title: The roles of Voltage-Gated Sodium Channels and β subunits in mechanosensitivity and neuronal electrical excitability
Creators: name: Maroni, Michele
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2024
Subjects: subject: Voltage Gated Sodium Channels
subject: Nav1.5
subject: Nav β Subunits
subject: Electrophysiology
subject: Induced Pluripotent Stem Cells
subject: Nociceptors
subject: Midbraind Dopaminergic Neurons

description: Background and aims: The voltage-gated sodium channels (Nav) are responsible for action potential generation and propagation in excitable cells like cardiac myocytes and neurons. The modulation of Navs affects the properties of action potentials and cellular excitability. The main Nav in cardiac myocytes is Nav1.5 and is modulated by mechanical stimuli. Previous studies showed that also Nav 1.4, Nav 1.6 and Nav1.7 are mechanosensitive, and a recent study showed that the Nav β1 subunit modulates the mechanosensitivity of Nav1.7. This work aimed to investigate whether the mechanosensitivity of Nav1.5 is regulated by β subunits. For this purpose, I characterized the gating and kinetic properties of Nav1.5 in the presence of β1 and β3 subunits in the HEK293 cell line. While cell lines offer an accessible and effective model for studying Nav properties and cellular excitability, they do not fully recapitulate the in vivo properties of human excitable cells like cardiomyocytes and neurons. Therefore, to better understand excitability in models of neurological diseases, I also conducted electrophysiological studies on human induced pluripotent stem cells (hiPSCs)-derived nociceptors and midbrain dopaminergic neurons. Methods and results: To investigate the modulation of Nav mechanosensitivity by β subunits, Nav1.5 was heterologously expressed in HEK293 cells, alone or combined with either β1 or β3 subunits. Patch-clamp recordings were performed in the Nav1.5, Nav1.5 β1, and Nav1.5 β3 expression groups, with or without mechanical stimulation. This study demonstrates that β1 and β3 subunits elicit specific differences in gating and kinetic properties of Na+ currents under mechanical stimulation. The β1 subunit enhanced the effect of mechanical stimulation on fast inactivation. In contrast, the β3 subunit showed a stronger effect on the kinetics of current decay under mechanical stimulation. Using homology modeling and residue interaction network (RIN) analysis, this study identified differences in the number, strength, and distribution of bonds that β1 and β3 subunits establish with DI, DIII, and DIV (Domain I-IV of Navs) of Nav1.5 channel. In the RIN analysis, β3 established a stronger binding to Nav1.5 compared to β1. These binding differences could account for the effects on fast inactivation and kinetics of current decay since it has been shown that the movement of the voltage sensing domain within the DIV of Nav channels is important for the onset of inactivation. Additionally, the electrophysiological properties of hiPSCs-derived midbrain dopaminergic neurons and nociceptors were analyzed using conventional patch-clamp and multi-electrode-arrays. These results show that both midbrain dopaminergic neurons and nociceptors differentiated from stem cells in dual-SMAD inhibition are electrically active. Finally, slow inactivation and modulation of electrical excitability by Lacosamide were evaluated using stem cell-derived nociceptors from an SFN patient bearing a Nav1.9 variant. Discussion: This study demonstrates that the mechanosensitivity of Navs and their subunits is subtype-specific. The study shows that Navs mechanosensitivity is differentially modulated by distinct β subunits and that one β subunit may have specific effects in modulating the mechanosensitivity of different Navs subtypes. These results provide insights into the role of Nav in the mechanosensitive regulation of cardiac function through mechano-electric feedback. The β1 and β3 subunits are differentially expressed in myofibroblasts and cardiomyocytes and are also expressed in different cellular compartments. This differential expression pattern offers a perspective on how electrical excitability is modulated mechanically by the β3 subunit at the plasma membrane and by β1 at intercalated disks and in myofibroblasts. The investigation of electrophysiological properties of hiPSCs-derived midbrain dopaminergic neurons and nociceptors showed that the obtained neurons are electrically active. Furthermore, no alteration in slow inactivation potentially contributing to the patient’s pain phenotype was detected in nociceptors derived from the SFN patient’s hiPSCs. Using multi-electrode-array, I show that Lacosamide normalizes the altered electrical activity observed in the patient’s nociceptors.