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

Abstract

In neurons, changes in Akt activity have been detected in response to the stimulation of transmembrane receptors. However, the mechanisms that lead to changes in neuronal function upon Akt inhibition are still poorly understood. In the present study, we interrogate how Akt inhibition could affect the activity of the neuronal Nav channels with while impacting intrinsic excitability. To that end, we employed voltage-clamp electrophysiological recordings in heterologous cells expressing the Nav1.6 channel isoform and in hippocampal CA1 pyramidal neurons in the presence of triciribine, an inhibitor of Akt. We showed that in both systems, Akt inhibition resulted in a potentiation of peak transient Na+ current (INa) density. Akt inhibition correspondingly led to an increase in the action potential firing of the CA1 pyramidal neurons that was accompanied by a decrease in the action potential current threshold. Complementary confocal analysis in the CA1 pyramidal neurons showed that the inhibition of Akt is associated with the lengthening of Nav1.6 fluorescent intensity along the axonal initial segment (AIS), providing a mechanism for augmented neuronal excitability. Taken together, these findings provide evidence that Akt-mediated signal transduction might affect neuronal excitability in a Nav1.6-dependent manner.

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

“…These studies are in line with the Shavkunov, Wildburger, Nenov et al, (2013) paper, demonstrating that pharmacological inhibition of GSK3 (GSK3 inhibitor XIII and CHIR99021) induces dissociation of FGF14 from the Na v channels at the AIS and redistribution of the complex in the somatodendritic compartment [90]. As mentioned earlier, pseudo-substrate inhibition of GSK3β via Ser9 phosphorylation can be prevented with compounds such as triciribine, allowing increased GSK3β activity and ultimately increasing Na v 1.6-mediated currents [160]. These results are consistent with the idea that GSK3β activity is directly connected to increased neuronal excitability.…”

TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria

“…Whole-cell voltage-clamp recordings to assess the Na v 1.6-mediated I Na of CA1 pyramidal cells in the slice preparation were performed as previously described [ 38 ]. Briefly, slices were transferred to a recording chamber perfused with continuously oxygenated and heated standard aCSF that was supplemented with 120 µM CdCl 2 , 1 µM ICA12142, and 10 nM Phrixotoxin3 to block currents mediated by voltage-gated Ca 2+ channels, Na v 1.1 channels, and Na v 1.2 channels, respectively.…”

“…After GΩ formation and entry into the whole-cell configuration, the following cocktail of synaptic blockers was perfused for 1 min to mitigate synaptic currents: 20 µM bicuculline, 20 µM NBQX, and 100 µM AP-5 (synaptic blockers were purchased from Tocris, Bristol, UK). I Na was elicited using the voltage-clamp protocols described elsewhere [ 39 , 40 ] and data analysis was performed as previously described [ 38 , 41 ].…”

“…Whole-cell current-clamp recordings to assess intrinsic firing properties of CA1 pyramidal cells were performed as previously described [ 38 ]. Briefly, slices were transferred to a recording chamber perfused with continuously oxygenated and heated standard aCSF.…”

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“…For voltage-clamp recordings, borosilicate glass pipettes (Harvard Apparatus, Holliston, MA, United States) with resistance of 1.5–3 MΩ filled with an internal solution comprised of the following salts were used: 100 mM Cs-gluconate (Hello Bio Inc., Princeton, NJ, United States); 10 mM tetraethylammonium chloride; 5 mM 4-aminopyridine; 10 mM EGTA; 1 mM CaCl 2 ; 10 mM HEPES; 4 mM Mg-ATP; 0.3 mM Na 3 -GTP; 4 mM Na 2 -phosphocreatine; and 4 mM NaCl (pH = 7.4 and osmolarity = 285 ± 5 mOsm/L; CsOH used to adjust pH and osmolarity; all salts except Cs-gluconate purchased from Sigma-Aldrich). After GΩ seal formation and entry into the whole-cell configuration, a cocktail of synaptic blockers (20 μM bicuculline, 20 μM NBQX, and 100 μM AP5; synaptic blockers purchased from Tocris, Bristol, United Kingdom) was perfused and two voltage-clamp protocols previously described were employed ( Dvorak et al, 2021 ; Marosi et al, 2022 ). Briefly, to assess the current–voltage relationship of I NaT elicited by MSNs and activation properties of I NaT , MSNs were subjected to voltage commands ranging from −90 mV to +30 mV (Δ = 5 mV) following a 5 ms pre-pulse at −35 mV to mitigate space clamp issues as previously described ( Milescu et al, 2010 ).…”