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 α 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.

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

Inheritable long-QT syndrome (LQTS) is a disease in which delayed ventricular repolarization leads to cardiac arrhythmias and the possibility of sudden death. In the chromosome 3-linked disease, one mutation of the cardiac Na+ channel gene results in a deletion of residues 1505 to 1507 (Delta KPQ), and two mutation result in substitutions (N1325S and R1644H). We compared all three mutant-channel phenotypes by heterologous expression in Xenopus oocytes. Each produced a late phase of inactivation-resistant, mexiletine- and tetrodotoxin-sensitive whole-cell currents, but the underlying mechanisms were different at the single-channel level. N1325S and R1644H showed dispersed reopenings after the initial transient, whereas Delta KPQ showed both dispersed reopenings and long-lasting bursts. Thus, two distinct biophysical defects underlie the in vitro phenotype of persistent current in Na+ channel-linked LQTS, and the additive effects of both are responsible for making the Delta KPQ phenotype the most severe.

Na+ channel inactivation, a critical determinant of refractoriness, differs in cardiomyocytes and neurons. In rat brain type IIa (rB2a) Na+ channels, a critical residue in the cytoplasmic linker between domains III and IV regulates fast inactivation such that a Phe-->Gln substitution (F1489Q) inhibits inactivation by at least 85%. Since this residue is conserved in voltage-gated Na+ channels, we tested whether F1485Q, the analogous mutation in human heart (hH1a) Na+ channels, has a similar functional effect. We found that fast inactivation in wild-type (WT) channels expressed in Xenopus oocytes was complete within 15 milliseconds at a test potential of 0 mV, and its time course was biexponential with time constants of 0.4 and 2 milliseconds. But in contrast to rB2a, the FQ mutation inhibited inactivation by < 50% and increased mean single-channel open time by only twofold. Residual fast inactivation was monoexponential, with a time constant similar to that of the slower phase of normal inactivation (2 milliseconds). In the mutant channels, unlike WT, null tracings were absent at holding potentials in the range of -140 to -120 mV, and the voltage range of steady-state inactivation coincided exactly with that of activation, suggesting that residual inactivation was tightly coupled to the open state. As in rB2a, simultaneous mutations of I1484Q and M1486Q, in addition to mutation F1485Q, completely inhibited fast inactivation. Our results show that in heart Na+ channels, the IFM cluster controls the stability of both open- and closed-channel inactivation in a manner qualitatively similar to that in the brain. Structural differences in the putative inactivation receptor may explain the distinct gating patterns in channel subtypes.

Ca 2+ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca 2+ release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca 2+ influx through L-type Ca 2+ channels (dihydropyridine receptors, DHPR), Ca 2+ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca 2+ together ensure the stability of this positive-feedback system of Ca 2+ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca 2+ ] i there is little clear evidence to date showing an increase in Ca 2+ spark rate. Indeed, there is some evidence that the SR Ca 2+ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca 2+ "leak" under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or "rogue" RyRs that may respond differently to local [Ca 2+ ] i in diastole and during the [Ca 2+ ] i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyperphosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca 2+ signaling.