Effects of FGF14 and NaVβ4 deletion on transient and resurgent Na current in cerebellar Purkinje neurons

Abstract

Voltage-gated Na channels of Purkinje cells are specialized to maintain high availability during high-frequency repetitive firing. They enter fast-inactivated states relatively slowly and undergo a voltage-dependent open-channel block by an intracellular protein (or proteins) that prevents stable fast inactivation and generates resurgent Na current. These properties depend on the pore-forming α subunits, as well as modulatory subunits within the Na channel complex. The identity of the factors responsible for open-channel block remains a question. Here we investigate the effects of genetic mutation of two Na channel auxiliary subunits highly expressed in Purkinje cells, NaVβ4 and FGF14, on modulating Na channel blocked as well as inactivated states. We find that although both NaVβ4 and the FGF14 splice variant FGF14-1a contain sequences that can generate resurgent-like currents when applied to Na channels in peptide form, deletion of either protein, or both proteins simultaneously, does not eliminate resurgent current in acutely dissociated Purkinje cell bodies. Loss of FGF14 expression does, however, reduce resurgent current amplitude and leads to an acceleration and stabilization of inactivation that is not reversed by application of the site-3 toxin, anemone toxin II (ATX). Tetrodotoxin (TTX) sensitivity is higher for resurgent than transient components of Na current, and loss of FGF14 preferentially affects a highly TTX-sensitive subset of Purkinje α subunits. The data suggest that NaV1.6 channels, which are known to generate the majority of Purkinje cell resurgent current, bind TTX with high affinity and are modulated by FGF14 to facilitate open-channel block.

Fibroblast growth factor homologous factors tune arrhythmogenic late NaV1.5 current in calmodulin binding–deficient channels

“…As such, we expect intact FHF binding to the F1759A mutant. Na V channels in Purkinje neurons exhibit a resurgent current that results from open-state block by intracellular endogenous proteins, including FGF14 and the cytosolic domain of the Na V β4 subunit ( 52 ). Interestingly, such endogenous open-channel blocking proteins antagonize lidocaine action, suggesting that the 2 sites may be coupled ( 53 ).…”

Mapping of the FGF14:Nav1.6 complex interface reveals FLPK as a functionally active peptide modulating excitability

“…Previous studies have shown that overexpression of FGF14-1b in hippocampal neurons potentiates transient Na + currents (Laezza et al, 2007), while genetic deletion of FGF14 leads to reduced Na + currents and firing in several neuron subtypes including MSNs in the NAc (Ali et al, 2018;Goldfarb et al, 2007;White et al, 2019). These phenotypes have been attributed to a role of FGF14 in preserving the pool of active channels, in part by minimizing inactivation and increasing channel availability (White et al, 2019). However, if FLPK acts as an inhibitor of the FGF14:Nav1.6 complex, as suggested by our LCA studies, the effect of the tetrapeptide on persistent Na + currents would be hard to reconcile.…”

“…accessory protein, excitability, inactivation, sodium channels 2020; White et al, 2019). These new studies identify the FLPK tetrapeptide as a useful tool to probe Nav1.6 channel function and a scaffold for future drug development towards treatment of a wide range of channelopathies associated with Nav channels (Alshammari et al, 2016;Chahine et al, 2008;Di Re et al, 2017;Eijkelkamp et al, 2012;Hsu et al, 2017).…”

“…Previous studies have provided evidence for FGF14 as an accessory subunit and a functional regulator of the Nav1.6 channel (Ali, Singh, & Laezza, 2016;Goetz et al, 2009;Laezza et al, 2009;Lou et al, 2005;Rush et al, 2006;White, Brown, Bozza, & Raman, 2019;Wittmack, 2004). Through direct protein:protein interactions (PPI) with the intracellular C-terminal tail of Nav1.6, the FGF14-1b isoform controls Nav1.6-mediated transient currents, kinetics of fast inactivation and voltage-dependence of activation and steady-state inactivation of the channel (Ali et al, 2016(Ali et al, , 2018.…”

Infant and adult SCA13 mutations differentially affect Purkinje cell excitability, maturation, and viability in vivo

“…Kv3.3 is co-expressed in Purkinje cells with the Na v 1.6 voltage-gated Na + channel ( Raman et al, 1997 ; Khaliq et al, 2003 ; Martina et al, 2003 ; Akemann and Knöpfel, 2006 ). During an action potential, Na v 1.6 is subject to open channel block conferred by an auxiliary subunit thought to be Na v β4 or FGF14 ( Grieco et al, 2005 ; White et al, 2019 ). Rapid repolarization mediated by Kv3.3 relieves open channel block of Na + channels, generating a resurgent Na + current in the interspike interval that triggers the next action potential ( Raman and Bean, 1997 ; Khaliq et al, 2003 ; Grieco et al, 2005 ).…”