The role of voltage-gated sodium channels in neurological diseases : lessons learned from pain disorders and Huntington’s disease

• Die Rolle spannungsgesteuerter Natriumkanäle bei neurologischen Erkrankungen: Lehren aus Schmerzerkrankungen und der Huntington-Krankheit

Le Cann, Kim; Rothermel, Markus (Thesis advisor); Lampert, Angelika (Thesis advisor); Spehr, Marc (Thesis advisor); Müller, Frank (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

Voltage-gated sodium channels (Navs) are large four-domain transmembrane proteins responsible for the initiation and the propagation of action potentials (APs) of electrically excitable cells. Nine distinct Nav  subunits have been characterized, that can be associated with one or two Nav  subunits. Nav  subunits can modulate Nav kinetics, gating properties and level of expression at the cell membrane. Mutations of these  and/or  subunits may lead to various diseases such as pain, epilepsy, cardiac failure, and psychiatric disorders. In this thesis, we focused on Nav1.7, a sodium channel highly involved in pain signaling. We were also interested in the function of Nav  and  subunits expressed in the striatal medium spiny neurons (MSNs), in the context of Huntington’s disease (HD).Whole-cell voltage-clamp recordings were performed in a heterologous expression system, here HEK cells, to investigate the gating properties of Nav1.7/N1245S, a Nav1.7 variant potentially responsible for chronic pain. To decipher the electrical firing of striatal neurons in both physiological and HD conditions, I generated human induced-pluripotent stem cell (iPS cell)-derived striatal MSNs and performed patch-clamp experiments.The Nav1.7/N1245S variant was identified in several patients suffering from burning pain in the extremities. Voltage-clamp recordings revealed that the N1245S variant channels activate and inactivate at the same voltages compared to Nav1.7/WT. They also inactivate and deactivate with the same kinetics and recover from fast inactivation at a similar time course. We found an enhanced slow inactivation for the N1245S variant, which does not support a Nav1.7 gain-of-function mutation often associated with pain. Homology modeling based on hNav1.7 cryo-electron microscopy structure pointed to one different amino acid interaction partner for S1245N, without any obvious change of the overall protein conformation. Taken together, these results suggest that the Nav1.7/N1245S variant may be part of a more complex pathophysiology underlying the pain phenotype of the patients, which potentially cannot be reproduced using the relatively simple HEK cell line expression system.Nav function was also investigated in a more physiological context in the study of HD, a genetic disorder characterized by a massive degeneration of striatal MSNs. The origin of this neuronal loss is still unknown and slows down the efforts to develop reliable therapeutics. Human post mortem studies revealed a down-regulation of the sodium channel 4 subunit specifically in these neurons of HD patients. We aimed to investigate the possible consequences of this down-regulation on Nav function and MSN excitability. We selected and established two differentiation protocols preported to generate human MSNs from iPS cells. Our immunocytochemical data revealed very low percentages of striatal neurons across genotypes and protocols, thus pointing to a lack of reliability and reproducibility in these protocols. Voltage-clamp recordings revealed inconsistencies between differentiation protocols rather than Nav gating changes between genotypes. Our results highlight the need to improve the efficiency of the currently existing differentiation protocols. We suggest comparing large datasets from different studies to interpret our findings more confidently. It seems that studying data obtained from only one differentiation protocol increases the risk of misinterpreting the results. Altogether, these results highlight the complexity of Nav function and their study in heterologous systems. They also direct attention on the hiPS cell technique that still requires methodological improvements before being powerful enough to study neuronal disorders in a more physiological human context.

Institutions

• Department of Biology [160000]
• Chemosensation Laboratory [163310]
• [512000-3]