Effects of familial hemiplegic migraine type 1 mutation T666M on voltage-gated calcium channel activities in trigeminal ganglion neurons.

Familial hemiplegic migraine type 1 (FHM-1), a rare hereditary form of migraine with aura and hemiparesis, serves as a good model for exploring migraine pathophysiology. The FHM-1 gene encodes the pore-forming Ca(V)2.1 subunit of human P/Q-type voltage-gated Ca(2+) channels (VGCCs). Some FHM-1 mutations result in a decrease of whole cell P/Q-type current density in transfected cells/neurons. Questions remain as to whether and how these mutations may increase the gain of the trigeminal nociceptive pathway underlying migraine headache. Here, we investigated the effects of T666M, the most frequently occurring FHM-1 mutation, on VGCC currents and neuronal excitability in trigeminal ganglion (TG) neurons. We expressed human wild-type and T666M Ca(V)2.1 subunits in cultured TG neurons from Ca(V)2.1 knockout mice and recorded whole cell VGCC currents in transfected neurons. Currents mediated by individual VGCC subtypes were dissected according to their pharmacological and biophysical properties. TG neurons were sorted into three subpopulations based on their soma size and their affinity to isolectin B4 (IB4). We found that the T666M mutation did not affect total or surface expression of Ca(V)2.1 proteins but caused a profound reduction of P/Q-type current in all subtypes of TG neurons. Interestingly, a compensatory increase in Ca(V)3.2-mediated low-voltage-activated T-type currents only occurred in small IB4-negative (IB4(-)) TG neurons expressing T666M subunits. Current-clamp recordings showed that the T666M mutation resulted in hyperexcitability of the small IB4(-) TG population. Taken together, our results suggest a possible scenario through which FHM-1 mutations might increase the gain of the trigeminal nociceptive pathway.

FGF14 N-terminal splice variants differentially modulate Nav1.2 and Nav1.6-encoded sodium channels.

The Intracellular Fibroblast Growth Factor (iFGF) subfamily includes four members (FGFs 11-14) of the structurally related FGF superfamily. Previous studies showed that the iFGFs interact directly with the pore-forming (alpha) subunits of voltage-gated sodium (Nav) channels and regulate the functional properties of sodium channel currents. Sequence heterogeneity among the iFGFs is thought to confer specificity to this regulation. Here, we demonstrate that the two N-terminal alternatively spliced FGF14 variants, FGF14-1a and FGF14-1b, differentially regulate currents produced by Nav1.2 and Nav1.6 channels. FGF14-1b, but not FGF14-1a, attenuates both Nav1.2 and Nav1.6 current densities. In contrast, co-expression of an FGF14 mutant, lacking the N-terminus, increased Nav1.6 current densities. In neurons, both FGF14-1a and FGF14-1b localized at the axonal initial segment, and deletion of the N-terminus abolished this localization. Thus, the FGF14 N-terminus is required for targeting and functional regulation of Nav channels, suggesting an important function for FGF14 alternative splicing in regulating neuronal excitability.

The FGF14(F145S) mutation disrupts the interaction of FGF14 with voltage-gated Na+ channels and impairs neuronal excitability.

Fibroblast growth factor 14 (FGF14) belongs to the intracellular FGF homologous factor subfamily of FGF proteins (iFGFs) that are not secreted and do not activate tyrosine kinase receptors. The iFGFs, however, have been shown to interact with the pore-forming (alpha) subunits of voltage-gated Na+ (Na(v)) channels. The neurological phenotypes seen in Fgf14-/- mice and the identification of an FGF14 missense mutation (FGF14(F145S)) in a Dutch family presenting with cognitive impairment and spinocerebellar ataxia suggest links between FGF14 and neuronal functioning. Here, we demonstrate that the expression of FGF14(F145S) reduces Na(v) alpha subunit expression at the axon initial segment, attenuates Na(v) channel currents, and reduces the excitability of hippocampal neurons. In addition, and in contrast with wild-type FGF14, FGF14(F145S) does not interact directly with Na(v) channel alpha subunits. Rather, FGF14(F145S) associates with wild-type FGF14 and disrupts the interaction between wild-type FGF14 and Na(v) alpha subunits, suggesting that the mutant FGF14(F145S) protein acts as a dominant negative, interfering with the interaction between wild-type FGF14 and Na(v) channel alpha subunits and altering neuronal excitability.

Electrical remodelling maintains firing properties in cortical pyramidal neurons lacking KCND2-encoded A-type K+ currents.

Considerable experimental evidence has accumulated demonstrating a role for voltage-gated K(+) (Kv) channel pore-forming (alpha) subunits of the Kv4 subfamily in the generation of fast transient outward K(+), I(A), channels. Immunohistochemical data suggest that I(A) channels in hippocampal and cortical pyramidal neurons reflect the expression of homomeric Kv4.2 channels. The experiments here were designed to define directly the role of Kv4.2 in the generation of I(A) in cortical pyramidal neurons and to determine the functional consequences of the targeted deletion of Kv4.2 on the resting and active membrane properties of these cells. Whole-cell voltage-clamp recordings, obtained from visual cortical pyramidal neurons isolated from mice in which the KCND2 (Kv4.2) locus was disrupted (Kv4.2-/- mice), revealed that I(A) is indeed eliminated. In addition, the densities of other Kv current components, specifically I(K) and I(ss), are increased significantly (P < 0.001) in most ( approximately 80%) Kv4.2-/- cells. The deletion of KCND2 (Kv4.2) and the elimination of I(A) is also accompanied by the loss of the Kv4 channel accessory protein KChIP3, suggesting that in the absence of Kv4.2, the KChIP3 protein is targeted for degradation. The expression levels of several Kv alpha subunits (Kv4.3, Kv1.4, Kv2.1, Kv2.2), however, are not measurably altered in Kv4.2-/- cortices. Although I(A) is eliminated in Kv4.2-/- pyramidal neurons, the mean +/- s.e.m. current threshold for action potential generation and the waveforms of action potentials are indistinguishable from those recorded from wild-type cells. Repetitive firing is also maintained in Kv4.2-/- cortical pyramidal neurons, suggesting that the increased densities of I(K) and I(ss) compensate for the in vivo loss of I(A).

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

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