The cerebellum is a region of the brain that is involved in motor control, and it contains a special type of nerve cells called Purkinje neurons. Messages travel along neurons as electrical signals carried by sodium ions, which have a positive electric charge. Normally, when a neuron is ‘at rest’, the plasma membrane that surrounds the neuron prevents the sodium ions outside the cell from entering. To send an electrical signal, voltage-sensitive proteins in the membrane called sodium channels open up. This allows the sodium ions to enter the cell by passing through a pore in the channel protein, thereby changing the voltage across the membrane.

Once sodium channels open, they rapidly become ‘locked’ in a closed state, which allows the membrane voltage to return to its original value before another signal can be sent. This locked state also prevents sodium channels from reopening quickly. As a consequence most neurons cannot send successive electrical signals rapidly. Purkinje neurons are unusual because they can send many electrical signals in quick succession—known as rapid firing—without having to be reset each time.

Rapid firing is possible in Purkinje neurons because the channel proteins can be reopened to allow more Na⁺ to enter the cell, but it is not clear how this is controlled. Now, based on experiments on Purkinje neurons isolated from mice, Yan et al. have shown that a protein called FGF14 that binds to the sodium channel proteins can help them to reopen quickly in order to allow rapid firing.

Spinocerebellar ataxia is a degenerative disease caused by damage to the cerebellum that leads to loss of physical coordination. Some patients suffering from this disease carry mutations in the gene that makes the FGF14 protein. Therefore, understanding the role of FGF14 in the rapid firing of Purkinje neurons may aid the development of new treatments for this disease.

Cerebellar Purkinje neurons, which provide the sole output from the cerebellar cortex, display a repetitive firing behavior driven by voltage-gated Na⁺ channels (Na_V channels). Fast repetitive firing in Purkinje neurons is promoted by unusual characteristics associated with Na_V channel inactivation, whereby channels recover unusually rapidly from inactivation and in doing so pass a ‘resurgent’ sodium current, which helps drive the cell to fire a subsequent action potential. The molecular components underlying the peculiar properties of Na_V channels in Purkinje neurons have not been definitively identified.

Several features of Na_V currents in cerebellar Purkinje neurons, however, set them apart from Na_V currents recorded in other neurons. First, more than half of the Na_V current in Purkinje neurons is carried by Na_V1.6, an isoform for which inactivation is less complete compared to other Na_V channels (Raman et al., 1997). Examination of Na_V currents in cerebellar Purkinje neurons from Scn8a^−/− mice showed that, compared to the other resident Na_V channels, the Scn8a-encoded Na_V1.6 channels have a relatively large ‘late’ (non-inactivating) component and Na_V1.6 channels have increased availability (depolarized V_1/2 for steady-state inactivation). Second, Na_V channels in cerebellar Purkinje neurons display an unusual transient re-opening during repolarization, which is identified as ‘resurgent’ Na_V current (Raman and Bean, 1997). This resurgent current derives from an unblocking of open channels by a peptide moiety that can be eliminated by the intracellular application of trypsin or chymotrypsin proteases into the cytoplasm (Grieco et al., 2005). The blocking particle acts only on open channels and competes with the inactivation process to prevent channels from entering an absorbed, inactivated state. During action potential repolarization, unblocking of these channels then allows Na⁺ influx that can initiate a subsequent action potential (Khaliq et al., 2003). Since the Na_V current in Purkinje neurons carried by Na_V1.6 is relatively resistant to inactivation, it is particularly susceptible to open block by the peptide moiety. Why Na_V1.6 channels in cerebellar Purkinje neurons are relatively resistant to inactivation, however, is unknown.

Here, we focused on the role of the ion channel regulator FGF14, which is enriched in cerebellar granule and Purkinje neurons (Wang et al., 2002). FGF14 is one of four fibroblast growth factor homologous factors (FHFs), members of the fibroblast growth factor (FGF) superfamily sharing a FGF-like core but having extended N- and C-termini not found in other FGFs. Individual FHFs undergo alternative splicing that generates distinct N-termini, none of which contain a signal sequence (Smallwood et al., 1996; Munoz-Sanjuan et al., 2000). Thus, unlike other FGFs they are not secreted and do not function as growth factors (Smallwood et al., 1996). Instead, FHFs remain intracellular and modulate various ion channels. FHFs can bind directly to the cytoplasmic C-terminal domains (CTDs) of Na_V channels (Liu et al., 2001) and regulate Na_V channel function (Lou et al., 2005). Further, FGF14 is a potent regulator of both Na_V1.1 and Na_V1.6 (Lou et al., 2005; Laezza et al., 2009), the two dominant Na_V channels in cerebellar Purkinje neurons (Raman et al., 1997; Kalume et al., 2007). FHFs also regulate voltage-gated Ca²⁺ channels through a mechanism that does not appear to involve a direct interaction (Hennessey et al., 2013; Yan et al., 2013). An ataxia phenotype in Fgf14^−/− mice highlights the role of FGF14 in cerebellar physiology and the development of spinocerebellar ataxia type 27 (SCA27) in patients with either FGF14 haploinsufficiency or a dominant negative FGF14 mutation (Wang et al., 2002; van Swieten et al., 2003; Dalski et al., 2005) underscores the role of FGF14 in disease.

Using shRNA knockdown of endogenous FGF14 in cultured cerebellar Purkinje neurons and replacement with informative mutants that blocked binding of FGF14 to Na_V CTDs and eliminated the FGF14 extended N-terminus, we found that FGF14, and especially the N-terminus of the FGF14b isoform exerts specific kinetic effects on cerebellar Na_V channels that support repetitive firing and therefore provides new information on the pathophysiology of SCA27.

To test whether FGF14 was a contributor to regulation of Na_V current in cerebellar Purkinje neurons, we first confirmed its expression in cerebellum and demonstrated that FGF14 co-immunoprecipitates with Na_V1.6 channels in mouse cerebellar lysates (Figure 1A). Although FGF14 was previously shown to co-immunoprecipitate with Na_V1.6 in a heterologous expression system (Laezza et al., 2009), co-immunoprecipitation between FGF14 and Na_V1.6 channels has not been reported from brain.

Figure 1

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To evaluate the consequences of FGF14 haploinsufficiency in SCA27, we used a previously validated shRNA (Yan et al., 2013) to knockdown endogenous FGF14 in cultured cerebellar Purkinje neurons. Knockdown of FGF14 did not affect Na_V current density compared to the Na_V current density recorded in neurons transfected with a control shRNA (Figure 1B). Because knockout of Fgf14 reduced spiking activity (Goldfarb et al., 2007) but Na_V current density was unaffected in Fgf14 knockout or knockdown, we hypothesized that the absence of FGF14 must affect Na_V kinetic properties in ways that prevent repetitive firing. We therefore examined Na_V kinetic properties in more detail.

Voltage dependence of activation was unaffected by FGF14 knockdown (Figure 2A), but the V_1/2 of steady-state inactivation was shifted about −10 mV, thereby decreasing channel availability (Figure 2B and Table 1). When we co-expressed a FGF14b cDNA with synonymous substitutions rendering it resistant to the FGF14 shRNA (FGF14b^WT), the V_1/2 of steady-state inactivation was restored to the control voltage; activation was unaffected (Figure 2A–B and Table 1). The ‘rescue’ of steady-state inactivation by expression of the shRNA-insensitive FGF14 provided additional demonstration of the specificity of our shRNA targeting strategy (and see similar rescue for additional parameters below). In addition to the decreased channel availability, we observed that FGF14 knockdown accelerated inactivation kinetics (Figure 2C–D). FGF14 knockdown also reduced the late Na_V current (Figure 2E–F). Thus, by several different kinetic measures, FGF14 knockdown in cerebellar Purkinje neurons biased the endogenous Na_V channels towards entering the inactivated state. We anticipated, therefore, that FGF14 knockdown should reduce Na_V resurgent current amplitude, a key feature that allows cerebellar Purkinje neurons to fire repetitively. Indeed, the amplitude of the resurgent Na_V current (Figure 2G–J) was markedly diminished using a well-established protocol to elicit Na_V resurgent current (Raman and Bean, 1997). As with the other kinetic properties of Na_V currents (above), co-expression of FGF14b^WT in the presence of the shRNA provided a complete rescue (Figure 2G–J). While FGF14 knockdown reduced the amplitude of resurgent Na_V current, it did not significantly affect the time to peak (Figure 2K).

Figure 2

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To determine how FGF14 regulates Na_V kinetics and Na_V resurgent current, we turned our focus to the FGF14 N-terminus. The alternatively spliced N-termini of various FHFs have been shown to exert specific effects on Na_V currents (Lou et al., 2005; Rush et al., 2006), including the regulation of inactivation kinetics (Laezza et al., 2009; Dover et al., 2010), but the role of the FGF14 N-termini have not been investigated in cerebellar Purkinje neurons. FGF14b is the predominant FGF14 splice variant expressed in brain (Wang et al., 2000, 2011) and the FGF14 splice variant found in the cytoplasm; FGF14a is localized to the nucleus (Wang et al., 2000). To test the specific roles of the FGF14b N-terminus, we expressed a FGF14 in which the N-terminus was deleted (FGF14Δ^NT). Because the interaction site for the Na_V CTD on FHFs lies within the conserved core domain (Wang et al., 2012), deletion of the N-terminus does not affect interaction with the Na_V CTD. Thus, the FGF14Δ^NT exerts a dominant negative effect by competing with endogenous FGF14 for interaction with the Na_V CTD (Figure 3A). Current density (Figure 3B) and the V_1/2 of activation (Figure 3C and Table 1) were unaffected. However, expression of FGF14Δ^NT shifted the V_1/2 of steady-state inactivation about −10 mV (Figure 3C), accelerated kinetics of inactivation (Figure 3D), reduced the late Na_V current (Figure 3E), and diminished resurgent current (Figure 3F) to a degree similar to or even greater than shRNA knockdown. FGF14Δ^NT diminished resurgent Na_V current even more effectively than shRNA knockdown of FGF14. As a control, we overexpressed the FGF14b^WT that had rescued the parameters affected by shRNA knockdown (Figure 1). Overexpression of FGF14b^WT here was without effect (Figure 3 and Table 1). Not only did this serve as a control for FGF14Δ^NT, but these data suggest that endogenous FGF14b is saturating. These data also suggest that FGF14b's main regulatory component for Purkinje neuron Na_V currents lies within the FGF14b N-terminus.

Figure 3

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The dominant negative effect exerted by FGF14Δ^NT as it replaced the endogenous FGF14 on the Na_V CTD implied that although FGF14 regulates Na_V current through its N-terminus, it may require that FGF14 be in direct interaction with the channel. To test this hypothesis specifically, we designed a FGF14b that was unable to bind to the channel CTD and examined whether expression of this mutant could restore the diminished resurgent current resulting from shRNA knockdown of FGF14. Structural determination of a ternary complex containing FGF13, the Na_V1.5 CTD, and calmodulin had identified key amino acids on the conserved FHF interaction surface that participated in the interaction with the conserved Na_V CTD surface (Wang et al., 2012). We focused on a highly conserved Arg in the FHF core domain (Arg57 in FGF13U, equivalent to Arg117 in FGF14b), which inserts into a deep pocket on the Na_V1.5 CTD binding surface (Figure 4A). We therefore generated the homologous R117A mutant (FGF14b^R/A) in the FGF14b shRNA-resistant cDNA background and tested whether FGF14b^R/A could bind to the intact Na_V1.6. Although immunoprecipitation of FGF14b^WT from lysates of HEK293 cells transfected with FGF14b^WT and Na_V1.6 showed co-immunoprecipitation of Na_V1.6, the mutant FGF14b^R/A was unable to co-immunoprecipitate Na_V1.6 (Figure 4B). Thus, mutation of R117A in FGF14b prevents interaction with Na_V1.6. We therefore transfected the shRNA-resistant FGF14b^R/A (or the shRNA-resistant FGF14b^WT) along with the FGF14 shRNA into cultured cerebellar Purkinje neurons to determine the effect of FGF14 interaction with Na_V1.6 on Na_V currents. Expression of FGF14b^R/A after shRNA knockdown of endogenous FGF14 had no effect on Na_V current density (Figure 4C). In contrast to the rescue afforded by FGF14b^WT, FGF14b^R/A did not rescue the key parameters that lead to increased inactivation observed after FGF14 shRNA. Na_V late current and the time constant of inactivation were not different from shRNA (Figure 4D–F). Consistent with these findings, we observed the expected reduction in resurgent Na_V current (Figure 4G–H). Thus, the FGF14 N-terminus must exert its key effects through FGF14's direct interaction with the Na_V C-terminus.

Figure 4

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Because the absence of the FGF14 or its N-terminus accelerated Na_V channel inactivation, diminished late current, and decreased resurgent current, we anticipated that the resultant bias towards Na_V inactivation would adversely affect excitability in cerebellar Purkinje neurons. We tested this hypothesis by determining the effects of the FGF14 and its N-terminus on action potential threshold and on the number of action potentials evoked with depolarizing current injections in current-clamp mode. Under our culture conditions (≤14 days in vitro), we found that 8–10% of cerebellar Purkinje neurons exhibit spontaneous action potentials (firing rate 5–20 Hz) with regular and irregular patterns. When neurons were cultured for extended periods (>20 days in vitro), we observed that ∼50% of Purkinje neurons spontaneously fired—more similar to acutely isolated Purkinje neurons (Raman and Bean, 1999) and cultured Purkinje neurons (Gruol and Franklin, 1987). These data suggest that spontaneous activity depends upon the maturity of the neurons in culture. Because of the difficulty in achieving voltage clamp on Purkinje neurons ≥14 days in vitro, our voltage-clamp experiments were performed on the younger cells (Figures 2–4). Thus, we continued with immature neurons for these experiments, and focused on measuring evoked action potentials rather than spontaneous action potentials. Single action potentials were evoked by a 10-ms current injection with 5-pA increments to determine the current threshold to initiate an action potential. Figure 5A–B and Table 2 show that FGF14 knockdown and expression of the dominant negative FGF14bΔ^NT markedly increased the current threshold to produce an action potential; other action potential parameters were not affected. Rescue of FGF14 knockdown by co-expression of FGF14b^WT restored the current threshold to control levels. Rescue with the non-interacting FGF14b^R/A did not. We also analyzed the number of actions evoked during 600 ms current injection over a wide range of current amplitudes. The number of action potentials evoked was significantly larger in control (scrambled shRNA) neurons than in neurons transfected with FGF14 shRNA. Rescue with FGF14b^WT, but not FGF14b^R/A, restored the number of evoked action potentials to control levels. Expression of the dominant negative FGF14bΔ^NT reduced the number of evoked action potentials to the level observed after FGF14 knockdown (Figure 5C–D). Thus, as observed for the individual Na_V channel kinetic parameters, FGF14b, and its N-terminus specifically, is a critical regulator of neuronal firing and intrinsic membrane properties.

Figure 5

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Although FGF14 haploinsufficiency is associated with spinocerebellar ataxia (Dalski et al., 2005) and intrinsic excitability of cerebellar Purkinje neurons is reduced in Fgf14^−/− mice (Shakkottai et al., 2009), the detailed multifactorial molecular mechanisms by which FGF14 affects neuronal excitability were not previously known. We had found that FGF14 knockdown in cerebellar granule neurons reduced presynaptic voltage-gated Ca²⁺ currents and thereby affected neurotransmission at the cerebellar granule neuron to Purkinje neuron synapse (Yan et al., 2013). Here, we found that FGF14b in cerebellar Purkinje neurons affects multiple kinetic parameters of Na_V channels, thereby reducing the resurgent Na_V current that underlies repetitive firing. Together, the accelerated inactivation, reduced channel availability, and decreased late current observed after FGF14 knockdown bias Na_V channels to the inactivated state. Thus, the blocking particle that competes with the inactivation gate for open Na_V channels is disadvantaged. Consequently, the resurgent current, and the resulting ability to support repetitive firing in Purkinje neurons, is reduced as we observed. Thus, our data show that an essential regulatory feature of FGF14 in cerebellar Purkinje neurons is to slow Na_V channel inactivation in order to foster resurgent current.

Resurgent current results from the actions on Na_V1.6 of a putative blocking particle, which is thought to be the cytoplasmic C-terminal peptide of the Na_V auxiliary Na_Vβ4 subunit (Grieco et al., 2005). Nevertheless, the actions of Na_Vβ4 are not sufficient to generate resurgent Na_V current (Chen et al., 2008; Aman et al., 2009), suggesting that other components of the Na_V channel complex may be necessary for proper regulation of resurgent Na_V current in cerebellar Purkinje neurons. Here, our knockdown experiments and expression of the dominant negative FGF14bΔ^NT show that FGF14b, and specifically its N-terminus, strongly influence resurgent Na_V current. FGF14 knockdown caused a ∼60% reduction. Expression of FGF14Δ^NT was even more efficient, resulting in a ∼85% reduction, perhaps because overexpression of FGF14Δ^NT was more efficient in replacing endogenous FGF14b than shRNA knockdown was in eliminating endogenous FGF14b. Thus, FGF14 may cooperate with Na_Vβ4 to generate resurgent current, perhaps by biasing Na_V1.6 channels towards inactivation and thereby disadvantaging the blocking particle. Our results examining effects on total Na_V current must be assessed in the context of FGF14 effects not only on Na_V1.6 channels but also on Na_V1.1 channel, the other major source of Na_V currents in cerebellar Purkinje neurons (Kalume et al., 2007). Those Na_V channels are also influence by FGF14 (Lou et al., 2005). Thus, the effects on resurgent current that we attribute to FGF14 may result from FGF14's overall influence of Na_V channel kinetics within Purkinje neurons.

Our results may also provide some insight into the more variable phenotypes and decreased penetrance in spinocerebellar ataxia patients with FGF14 haploinsufficiency (Dalski et al., 2005; Coebergh et al., 2013) vs patients with a FGF14 mutation (FGF14b^F150S) found in a large Dutch spinocerebellar ataxia kindred (van Swieten et al., 2003). Studies in hippocampal neurons suggest that the FGF14b^F150S mutant exerts a dominant negative mechanism in which the axon initial segment is depleted of Na_V channels and Na_V current density is reduced (Laezza et al., 2007). Here, we found no effect on Na_V current density in cerebellar Purkinje neurons after FGF14 knockdown. A reduction in Na_V current density (with the FGF14b^F150S mutation) compared to an effect solely on Na_V channel kinetics (with FGF14 haploinsufficiency) may explain the higher penetrance and decreased phenotypic variability in patients with the FGF14b^F150S mutation.

An important aspect of this study examining effects on Na_V channel kinetics was that our analyses were performed in Purkinje neurons. Studying FHFs in their native cellular context appears to be crucial for defining their specific roles. Previous investigations of FGF14-dependent regulation of neuronal Na_V channels in heterologous expression systems led to different conclusions from experiments in neurons. For example, expression of Na_V1.6 and FGF14b^WT in ND7/23 cells almost eliminated Na_V current density (Laezza et al., 2009) while overexpression of FGF14b^WT in hippocampal neurons increased Na_V current density (Laezza et al., 2007). Similarly, expression of FGF14Δ^NT in ND7/23 cells increased current density, while here we observed that FGF14Δ^NT expression in cerebellar Purkinje neurons exerted dominant-negative effects upon Na_V channel kinetics, but no consequences for Na_V current density. While the etiology of the different results in neurons compared to heterologous expression systems is not known, we speculate that additional regulatory factors present in neurons are necessary for physiologic regulation of Na_V channels by FHFs. Indeed, we have observed similarly disparate results for the related FGF13 and the cardiac Na_V1.5 channel. In HEK293 cells, FGF13 co-expression reduces Na_V1.5 current density (not shown), but we showed that the role FGF13 in cardiomyocytes is to increase Na_V current density (Wang et al., 2011; Hennessey et al., 2013).

Finally, our data also provide information about possible consequences of FGF14 overexpression, hypothesized as pathologic in rare cases. Some patients with spinocerebellar ataxia, but not controls, harbor synonymous variants in FGF14. These variants encode a more frequently used codon than the wild type sequence, leading to the hypothesis that patients with these variants would overexpress FGF14 as a cause of disease (Dalski et al., 2005). We found, however, that overexpression of FGF14b^WT did not affect current density or any of the measured Na_V kinetic properties (Figure 3 and Table 1). Thus, if these variants identified in spinocerebellar ataxia patients are truly associated with disease, our data suggest mechanisms other than FGF14 overexpression.

In summary, these data add to a growing appreciation of physiologic and pathophysiologic roles for FHFs in neurons. In the case of spinocerebellar ataxia, our data suggest that FGF14 is a critical regulator of the ability of cerebellar Purkinje neuron Na_V channels to support resurgent Na_V current and thereby allow Purkinje neurons to fire repetitively. Thus, we provide a molecular understanding for the observation that FGF14 regulates Purkinje neuron intrinsic excitability (Shakkottai et al., 2009). In combination with our data showing that FGF14 also regulates presynaptic voltage-gated Ca²⁺ channels at the cerebellar granule cell to Purkinje cell synapse, these data pinpoint several specific mechanisms by which mutations in FGF14 underlie spinocerebellar ataxia.

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Author details

1. Haidun Yan

   1. Department of Medicine, Duke University Medical Center, Durham, United States
   2. Ion Channel Research Unit, Duke University Medical Center, Durham, United States

   Contribution

   HY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Juan L Pablo

   1. Ion Channel Research Unit, Duke University Medical Center, Durham, United States
   2. Department of Neurobiology, Duke University Medical Center, Durham, United States

   Contribution

   JLP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Chaojian Wang

   1. Department of Medicine, Duke University Medical Center, Durham, United States
   2. Ion Channel Research Unit, Duke University Medical Center, Durham, United States

   Contribution

   CW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Geoffrey S Pitt

   1. Department of Medicine, Duke University Medical Center, Durham, United States
   2. Ion Channel Research Unit, Duke University Medical Center, Durham, United States
   3. Department of Neurobiology, Duke University Medical Center, Durham, United States

   Contribution

   GSP, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   geoffrey.pitt@duke.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-2246-0289

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