Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex

Abstract

Voltage-gated Na+ (Nav) channels are a primary molecular determinant of the action potential (AP). Despite the canonical role of the pore-forming α subunit in conferring this function, protein–protein interactions (PPI) between the Nav channel α subunit and its auxiliary proteins are necessary to reconstitute the full physiological activity of the channel and to fine-tune neuronal excitability. In the brain, the Nav channel isoforms 1.2 (Nav1.2) and 1.6 (Nav1.6) are enriched, and their activities are differentially regulated by the Nav channel auxiliary protein fibroblast growth factor 14 (FGF14). Despite the known regulation of neuronal Nav channel activity by FGF14, less is known about cellular signaling molecules that might modulate these regulatory effects of FGF14. To that end, and building upon our previous investigations suggesting that neuronal Nav channel activity is regulated by a kinase network involving GSK3, AKT, and Wee1, we interrogate in our current investigation how pharmacological inhibition of Wee1 kinase, a serine/threonine and tyrosine kinase that is a crucial component of the G2-M cell cycle checkpoint, affects the Nav1.2 and Nav1.6 channel macromolecular complexes. Our results show that the highly selective inhibitor of Wee1 kinase, called Wee1 inhibitor II, modulates FGF14:Nav1.2 complex assembly, but does not significantly affect FGF14:Nav1.6 complex assembly. These results are functionally recapitulated, as Wee1 inhibitor II entirely alters FGF14-mediated regulation of the Nav1.2 channel, but displays no effects on the Nav1.6 channel. At the molecular level, these effects of Wee1 inhibitor II on FGF14:Nav1.2 complex assembly and FGF14-mediated regulation of Nav1.2-mediated Na+ currents are shown to be dependent upon the presence of Y158 of FGF14, a residue known to be a prominent site for phosphorylation-mediated regulation of the protein. Overall, our data suggest that pharmacological inhibition of Wee1 confers selective modulatory effects on Nav1.2 channel activity, which has important implications for unraveling cellular signaling pathways that fine-tune neuronal excitability.

Inhibition of the Akt/PKB Kinase Increases Nav1.6-Mediated Currents and Neuronal Excitability in CA1 Hippocampal Pyramidal Neurons

“…Pertinent to the present investigation, we have previously shown that the genetic and pharmacological manipulation of glycogen synthase kinase 3 (GSK3), a kinase whose activity is decreased upon phosphorylation by Akt, confers changes in the activity of the Na v channels and neuronal excitability [52]. Related to this, we have shown that the pharmacological manipulation of Wee1, a kinase whose activity potentially increases Akt activity, also confers changes in Na v channel activity [49,54]. Despite these reported regulatory effects on Na v channel activity conferred by manipulating the kinases that control Akt activity, the effects of directly altering Akt activity on Na v channel activity have been less well described.…”

Pharmacologically Targeting the Fibroblast Growth Factor 14 Interaction Site on the Voltage-Gated Na+ Channel 1.6 Enables Isoform-Selective Modulation

“…However, the opposite effects of FGF14, the protein from which PW201 is derived, in heterologous cells versus neurons is widely recognized [12,17,24]. Specifically, co-expression of FGF14 with the Na v 1.6 channel in heterologous systems has previously been shown to suppress Na v 1.6-mediated I Na [12,15,24,38,40,41,60,61], whereas over-expression of FGF14 in neurons has been shown to increase I Na [17]. As such, these opposite effects observed for PW201 in heterologous cells versus in neurons are unsurprising and provide supporting evidence for the compound functioning as a partial pharmacological mimic of FGF14.…”

Glycogen Synthase Kinase 3: Ion Channels, Plasticity, and Diseases

“…Wee1 may also activate AKT (PkB), which, as mentioned earlier, can inactivate GSK3β through S9 pseudo-substrate phosphorylation [163]. Not only is the Wee1 kinase a crucial component of the G2-M cell cycle checkpoint, and its activity is modulated by GSK3 [164], but also it has been shown to confer isoform-specific effects on Na v channels via PPIs regulation [165].…”