The effects of FGF14 coexpression on the voltage-dependent properties of Nav1.5 and Nav1.1 were also measured (Fig. 2C and F). The voltage dependences of INa activation were determined by plotting normalized conductance as a function of test potential. Although FGF14 coexpression had no measurable effects on INa activation in HEK-Nav1.5 cells (Fig. 2C, Table 1), coexpression of FGF14-1a–GFP or GFP–FGF14NT resulted in small depolarizing and hyperpolarizing, respectively, shifts in the voltage dependence of Nav1.1 activation (Fig. 2F, Table 1). To examine steady-state inactivation of INa, a two-pulse voltage protocol was used (see Methods). In HEK-Nav1.5 cells, coexpression of FGF14-1b–GFP had a negligible effect on the half-maximal voltage (Vh) of inactivation (Table 1), whereas in HEK-Nav1.1 cells, coexpression of FGF14-1b–GFP resulted in a small, but statistically significant (P < 0.05), depolarizing shift in Vh (Fig. 2F, Table 1). In contrast to FGF14-1b, the coexpression of FGF14-1a or FGF14NT resulted in depolarizing or hyperpolarizing, respectively, shifts in the voltage dependences of Nav1.5 (Fig. 2C) and Nav1.1 (Fig. 2F) currents. No significant changes in the slope factors of INa activation or inactivation were observed (Table 1). These results indicate that the marked changes in INa densities in HEK-Nav1.5 and HEK-Nav1.1 cells coexpressing FGF14-1b do not reflect changes in Nav channel availability. Taken together, the results presented in Fig. 2 also suggest that the functional effects of the various FGF14 isoforms on the channels formed by different Nav subunits (Nav1.1 and Nav1.5) are distinct.
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FGF14-1b interacts with endogenous Nav subunits and modulates INa in Neuro2A cells
The results presented above demonstrate that FGF14 interacts strongly with recombinant heterologously expressed Nav channel subunits. Subsequent experiments were focused on exploring the possibility that FGF14 also interacts with, and modulates, the activity of Nav channels endogenously expressed in neuronal cells. Specifically, we examined the ability of FGF14-1b-Myc to interact with native Nav channels expressed in the murine neuroblastoma cell line, Neuro2A. Neuro2A cells are known to have endogenous voltage-gated Na+ currents and can be induced to differentiate into neurone-like cells (Hamasaki et al. 1996; Carpaneto et al. 1999). Because the Nav subunits expressed in Neuro2A cells had not been previously identified, initial experiments used RT-PCR to examine the expression of individual Nav channel - and subunits (Fig. 3A). Unique primers for each of the murine Nav channel (Nav1.1–Nav1.9) and (1–4) subunits were used to amplify cDNA generated from Neuro2A cells (see Methods). Control (murine) cDNA samples from adult brain, dorsal root ganglion and embryonic heart were used as positive controls. All Nav channel primer sets produced amplicons of the predicted size in at least one of the control cDNA samples, but were absent from others, indicating tissue specific expression of Nav channel and subunits consistent with published expression patterns. As illustrated in Fig. 3A, Neuro2A cells expressed multiple Nav channel subunits, including Nav1.2, 1.3, 1.4 and 1.7 and two subunits, 1 and 3. The Nav1.7 subunit, which is primarily expressed in the peripheral nervous system, appears to be the predominant Nav subunit in Neuro2A cells (Fig. 3A).
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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 1-4 and Gapdh; E15 heart cDNA is positive for Nav1.4 and 1.5 and for 1–4; and adult DRG cDNA is positive for Nav1.7, 1.8, 1.9 and 1–4. In Neuro2A cells, Nav1.7 is robustly expressed; Nav1.2, Nav1.3 and Nav1.4 are also detected. Note selective expression of 1 and 3 in Neuro2A cells. B, expression of endogenous Nav 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 subunit expression was detected using an anti-pan-Nav 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 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.
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To determine if FGF14-1b interacts with endogenous Nav channels, a Myc tagged construct, Fgf14-1b–Myc, and a Myc tagged control construct, hSpry–Myc, were individually transfected into Neuro2A cells. Cell lysates were prepared and used for Western blot analysis or were immunoprecipitated with anti-Myc beads. Immunoblots using an anti-Myc or an anti-pan-Nav subunit antibody revealed expression of the Myc tagged constructs and robust endogenous Nav channel subunit expression (Fig. 3B). Western blots of proteins immunoprecipitated with anti-Myc revealed that Nav subunits coimmunoprecipitate with FGF14-1b–Myc, but not with the Myc-tagged hSpry–Myc control protein (Fig. 3C).
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The effects of FGF14 coexpression on Nav channel function was assessed in whole-cell recordings from Neuro2A cells transiently transfected with GFP, Fgf14-1a–GFP or Fgf14-1b–GFP. Whole-cell recordings from GFP-positive control cells revealed robust voltage-dependent inward Na+ currents (Fig. 3C), consistent with the expression of Nav subunits shown by RT-PCR (Fig. 3A) and immunoblot (Fig. 3B) analyses. In FGF14-1a–GFP-expressing cells, INa densities were attenuated but not statistically significantly (P= 0.07) lower than in cells expressing GFP alone (Fig. 3C). In FGF14-1b–GFP-expressing cells, INa densities were significantly (P < 0.005) lower than in cells expressing GFP alone (Fig. 3C); current densities in individual cells are plotted in Fig. 3D. The mean current density in FGF14-1b–GFP-expressing cells was 10 ± 3% of control cells (Table 1).
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The voltage dependences of Nav channel activation and inactivation in Neuro2A cells were also examined and are plotted in Fig. 3E. Analysis of the properties of the currents revealed a large (13 or 15 mV) depolarizing shift in Vh in FGF14-1a–GFP- or FGF14-1b–GFP-expressing cells compared to wild-type Neuro2A cells expressing GFP alone (Table 1). Interestingly, the magnitude of the shift in Vh in Neuro2A cells is substantially larger than that seen for Nav1.5 or Nav1.1-encoded currents in HEK-293 cells, suggesting that the modulatory effects of the two splice variants of FGF14 are Nav channel subunit-specific and/or cell type-specific. Importantly, however, and similar to the findings for Nav1.1 and Nav1.5 currents, the marked reductions in endogenous INa caused by FGF14-1b isoform cannot be attributed to changes in the voltage-dependent properties of the currents. Indeed, FGF14-1b markedly increased (not decreased) Nav channel availability at more hyperpolarized potentials (Fig. 3E).
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FGF14-1b modulates INa and co-localizes with native Nav channel subunits in hippocampal neurones
The results presented above demonstrate that FGF14 can bind to, and modulate, the properties of recombinant Nav channels expressed in HEK293 cells and endogenously expressed Nav channels in Neuro2A cells. Previous studies suggest that Fgf14-1b is highly expressed in the central nervous system (Wang et al. 2000). Genetic ablation of Fgf14 or missense mutations in the Fgf14 gene are linked to neurological phenotypes associated with disorders of the central nervous system, including cognitive dysfunction in humans and learning and memory deficits in mice (M. Xiao, D. Wozniak & D. Ornitz, unpublished data). In the hippocampus, Fgf14 is highly expressed in pyramidal cells in the CA1 and CA3 regions, and in granule cells of the dentate gyrus (Fig. 4A). Fgf14 is also expressed in isolated hippocampal neurones in vitro (Fig. 4B). We therefore investigated whether FGF14-1a and FGF14-1b could affect the properties of the Nav channels expressed in hippocampal neurones. FGF14-1a–GFP or FGF14-1b–GFP was transfected into freshly isolated (E18) rat hippocampal neurones prior to plating, and whole cell INa were recorded from GFP-positive cells within 18 h of plating. In a previous study, quantitative RT-PCR analysis indicated that hippocampal neurones at this stage (P1) express Nav1.1, Nav1.2, Nav1.3 and Nav1.6 subunits (Schaller & Caldwell, 2000). Hippocampal neurones at this developmental stage in vitro lack neurites, allowing good spatial control of the membrane voltage and the clear resolution of INa. Interestingly, INa densities were significantly higher in both FGF14-1a–GFP- and FGF14-1b–GFP-expressing neurones (Fig. 4D), compared to GFP-expressing control neurones (Fig. 4C). Although INa densities in individual cells were quite variable (Fig. 4E), mean INa density was 83 ± 30% and 54 ± 17% higher in cells expressing, respectively, FGF14-1a or FGF14-1b (P < 0.05, Table 1). Small, but statistically significant, differences in the voltage dependences of INa activation and inactivation were also observed in the cells expressing FGF14-1a or FGF14-1b, compared with control (GFP-expressing) neurones (Fig. 4F). Taken together, these results demonstrate that both FGF14 isoforms can modulate properties of native Nav channels expressed in central (hippocampal pyramidal) neurones.
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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.
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In transfected hippocampal pyramidal neurones maintained in vitro for several days, both FGF14-1a–GFP and FGF14-1b–GFP were found to be enriched in the proximal regions of axons (Fig. 5). In these experiments, axons were identified as neuronal processes devoid of the microtubule-associated protein 2 (MAP2), a somato-dendritic marker (Kanai & Hirokawa, 1995). Immunolabelling with a Nav1.2 or a pan-Nav subunit-specific antibody revealed that Nav channel expression was high in the region of high FGF14-1a–GFP or FGF14-1b–GFP localization (n > 50, Fig. 5A–J). Previous studies have shown that the region of high Nav channel density corresponds to the axon initial segment (Garrido et al. 2003). In contrast, in cells expressing GFP, the GFP was not enriched in the Nav-positive (MAP2-negative) regions (n > 50, Fig. 5K–O). Quantification of fluorescence intensity showed that FGF14-1a–GFP and FGF14-1b–GFP were 3-fold more concentrated in the AIS than in dendrites, compared to GFP (Fig. 5S). Analysis of (0.6 μm) optical sections through the region of high Nav channel expression demonstrated colocalization of native Nav channels (anti pan-Nav channel antibody) and FGF14-1b–GFP (Fig. 5P–R). The findings that FGF14-1a–GFP and FGF14-1b–GFP modulate INa densities in hippocampal neurones and colocalize with endogenous Nav channels in the axon initial segment suggest that both FGF14 isoforms are likely to play a role in the regulation of neuronal membrane excitability.
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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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Discussion
FGF14 binds directly to Nav subunits
The intracellular subfamily of FGF-like molecules (FGFs11–14) are highly expressed in neuronal tissues. Fgf14 is particularly interesting in that it is expressed during development and in the adult CNS. Targeted deletion studies in mice and the discovery of mutations in Fgf14 in humans with autosomal dominant SCA revealed an essential role for FGF14 in the regulation of neuronal functioning (Wang et al. 2002; Van Swieten et al. 2003; Dalski et al. 2005). Previous reports that FGF12-1b and FGF13-1b bind to and influence the properties of Nav1.5, Nav1.6 and Nav1.9 channels established a link between this subfamily of FGFs and Nav channel function (Liu et al. 2001, 2003; Dib-Hajj et al. 2002; Wittmack et al. 2004). As a beginning step to understanding the mechanisms underlying the observed phenotypes in Fgf14-null mice and humans with missense mutations in Fgf14, studies were initiated to test the hypothesis that FGF14 interacts with Nav channel subunits and modulates Nav channel function. The data presented here demonstrate that FGF14-1a and FGF14-1b coimmunoprecipitate with both Nav1.1 and Nav1.5, suggesting a generalized biochemical interaction between FGF14 and members of the Nav channel family. We have also shown that FGF14 will coimmunoprecipitate with the carboxy terminal tail of Nav1.6 (data not shown), further supporting an interaction between FGF14 and neuronal sodium channels.
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FGF14 modulates heterologously expressed Nav channels
Experiments focused on examining the effects of FGF14 in murine neuroblastoma (Neuro2A) cells, which express multiple neuronal and non-neuronal Nav channel subunits and at least two of the Nav channel subunits, revealed that INa densities in these cells are reduced significantly with heterologous expression of FGF14-1b. Additional experiments also demonstrated that FGF14 modulates the properties of heterologously expressed (in HEK293 cells) Nav1.1 and Nav1.5 channels.
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In contrast to the reported hyperpolarizing shift in the voltage dependence of Nav1.5 channel inactivation produced on coexpression of FGF12-1b/FHF1B (Liu et al. 2003), the coexpression of FGF14-1b resulted in a dramatic voltage-independent suppression of Nav1.5 current density. The magnitude of the effect on current density depends on the 1b N-terminal domain; the current reduction by the 1a N-terminus is substantially less and FGF14NT showed no activity. Importantly, the N-terminal domain of FGF14-1b is unique to FGF14 and homologous sequences are not found in any other gene in the mouse or human genome databases. The marked reduction in INa (with FGF14-1b expression) is not accounted for by the presence of the GFP-tag, variations in cell size or alterations in the voltage dependence of channel activation or inactivation. Rather, it appears that FGF14-1b modulates functional Na+ current densities directly, probably by altering biophysical properties of individual Nav channels or by modulating channel trafficking or cell surface expression.
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Nav1.1-encoded current densities were also reduced significantly in cells cotransfected with FGF14-1b–GFP, whereas in cells coexpressing GFP–FGF14NT or FGF14-1a–GFP, INa densities were not significantly different from controls. These observations again point to an important functional role for the unique N-terminus of FGF14-1b in some cell types. In addition, these results suggest the presence of sequence-specific, and perhaps unique, interactions between different Nav subunits and the various FGF14 splice variants. Alternatively, there may be additional channel accessory subunits and/or channel regulatory proteins that are Nav subunit specific and may modulate the regulatory effects of the FGF14 variants on Nav activity.
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FGF14 and the regulation of neuronal membrane excitability
To investigate whether FGF14 affects the activity of Nav channels in primary neurones, rat hippocampal neurones were transfected with FGF14–GFP. Interestingly, compared to control transfected neurones, peak INa densities were increased in freshly isolated hippocampal neurones over-expressing FGF14-1a–GFP or FGF14-1b–GFP. Furthermore, small, but statistically significant, shifts in the voltage dependences of INa activation (–5 mV) and inactivation (+6 mV) were observed with FGF14-1a or FGF14-1b coexpression. The molecular basis of the distinct effects of FGF14-1a and FGF14-1b on INa in primary hippocampal neurones, compared to Neuro2A cells and HEK293 cells, remains to be determined. However, several possibilities could be considered: (1) FGF14 might have variable effects on the activity of Nav channels depending on the phosphorylation states of the Nav (or ) subunit (Gasparini & Magee, 2002); (2) FGF14 may suppress INa in channels encoded by some Nav subunits while increasing the activity of other Nav channel types; (3) the activity of FGF14 may be modified by other proteins expressed in primary neurones that are either not expressed or are not modified in Neuro2A cells or HEK293 cells; and/or (4) hippocampal neurones already express high levels of endogenous FGF14 and may therefore also express other proteins that modify the ability of FGF14 to interact with, and modulate, Nav channel function.
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Of the above-mentioned possibilities, the simplest explanation of the results reported here is that FGF14 differentially affects Nav channels encoded by different Nav (and perhaps ) subunits. It might be that the effects of FGF14 in hippocampal neurones reflect the ability of FGF14 to directly interact with the carboxy terminal tail of Nav1.6 (data not shown), a neuronal sodium channel that is found abundantly in central neurones, including the somatodendritic and axonal domains of hippocampal neurones (Garrido et al. 2001, 2003). Interestingly, FGF13-1b has been shown to increase the density of Nav1.6-encoded currents when coexpressed in neuroblastoma cells (Wittmack et al. 2004). Future studies focused on exploring the role of Nav1.6 and the modulatory effects of FGF14 on Nav1.6-encoded INa are clearly warranted.
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The observation that FGF14-1a–GFP and FGF14-1b–GFP are preferentially colocalized with Nav channels at the axon initial segment of hippocampal neurones further suggests that FGF14 may be an important regulator of neuronal excitability. Although initially thought to be a critical region for action potential generation (Colbert & Johnston, 1996), more recent studies suggest that the axon initial segment more likely functions to ensure back propagation of action potentials (initiated at more distal segments of the axons) to the cell bodies and dendrites (Colbert & Pan, 2002). The increased concentration of FGF14 in this region and the ability of FGF14 to interact with several different Nav subunits suggest that FGF14 may play a pivotal role in regulating neuronal excitability and plasticity.
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Relationship to previous studies
Sequences in the N-terminal half of the conserved FGF domain (amino acid residues 1–41 in FGF12-1b) are essential for interactions with Nav subunits (Liu et al. 2001). This sequence is highly conserved throughout the FGF 11–14 subfamily. It is likely that this domain serves to anchor FGF14 to the C-terminal region of Nav subunits. Additionally, FGF14 could potentially compete with other proteins binding to the Nav subunit C-terminus. We propose a model in which multiple domains of FGF14 could interact with multiple domains of Nav channels. In this model, the unique 65 amino acid N-terminus of FGF14-1a or 69 amino acid N-terminus of FGF14-1b may function by interacting with other regions of the Nav subunit, or with other proteins in complex with the Nav subunit, to modulate INa. This interaction may lead to channel modifications such as phosphorylation, involving residues critical for channel availability (Ratcliffe et al. 2000; Cantrell et al. 2002; Carr et al. 2003) or direct interference with the channel pore. Interestingly, several members of the FGF 11–14 subfamily have been shown to interact with islet/brain 2, a mitogen-activated protein kinase scaffolding protein (Goldfarb, 2001; Schoorlemmer & Goldfarb, 2001, 2002) (Q. Wang, J. Lou & D. Ornitz, unpublished data.). FGFs 11–14 may thus serve to link kinase signalling complexes with Nav subunits. Another possibility is that the unique FGF14-1b N-terminus might sterically hinder the inward flow of Na+ ions near the intracellular vestibule of the pore. In addition, the fact that the FGF14-1b N-terminus contains two leucine-based motifs which have been implicated in protein trafficking (Bonifacino & Traub, 2003) raises the interesting possibility that the FGF14-1b splice isoform functions to regulate peak current density by affecting Nav channel subcellular localization and/or trafficking. Further studies to test each of these possibilities are clearly warranted.
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A recently identified missense mutation (F149S) in FGF14 is the aetiology of an autosomal dominant spinocerebellar ataxia (SCA) and mental retardation syndrome in humans (Van Swieten et al. 2003). Patients with this mutation develop early onset tremors and progressive ataxia in a manner strikingly similar to the phenotype of FGF14-null mice (Wang et al. 2002). Cognitive disturbances have also been found in FGF14-null mice (M. Xiao, D. Wozniak & D. Ornitz, unpublished observation). Although it was proposed that the F149S missense mutation destabilizes the FGF14 protein (Van Swieten et al. 2003), the dominant pattern of inheritance of the human syndrome and the recessive nature of the knockout mouse phenotype suggests that the F149S mutation may produce a protein that functions as a dominant negative. Clearly, further experiments focused on exploring the molecular mechanisms involved in mediating the modulatory effects of FGF14 on Nav channel functioning and/or determining the effects of disease mutations in Fgf14 will be of considerable interest.
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2 Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA
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 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 (INa) 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 INa, suggesting an important role for the amino terminus domain of FGF14 in the regulation of Nav 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 INa 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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Introduction
Voltage-gated Na+ (Nav) channels are responsible for the rapidly rising phase of action potentials in neurones and myocytes. Nav channels are heteromeric, consisting of a pore-forming subunit (Nav1.x), associated with subunits (1 to 4) (Dib-Hajj et al. 1998; Smith et al. 1998; Qu et al. 1999; Catterall, 2000; Yu et al. 2003; Grieco et al. 2005). To date, 10 subunit genes have been identified: Nav1.1–Nav1.9 and NaX. Heterologous expression of any one of the Nav1.1–1.9 subunits alone results in functional channels with rapidly inactivating currents (INa), each with distinct biophysical properties and pharmacological sensitivities (Goldin, 1999, 2000). Nav1.x subunits are differentially expressed, with Nav1.1, 1.2, 1.3 and 1.6 being primarily expressed in the central and peripheral nervous systems (Schaller et al. 1995; Schaller & Caldwell, 2000), and Nav1.7, 1.8 and 1.9 being expressed preferentially in the peripheral nervous system (Toledo-Aral et al. 1997; Vijayaragavan et al. 2004). Nav1.4 is expressed in adult skeletal muscle and Nav1.5 is expressed in cardiac muscle (Goldin, 2001). In neurones, Nav channels are concentrated at the axon initial segments (AIS) and at the nodes of Ranvier, specialized domains formed by a complex matrix of proteins (Salzer, 2002). Interactions of the intracellular regions of Nav channels with other proteins are required to maintain their functional activity and proper subcellular localization (Peles & Salzer, 2000).
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Intracellular fibroblast growth factors (FGFs) 11–14 lack amino (N)-terminal secretory signal peptides yet they retain the conserved FGF domain primary sequence and the FGF -trefoil structure (Fig. 1) (Munoz-Sanjuan et al. 2000; Olsen et al. 2003). Two members of this FGF subfamily have been found to interact with, and to modify, the properties of heterologously expressed Nav channels (Liu et al. 2001, 2003; Wittmack et al. 2004). FGF12-1b/FHF1B binds Nav1.5 and induces a hyperpolarizing shift in the voltage dependence of Nav1.5 channel inactivation (Liu et al. 2003). FGF13-1b/FHF2B increases Nav1.6 current density and induces a depolarizing shift in voltage dependence of Nav1.6 channel inactivation (Wittmack et al. 2004).
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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, FGF14NT–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).
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Two alternatively spliced FGF14 isoforms, FGF14-1a and FGF14-1b, are highly homologous to both FGF12-1b and FGF13-1b but contain divergent amino termini (Fig. 1). FGF14-1b is abundantly expressed in the central nervous system and FGF14-1a is prominently expressed in the thymus and, at low levels, in the central nervous system (Smallwood et al. 1996; Wang et al. 2000). Mice deficient in FGF14 develop ataxia and dystonia early in postnatal development and, as adults, show reduced response to dopamine agonists and increased sensitivity to epileptogenic compounds (Wang et al. 2002). Humans harbouring mutations in FGF14 develop early onset spinocerebellar ataxia (SCA) and have cognitive impairments (Van Swieten et al. 2003; Dalski et al. 2005).
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Here, we demonstrate a direct interaction with, and a unique ability to inhibit, cardiac and neuronal Nav channel subunits by both FGF14 isoforms in heterologous expression systems. We show that removal of the FGF14-1b N-terminus or expression of the Fgf14 splice isoform, Fgf14–1a, modifies the ability of FGF14 to suppress INa. In addition, FGF14-1b directly interacts with native Nav channels in neuroblastoma cells, resulting in markedly reduced INa densities. Overexpression of either FGF14-1a–green fluorescent protein (GFP) or FGF14-1b–GFP in freshly isolated hippocampal neurones, however, results in increased Nav current densities, an effect that is expected to increase the excitability of these cells. In mature hippocampal neurones maintained for several days in vitro, FGF14-1a–GFP and FGF14-1b–GFP are both enriched at the axonal initial segment and colocalized with native neuronal Nav channels.
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Methods
Plasmids and antibodies
Fgf14–1a, Fgf14–1b and Fgf14NT were amplified by PCR from a human brain cDNA library and cloned into pCS2-MT (Roth et al. 1991). GFP fusion proteins were made by subcloning Fgf14 from pCS2-MT into pQBI25-fC or -fN vectors (QBiogene, Irvine, CA, USA). Fgf14–1b was also subcloned into pIRES2-EGFP (Clontech, Palo Alto, CA, USA) to generate the bicistronic construct Fgf14–1b-IRES-EGFP (Fig. 1B). To provide FGF14NT with a transcription initiation codon, the amino acid sequence ‘MESK’ was added using PCR. The MESK sequence corresponds to the amino-terminal sequence encoded by the first exon of FGF12-1b. This sequence was not included in the construction of GFP–FGF14NT. hSpry-Myc was made by cloning the human Sprouty 2 cDNA into the pCS2-MT vector. Full-length human Nav1.1 expression vector (pScn1a) was obtained from A. George (Vanderbilt University, Nashville, TN, USA).
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Monoclonal anti-Myc, anti-HA and goat anti-mouse–horseradish peroxidase (HRP) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A monoclonal anti-pan-Nav channel antibody was purchased from Sigma (St Louis, MO, USA). Rabbit anti-Nav1.2 was purchased from Upstate Signalling (Charlottesville, VA, USA). Monoclonal mouse IgG1 anti-MAP2 was purchased from Chemicon (Temecula, CA, USA). Alexa fluorescent-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) were used as needed.
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Cell culture and transient transfection
All reagents were purchased from Sigma unless otherwise noted. HEK293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 4 mML-glutamine and 10% fetal bovine serum, 100 U ml–1 penicillin and 100 μg ml–1 streptomycin and incubated at 37°C with 5% CO2. The stable cell line HEK-hNav1.5 was maintained similarly, but media contained 500 μg ml–1 G418 (Invitrogen). Cells were transfected at 90–100% confluency using Lipofectamine and PLUS reagent (Invitrogen) according to the manufacturer's instructions.
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Rat hippocampal cultures were prepared from 18-day-old rat embryos by previously described methods (Goslin et al. 1998). Time mated female rats were killed by carbon dioxide asphyxiation using a protocol approved by the Washington University Animal Studies Committee. Embryos were isolated, placed on ice, decapitated, and hippocampi were dissected and dissociated using trypsin and trituration through a Pasteur pipette. Neurones were plated at low density (1–5 x 105 cells/dish) on poly L-lysine-coated coverslips in 60 mm culture dishes in minimum essential medium (MEM) supplemented with 10% horse serum. After 2–4 h, coverslips containing neurones were inverted over a glial feeder layer in serum-free MEM with N2 supplements, 0.1% ovalbumin, and 1 mM pyruvate (N2.1 medium; components from Invitrogen). The neurones grew over the feeder layer but were kept separate from the glia by wax dots on the neuronal side of the coverslips. To prevent the overgrowth of the glia, neurone cultures were treated with cytosine arabinoside (5 μM; Calbiochem, La Jolla, CA, USA) for 3 days in vitro (DIV). Cultures were maintained in N2.1 medium for up to 10 days. Neurones were chronically treated with 100 μMD,L-2amino-5-phosphonovaleric acid (APV, Research Biochemicals, Natick, MA, USA). Transfections were performed at DIV9 for imaging and at DIV0 for electrophysiology using Lipofectamine 2000 (Invitrogen).
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Immunofluorescence
Rat hippocampal neurones (DIV10) were fixed in freshly made 4% paraformaldehyde and 10% sucrose in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.25% Triton X-100. After blocking with 10% BSA for 30 min at 37°C, neurones were incubated at room temperature for 12–16 h with the following primary antibodies: polyclonal rabbit anti-Nav1.2 (1 : 1000), monoclonal IgG1 anti-pan-Nav (1 : 100) and monoclonal mouse IgG1 anti-Map2 (1 : 2000), diluted in PBS containing 3% BSA. Neurones were then washed three times in PBS and incubated for 2 h at 37°C with appropriate secondary antibodies: Alexa 568 anti-rabbit or Alexa 647 anti-IgG1 (both at 1 : 500) together with aminomethylcoumarin (AMCA) anti-rabbit (1 : 100, Vector Laboratories, Burlingame, CA, USA). Neurones were then washed three times with PBS and coverslips were mounted in elvanol (Tris-HCl, glycerol, and polyvinyl alcohol with 2% 1,4-diazabicyclo[2,2,2]octane). Images were acquired using an Axoplan 2 epifluorescence microscope (Zeiss, Oberkochen, Germany) with a 63x (1.4 NA) objective or an Olympus FV500 confocal microscope with 488 nm and 633 nm laser lines with a 60x objective (1.4 NA objective). Images were acquired with a CCD camera and MetaVue Software for the epifluorescence microscope (Universal Imaging Corp., Downingtown, PA, USA) and with Fluoview Software for the confocal microscope. Optical sections of 0.2 μm were acquired and averaged in a stack of three (final thickness = 0.6 μm).
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Image quantification
For the analysis of the AIS enrichment of FGF14-1a–GFP and FGF14-1b–GFP, samples were colabelled with anti-Nav1.2 (AIS marker) and anti MAP2 (dendritic marker) antibodies. Images were acquired using an Axoplan 2 epifluorescence microscope (Zeiss) with a 63x (1.4 NA) objective. A mask of the axonal initial segment and a mask of the dendrites were first generated by thresholding, respectively, the Nav1.2 or MAP2 images. The masks of the corresponding regions were then applied to the unthresholded GFP images. The AIS enrichement index (AEI) was defined as the ratio of total fluorescence intensity/area(AIS) divided by the total fluorescence intensity/area(dendrites). GFP had an AEI of 1, as expected for a uniformly distributed non-polarized protein (Rivera et al. 2003). Analysis was done using MetaMorph software (Universal Imaging).
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Immunoprecipitations
Transfected HEK293 cells or Neuro2A cells were washed twice with PBS and lysed in modified RIPA buffer (mM): 10 Tris-HCl, 150 NaCl, 1 EDTA, 1% NP-40, 0.25% sodium deoxycholate. Protease inhibitor cocktail and phenylmethanesulphonyl fluoride (PMSF) were added immediately before cell lysis. Cell extracts were collected and sonicated for 20 s, incubated for 10 min and centrifuged at 4°C, 15 000 g for 15 min. Supernatants were collected and precleared with non-immune rabbit-IgG-agarose beads for 1 h at 4°C. Rabbit anti-Myc-agarose beads (Sigma) were incubated with the supernatants overnight at 4°C with agitation and then washed 5 times with lysis buffer before adding 6x sample buffer containing 5 mM tris(2-carboxyethyl)phosphine (TCEP). Mixtures were heated for 5 min at 95°C and resolved on 4–15% polyacrylamide gradient gels (Bio-Rad, Hercules, CA, USA). Resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) overnight at 4°C and blocked in Tris-buffered saline (TBS) with 5% skimmed milk and 0.1% Tween-20. Membranes were then incubated in blocking buffer containing monoclonal anti-Myc (1 : 1000) or anti-pan-Nav channel (1 : 5000) antibodies for 1 h. Washed membranes were incubated with goat anti-mouse-HRP (1 : 5000) detected with SuperSignal Pico chemiluminescent substrate (Pierce, Rockford, IL, USA).
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Nav subunit expression
RNA was prepared from Neuro2A cells, E13.5 heart, E16.5 whole embryo, adult dorsal root ganglion (DRG) and adult brain. cDNA was prepared using the ProMega Reverse Transcription System according to the manufacturer's protocol. PCR amplification used 40 ng cDNA in a 20 μl reaction volume with the primers listed below. PCR cycle conditions: 68°C, 5 min; 35 cycles (93°C, 40 s; 68°C, 30 s); 68°C, 10 min. For primers and amplicon sizes see Supplemental material. Note that amplicon sizes for each Nav subunit are unique and that primers were chosen to minimize the chance of non-specific amplification of an incorrect Nav subunit.
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In situ Hybridization
A 550 bp mouse Fgf14 probe (Wang et al. 2000) was labelled with digoxigenin following the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN, USA). Free-floating brain sections (30 μm) were washed twice in PBS and treated with freshly prepared 10 μg ml–1 proteinase K (Invitrogen) at 37°C. After acetylation, sections were incubated in hybridization buffer containing 0.2 μg ml–1 digoxigenin-labelled riboprobe at 43°C overnight. Hybridized sections were washed by successively immersing in 4x SSC (mM: 150 NaCl and 15 sodium citrate, pH 7.0, room temperature), 2x SSC containing 50% formamide (50°C, 30 min), 2x SSC (37°C, 10 min), 2x SSC containing 20 μg ml–1 RNase A (37°C, 30 min), 2x SSC (37°C, 20 min), and 0.1 x SSC (room temperature, 10 min). The hybridization signals were detected with the digoxigenin detection reagents (Roche Diagnostics) and photographed on a Zeiss Axioskop microscope.
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Electrophysiology
Transfected cells were dissociated and re-plated at low-density approximately 12 h post-transfection. Recordings were performed at room temperature (20–22°C) 12–18 h post-transfection (for both rat hippocampal neurones and mammalian cell lines) using an Axopatch 1D amplifier (Axon Instruments, Union City, CA, USA). Borosilicate glass pipettes with resistance of 1–2 M (HEK293 and Neuro2A) or 2.5–5 M (hippocampal neurones) were made using a P-87 Micropipette Puller (Sutter Instruments, Novato, CA, USA). The recording solutions were as follows: extracellular (mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 20 Hepes, pH 7.3; intracellular: 140 CsF, 1 EGTA, 10 NaCl, 10 Hepes, pH 7.3. After seal formation and cell break-in, membrane capacitance was calculated by integrating the area under the capacitative transients recorded in response to a 5 ms hyperpolarizing test pulse from –70 mV to –80 mV. Capacitative transients and series resistances were compensated electronically by 80–90%. Data were acquired at 50 kHz and filtered at 5 kHz prior to digitization and storage. All experimental parameters were controlled by Clampex 9.2 software (Axon Instruments) and interfaced to the electrophysiological equipment using a Digidata 1322A analog–digital interface (Axon Instruments).
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Voltage-dependent inward currents were evoked by depolarizations to test potentials between –100 mV and +60 mV from a holding potential (between –130 mV and –90 mV). Steady-state fast inactivation of Nav channels was measured with a paired-pulse protocol. From the holding potential, cells were stepped to varying test potentials between –130 mV and –10 mV (prepulse) prior to a test pulse to –10 mV or –20 mV. The extracellular solution used to record INa in neurones contained bicuculline (10 μM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 μM) and D,L-2amino-5-phosphonovaleric acid (APV, 100 μM) to block synaptic activity mediated, respectively, by GABA, AMPA and NMDA receptors.
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Data analysis
Current densities were obtained by dividing INa amplitude by membrane capacitance. Current–voltage relationships were generated by plotting current density as a function of the test potential. Conductance GNa was calculated by the following equation:
where INa is the current amplitude at voltage Vm, and Erev is the Na+ reversal potential. Steady-state activation curves were derived by plotting normalized GNa as a function of test potential and fitted using the Boltzmann equation:
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where GNa,Max is the maximum conductance, Va is the membrane potential of half-maximal activation, Vm is the membrane voltage and k is the slope factor.
For steady-state inactivation, normalized current amplitude (INa/INa,Max) at the test potential was plotted as a function of prepulse potential (Vm) and fitted using the Boltzmann equation:
where Vh is the potential of half-maximal inactivation and k is the slope factor.
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Data analysis was performed using Clampfit 9.2 software (Axon Instruments) and Prism 4 software (GraphPad, San Diego, CA, USA). Results are presented as means ±S.E.M. and the statistical significance of differences between groups was assessed using Student's t test and was set at P < 0.05.
Results
FGF14 interacts with multiple Nav channel subunits
Primary sequence comparison showed a high degree of sequence similarity between the core domain of FGF14 and two previously described intracellular FGFs, FGF12-1b and FGF13-1b (Fig. 1A) (Liu et al. 2001, 2003; Wittmack et al. 2004). The Fgf14 gene is alternatively spliced and encodes proteins with distinct amino-termini, FGF14-1a and FGF14-1b (65 and 69 amino acid residues, respectively) but are otherwise identical in the core FGF domain and carboxy-terminus (Wang et al. 2002). Two recently reported mutations in Fgf14, linked to autosomal dominant spinocerebellar ataxia (SCA), occur in the conserved FGF domain (Fig. 1A) (Van Swieten et al. 2003; Dalski et al. 2005).
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The Nav channel-interacting domain on FGF12-1b is localized to the amino terminal end of the FGF domain, amino acid residues 1–41 (Fig. 1A, underline), and shows 68% amino acid identity with the corresponding regions of FGF14-1a and FGF14-1b and 76% amino acid identity with FGF13-1b (Liu et al. 2001; Liu et al. 2003). Initial experiments were therefore focused on determining if FGF14 also interacts directly with Nav subunits using biochemical assays. For these experiments, FGF14-1a, FGF14-1b and an N-terminal deletion mutant (FGF14NT) were fused with six copies of the Myc epitope tag to generate FGF14-1a–Myc, FGF14-1b–Myc, and FGF14NT–Myc, respectively. We tested the ability of these tagged FGF14 molecules to interact with full-length cardiac and neuronal Nav subunits in HEK293 cells stably expressing human Nav1.5 and in wild-type HEK293 cells transiently expressing human Nav1.1 (Fig. 1B). Approximately 24 h after cotransfection with FGF14-1a–Myc, FGF14-1b–Myc, FGF14NT–Myc and hSpry–Myc, cell lysates were collected and immunoprecipitated with an anti-Myc antibody (Fig. 1B). Expression of Nav1.5 and Nav1.1 were readily detected using an anti-pan-Nav subunit antibody directed against the intracellular loop between domains III and IV. As illustrated in Fig. 1B, Nav1.5 and Nav1.1 coimmunoprecipitated with both FGF14-1a-Myc and FGF14-1b-Myc. Interestingly, deletion of the FGF14 N-terminus abolished binding to Nav1.5, but not to Nav1.1. These data demonstrate that FGF14 can interact with several Nav subunits and suggest that the amino terminal domain(s) of FGF14 may modulate these interactions.
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FGF14-1b inhibits INa in HEK293 cells expressing Nav1.5 or Nav1.1
To explore the functional significance of the biochemical interaction between FGF14 and Nav subunits, whole-cell voltage-clamp recordings were obtained from HEK293 cells coexpressing Nav subunits and FGF14. In initial experiments, GFP (control) or one of the GFP-tagged Fgf14 constructs (Fgf14-1a–GFP, Fgf14-1b–GFP or GFP-Fgf14NT) were transfected into HEK293 cells stably expressing human Nav1.5 (HEK-Nav1.5). Approximately 12–18 h after transfection, fluorescent cells were identified and whole-cell recordings were obtained (Fig. 2A and D). Robust, rapidly activating and inactivating inward currents (INa) were observed in GFP-expressing (control) cells, similar to non-fluorescent cells (not shown). INa amplitudes in HEK-Nav1.5 cells were markedly lower in cells coexpressing FGF14-1a–GFP or FGF14-1b–GFP compared with cells expressing GFP alone (Fig. 2A). In all experiments, INa was stable for the duration (5–10 min) of the recordings regardless of the coexpressed fluorescent protein. In addition, INa in all transfection groups exhibited typical current–voltage relationships, i.e. maximal INa was observed to occur at approximately –20 mV and the currents reversed at approximately 45 mV (not shown).
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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-FGF14NT; 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.
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Although there was considerable variability in peak INa densities recorded from individual Nav1.5 + GFP-expressing cells (Fig. 2B), coexpression of FGF14-1b–GFP or FGF14-1a–GFP led to a 91 ± 3% (P < 0.0005) and a 45 ± 8% (P < 0.005), respectively, decrease in mean current density. Co-expression of GFP–FGF14NT did not measurably affect peak INa densities in Nav1.5-expressing cells (Fig. 2B). To exclude the possibility that the C-terminal GFP tag on FGF14-1b contributed to the observed attenuation of INa, an FGF14-1b construct with an N-terminal GFP tag (GFP–FGF14-1b) and an untagged FGF14-1b (using a bicistronic vector, FGF14-1b-IRES–GFP) were also generated. Expression of either of these constructs in HEK-Nav1.5 cells resulted in mean ±S.E.M. peak INa densities not significantly different from those determined in cells expressing FGF14-1b–GFP. The mean ±S.E.M. peak INa densities were 103 ± 34 pA pF–1 for GFP–FGF14-1b (n= 10) and 103 ± 29 pA pF–1 for FGF14-1b-IRES–GFP (n= 12). Co-expression of FGF14-1b–GFP in cells expressing Nav1.1 also resulted in marked attenuation in peak INa (Fig. 2D). In contrast to the findings for Nav1.5, Nav1.1 cells coexpressing FGF14-1a–GFP were indistinguishable from controls (Fig. 2D). INa amplitudes in Nav1.1-expressing cells transfected with GFP-Fgf14NT were also not measurably different from control cells (Fig. 2E).
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The effects of FGF14 coexpression on the voltage-dependent properties of Nav1.5 and Nav1.1 were also measured (Fig. 2C and F). The voltage dependences of INa activation were determined by plotting normalized conductance as a function of test potential. Although FGF14 coexpression had no measurable effects on INa activation in HEK-Nav1.5 cells (Fig. 2C, Table 1), coexpression of FGF14-1a–GFP or GFP–FGF14NT resulted in small depolarizing and hyperpolarizing, respectively, shifts in the voltage dependence of Nav1.1 activation (Fig. 2F, Table 1). To examine steady-state inactivation of INa, a two-pulse voltage protocol was used (see Methods). In HEK-Nav1.5 cells, coexpression of FGF14-1b–GFP had a negligible effect on the half-maximal voltage (Vh) of inactivation (Table 1), whereas in HEK-Nav1.1 cells, coexpression of FGF14-1b–GFP resulted in a small, but statistically significant (P < 0.05), depolarizing shift in Vh (Fig. 2F, Table 1). In contrast to FGF14-1b, the coexpression of FGF14-1a or FGF14NT resulted in depolarizing or hyperpolarizing, respectively, shifts in the voltage dependences of Nav1.5 (Fig. 2C) and Nav1.1 (Fig. 2F) currents. No significant changes in the slope factors of INa activation or inactivation were observed (Table 1). These results indicate that the marked changes in INa densities in HEK-Nav1.5 and HEK-Nav1.1 cells coexpressing FGF14-1b do not reflect changes in Nav channel availability. Taken together, the results presented in Fig. 2 also suggest that the functional effects of the various FGF14 isoforms on the channels formed by different Nav subunits (Nav1.1 and Nav1.5) are distinct.
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FGF14-1b interacts with endogenous Nav subunits and modulates INa in Neuro2A cells
The results presented above demonstrate that FGF14 interacts strongly with recombinant heterologously expressed Nav channel subunits. Subsequent experiments were focused on exploring the possibility that FGF14 also interacts with, and modulates, the activity of Nav channels endogenously expressed in neuronal cells. Specifically, we examined the ability of FGF14-1b-Myc to interact with native Nav channels expressed in the murine neuroblastoma cell line, Neuro2A. Neuro2A cells are known to have endogenous voltage-gated Na+ currents and can be induced to differentiate into neurone-like cells (Hamasaki et al. 1996; Carpaneto et al. 1999). Because the Nav subunits expressed in Neuro2A cells had not been previously identified, initial experiments used RT-PCR to examine the expression of individual Nav channel - and subunits (Fig. 3A). Unique primers for each of the murine Nav channel (Nav1.1–Nav1.9) and (1–4) subunits were used to amplify cDNA generated from Neuro2A cells (see Methods). Control (murine) cDNA samples from adult brain, dorsal root ganglion and embryonic heart were used as positive controls. All Nav channel primer sets produced amplicons of the predicted size in at least one of the control cDNA samples, but were absent from others, indicating tissue specific expression of Nav channel and subunits consistent with published expression patterns. As illustrated in Fig. 3A, Neuro2A cells expressed multiple Nav channel subunits, including Nav1.2, 1.3, 1.4 and 1.7 and two subunits, 1 and 3. The Nav1.7 subunit, which is primarily expressed in the peripheral nervous system, appears to be the predominant Nav subunit in Neuro2A cells (Fig. 3A).
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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 1-4 and Gapdh; E15 heart cDNA is positive for Nav1.4 and 1.5 and for 1–4; and adult DRG cDNA is positive for Nav1.7, 1.8, 1.9 and 1–4. In Neuro2A cells, Nav1.7 is robustly expressed; Nav1.2, Nav1.3 and Nav1.4 are also detected. Note selective expression of 1 and 3 in Neuro2A cells. B, expression of endogenous Nav 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 subunit expression was detected using an anti-pan-Nav 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 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.
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To determine if FGF14-1b interacts with endogenous Nav channels, a Myc tagged construct, Fgf14-1b–Myc, and a Myc tagged control construct, hSpry–Myc, were individually transfected into Neuro2A cells. Cell lysates were prepared and used for Western blot analysis or were immunoprecipitated with anti-Myc beads. Immunoblots using an anti-Myc or an anti-pan-Nav subunit antibody revealed expression of the Myc tagged constructs and robust endogenous Nav channel subunit expression (Fig. 3B). Western blots of proteins immunoprecipitated with anti-Myc revealed that Nav subunits coimmunoprecipitate with FGF14-1b–Myc, but not with the Myc-tagged hSpry–Myc control protein (Fig. 3C).
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The effects of FGF14 coexpression on Nav channel function was assessed in whole-cell recordings from Neuro2A cells transiently transfected with GFP, Fgf14-1a–GFP or Fgf14-1b–GFP. Whole-cell recordings from GFP-positive control cells revealed robust voltage-dependent inward Na+ currents (Fig. 3C), consistent with the expression of Nav subunits shown by RT-PCR (Fig. 3A) and immunoblot (Fig. 3B) analyses. In FGF14-1a–GFP-expressing cells, INa densities were attenuated but not statistically significantly (P= 0.07) lower than in cells expressing GFP alone (Fig. 3C). In FGF14-1b–GFP-expressing cells, INa densities were significantly (P < 0.005) lower than in cells expressing GFP alone (Fig. 3C); current densities in individual cells are plotted in Fig. 3D. The mean current density in FGF14-1b–GFP-expressing cells was 10 ± 3% of control cells (Table 1).
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The voltage dependences of Nav channel activation and inactivation in Neuro2A cells were also examined and are plotted in Fig. 3E. Analysis of the properties of the currents revealed a large (13 or 15 mV) depolarizing shift in Vh in FGF14-1a–GFP- or FGF14-1b–GFP-expressing cells compared to wild-type Neuro2A cells expressing GFP alone (Table 1). Interestingly, the magnitude of the shift in Vh in Neuro2A cells is substantially larger than that seen for Nav1.5 or Nav1.1-encoded currents in HEK-293 cells, suggesting that the modulatory effects of the two splice variants of FGF14 are Nav channel subunit-specific and/or cell type-specific. Importantly, however, and similar to the findings for Nav1.1 and Nav1.5 currents, the marked reductions in endogenous INa caused by FGF14-1b isoform cannot be attributed to changes in the voltage-dependent properties of the currents. Indeed, FGF14-1b markedly increased (not decreased) Nav channel availability at more hyperpolarized potentials (Fig. 3E).
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FGF14-1b modulates INa and co-localizes with native Nav channel subunits in hippocampal neurones
The results presented above demonstrate that FGF14 can bind to, and modulate, the properties of recombinant Nav channels expressed in HEK293 cells and endogenously expressed Nav channels in Neuro2A cells. Previous studies suggest that Fgf14-1b is highly expressed in the central nervous system (Wang et al. 2000). Genetic ablation of Fgf14 or missense mutations in the Fgf14 gene are linked to neurological phenotypes associated with disorders of the central nervous system, including cognitive dysfunction in humans and learning and memory deficits in mice (M. Xiao, D. Wozniak & D. Ornitz, unpublished data). In the hippocampus, Fgf14 is highly expressed in pyramidal cells in the CA1 and CA3 regions, and in granule cells of the dentate gyrus (Fig. 4A). Fgf14 is also expressed in isolated hippocampal neurones in vitro (Fig. 4B). We therefore investigated whether FGF14-1a and FGF14-1b could affect the properties of the Nav channels expressed in hippocampal neurones. FGF14-1a–GFP or FGF14-1b–GFP was transfected into freshly isolated (E18) rat hippocampal neurones prior to plating, and whole cell INa were recorded from GFP-positive cells within 18 h of plating. In a previous study, quantitative RT-PCR analysis indicated that hippocampal neurones at this stage (P1) express Nav1.1, Nav1.2, Nav1.3 and Nav1.6 subunits (Schaller & Caldwell, 2000). Hippocampal neurones at this developmental stage in vitro lack neurites, allowing good spatial control of the membrane voltage and the clear resolution of INa. Interestingly, INa densities were significantly higher in both FGF14-1a–GFP- and FGF14-1b–GFP-expressing neurones (Fig. 4D), compared to GFP-expressing control neurones (Fig. 4C). Although INa densities in individual cells were quite variable (Fig. 4E), mean INa density was 83 ± 30% and 54 ± 17% higher in cells expressing, respectively, FGF14-1a or FGF14-1b (P < 0.05, Table 1). Small, but statistically significant, differences in the voltage dependences of INa activation and inactivation were also observed in the cells expressing FGF14-1a or FGF14-1b, compared with control (GFP-expressing) neurones (Fig. 4F). Taken together, these results demonstrate that both FGF14 isoforms can modulate properties of native Nav channels expressed in central (hippocampal pyramidal) neurones.
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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.
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In transfected hippocampal pyramidal neurones maintained in vitro for several days, both FGF14-1a–GFP and FGF14-1b–GFP were found to be enriched in the proximal regions of axons (Fig. 5). In these experiments, axons were identified as neuronal processes devoid of the microtubule-associated protein 2 (MAP2), a somato-dendritic marker (Kanai & Hirokawa, 1995). Immunolabelling with a Nav1.2 or a pan-Nav subunit-specific antibody revealed that Nav channel expression was high in the region of high FGF14-1a–GFP or FGF14-1b–GFP localization (n > 50, Fig. 5A–J). Previous studies have shown that the region of high Nav channel density corresponds to the axon initial segment (Garrido et al. 2003). In contrast, in cells expressing GFP, the GFP was not enriched in the Nav-positive (MAP2-negative) regions (n > 50, Fig. 5K–O). Quantification of fluorescence intensity showed that FGF14-1a–GFP and FGF14-1b–GFP were 3-fold more concentrated in the AIS than in dendrites, compared to GFP (Fig. 5S). Analysis of (0.6 μm) optical sections through the region of high Nav channel expression demonstrated colocalization of native Nav channels (anti pan-Nav channel antibody) and FGF14-1b–GFP (Fig. 5P–R). The findings that FGF14-1a–GFP and FGF14-1b–GFP modulate INa densities in hippocampal neurones and colocalize with endogenous Nav channels in the axon initial segment suggest that both FGF14 isoforms are likely to play a role in the regulation of neuronal membrane excitability.
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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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Discussion
FGF14 binds directly to Nav subunits
The intracellular subfamily of FGF-like molecules (FGFs11–14) are highly expressed in neuronal tissues. Fgf14 is particularly interesting in that it is expressed during development and in the adult CNS. Targeted deletion studies in mice and the discovery of mutations in Fgf14 in humans with autosomal dominant SCA revealed an essential role for FGF14 in the regulation of neuronal functioning (Wang et al. 2002; Van Swieten et al. 2003; Dalski et al. 2005). Previous reports that FGF12-1b and FGF13-1b bind to and influence the properties of Nav1.5, Nav1.6 and Nav1.9 channels established a link between this subfamily of FGFs and Nav channel function (Liu et al. 2001, 2003; Dib-Hajj et al. 2002; Wittmack et al. 2004). As a beginning step to understanding the mechanisms underlying the observed phenotypes in Fgf14-null mice and humans with missense mutations in Fgf14, studies were initiated to test the hypothesis that FGF14 interacts with Nav channel subunits and modulates Nav channel function. The data presented here demonstrate that FGF14-1a and FGF14-1b coimmunoprecipitate with both Nav1.1 and Nav1.5, suggesting a generalized biochemical interaction between FGF14 and members of the Nav channel family. We have also shown that FGF14 will coimmunoprecipitate with the carboxy terminal tail of Nav1.6 (data not shown), further supporting an interaction between FGF14 and neuronal sodium channels.
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FGF14 modulates heterologously expressed Nav channels
Experiments focused on examining the effects of FGF14 in murine neuroblastoma (Neuro2A) cells, which express multiple neuronal and non-neuronal Nav channel subunits and at least two of the Nav channel subunits, revealed that INa densities in these cells are reduced significantly with heterologous expression of FGF14-1b. Additional experiments also demonstrated that FGF14 modulates the properties of heterologously expressed (in HEK293 cells) Nav1.1 and Nav1.5 channels.
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In contrast to the reported hyperpolarizing shift in the voltage dependence of Nav1.5 channel inactivation produced on coexpression of FGF12-1b/FHF1B (Liu et al. 2003), the coexpression of FGF14-1b resulted in a dramatic voltage-independent suppression of Nav1.5 current density. The magnitude of the effect on current density depends on the 1b N-terminal domain; the current reduction by the 1a N-terminus is substantially less and FGF14NT showed no activity. Importantly, the N-terminal domain of FGF14-1b is unique to FGF14 and homologous sequences are not found in any other gene in the mouse or human genome databases. The marked reduction in INa (with FGF14-1b expression) is not accounted for by the presence of the GFP-tag, variations in cell size or alterations in the voltage dependence of channel activation or inactivation. Rather, it appears that FGF14-1b modulates functional Na+ current densities directly, probably by altering biophysical properties of individual Nav channels or by modulating channel trafficking or cell surface expression.
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Nav1.1-encoded current densities were also reduced significantly in cells cotransfected with FGF14-1b–GFP, whereas in cells coexpressing GFP–FGF14NT or FGF14-1a–GFP, INa densities were not significantly different from controls. These observations again point to an important functional role for the unique N-terminus of FGF14-1b in some cell types. In addition, these results suggest the presence of sequence-specific, and perhaps unique, interactions between different Nav subunits and the various FGF14 splice variants. Alternatively, there may be additional channel accessory subunits and/or channel regulatory proteins that are Nav subunit specific and may modulate the regulatory effects of the FGF14 variants on Nav activity.
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FGF14 and the regulation of neuronal membrane excitability
To investigate whether FGF14 affects the activity of Nav channels in primary neurones, rat hippocampal neurones were transfected with FGF14–GFP. Interestingly, compared to control transfected neurones, peak INa densities were increased in freshly isolated hippocampal neurones over-expressing FGF14-1a–GFP or FGF14-1b–GFP. Furthermore, small, but statistically significant, shifts in the voltage dependences of INa activation (–5 mV) and inactivation (+6 mV) were observed with FGF14-1a or FGF14-1b coexpression. The molecular basis of the distinct effects of FGF14-1a and FGF14-1b on INa in primary hippocampal neurones, compared to Neuro2A cells and HEK293 cells, remains to be determined. However, several possibilities could be considered: (1) FGF14 might have variable effects on the activity of Nav channels depending on the phosphorylation states of the Nav (or ) subunit (Gasparini & Magee, 2002); (2) FGF14 may suppress INa in channels encoded by some Nav subunits while increasing the activity of other Nav channel types; (3) the activity of FGF14 may be modified by other proteins expressed in primary neurones that are either not expressed or are not modified in Neuro2A cells or HEK293 cells; and/or (4) hippocampal neurones already express high levels of endogenous FGF14 and may therefore also express other proteins that modify the ability of FGF14 to interact with, and modulate, Nav channel function.
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Of the above-mentioned possibilities, the simplest explanation of the results reported here is that FGF14 differentially affects Nav channels encoded by different Nav (and perhaps ) subunits. It might be that the effects of FGF14 in hippocampal neurones reflect the ability of FGF14 to directly interact with the carboxy terminal tail of Nav1.6 (data not shown), a neuronal sodium channel that is found abundantly in central neurones, including the somatodendritic and axonal domains of hippocampal neurones (Garrido et al. 2001, 2003). Interestingly, FGF13-1b has been shown to increase the density of Nav1.6-encoded currents when coexpressed in neuroblastoma cells (Wittmack et al. 2004). Future studies focused on exploring the role of Nav1.6 and the modulatory effects of FGF14 on Nav1.6-encoded INa are clearly warranted.
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The observation that FGF14-1a–GFP and FGF14-1b–GFP are preferentially colocalized with Nav channels at the axon initial segment of hippocampal neurones further suggests that FGF14 may be an important regulator of neuronal excitability. Although initially thought to be a critical region for action potential generation (Colbert & Johnston, 1996), more recent studies suggest that the axon initial segment more likely functions to ensure back propagation of action potentials (initiated at more distal segments of the axons) to the cell bodies and dendrites (Colbert & Pan, 2002). The increased concentration of FGF14 in this region and the ability of FGF14 to interact with several different Nav subunits suggest that FGF14 may play a pivotal role in regulating neuronal excitability and plasticity.
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Relationship to previous studies
Sequences in the N-terminal half of the conserved FGF domain (amino acid residues 1–41 in FGF12-1b) are essential for interactions with Nav subunits (Liu et al. 2001). This sequence is highly conserved throughout the FGF 11–14 subfamily. It is likely that this domain serves to anchor FGF14 to the C-terminal region of Nav subunits. Additionally, FGF14 could potentially compete with other proteins binding to the Nav subunit C-terminus. We propose a model in which multiple domains of FGF14 could interact with multiple domains of Nav channels. In this model, the unique 65 amino acid N-terminus of FGF14-1a or 69 amino acid N-terminus of FGF14-1b may function by interacting with other regions of the Nav subunit, or with other proteins in complex with the Nav subunit, to modulate INa. This interaction may lead to channel modifications such as phosphorylation, involving residues critical for channel availability (Ratcliffe et al. 2000; Cantrell et al. 2002; Carr et al. 2003) or direct interference with the channel pore. Interestingly, several members of the FGF 11–14 subfamily have been shown to interact with islet/brain 2, a mitogen-activated protein kinase scaffolding protein (Goldfarb, 2001; Schoorlemmer & Goldfarb, 2001, 2002) (Q. Wang, J. Lou & D. Ornitz, unpublished data.). FGFs 11–14 may thus serve to link kinase signalling complexes with Nav subunits. Another possibility is that the unique FGF14-1b N-terminus might sterically hinder the inward flow of Na+ ions near the intracellular vestibule of the pore. In addition, the fact that the FGF14-1b N-terminus contains two leucine-based motifs which have been implicated in protein trafficking (Bonifacino & Traub, 2003) raises the interesting possibility that the FGF14-1b splice isoform functions to regulate peak current density by affecting Nav channel subcellular localization and/or trafficking. Further studies to test each of these possibilities are clearly warranted.
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A recently identified missense mutation (F149S) in FGF14 is the aetiology of an autosomal dominant spinocerebellar ataxia (SCA) and mental retardation syndrome in humans (Van Swieten et al. 2003). Patients with this mutation develop early onset tremors and progressive ataxia in a manner strikingly similar to the phenotype of FGF14-null mice (Wang et al. 2002). Cognitive disturbances have also been found in FGF14-null mice (M. Xiao, D. Wozniak & D. Ornitz, unpublished observation). Although it was proposed that the F149S missense mutation destabilizes the FGF14 protein (Van Swieten et al. 2003), the dominant pattern of inheritance of the human syndrome and the recessive nature of the knockout mouse phenotype suggests that the F149S mutation may produce a protein that functions as a dominant negative. Clearly, further experiments focused on exploring the molecular mechanisms involved in mediating the modulatory effects of FGF14 on Nav channel functioning and/or determining the effects of disease mutations in Fgf14 will be of considerable interest.
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