Biology subjects

Hautvast, P.

Publications and source records attributed to Hautvast, P..

3 recordsLinked to original sources

bioRxiv2026

Polyclonal sensory neuron derivation from iPSCs as an efficient alternative to single clone strategies for pain relevant in vitro models

Induced pluripotent stem cell (iPSC) workflows typically rely on monoclonal cultures, requiring multiple independent cell lines to compensate for clonal variability. This increases workload, cost, and limits scalability for translational applications. Here, we establish polyclonal reprogramming strategies using Sendai virus to generate clonally diverse iPSC cultures already during reprogramming through FACS, MACS, manual selection, or prolonged culture. Both monoclonal and FACS-derived polyclonal cultures reached pluripotency. Reprogramming transgenes were silenced, whereas Sendai virus (SeV) mRNA persisted across passages in both culture types; heat treatment at 38.5C markedly reduced SeV levels. Monoclonal and polyclonal cultures differentiated efficiently into neural crest-like cells and sensory neurons. As a second polyclonal strategy, monoclonal cultures were pooled at day5 of sensory neuron differentiation to generate percentage-controlled progenitor mixtures. TRPA1 protein expression has not been shown reliably in iPS-derived sensory neurons. We hypothesized that increased clonal diversity might facilitate detection of TRPA1 protein expression. Despite rigorous antibody validation, TRPA1 protein expression was detectable only diffusely across the whole cell area. Together, our results show that polyclonal iPSC strategies enable clonally inclusive generation of patient-specific sensory neurons. These workflows reduce early workload and provide a robust foundation for scalable drug-screening and patient-in-a-dish applications.

neuroscience↗

bioRxiv2026

Outer Pore Collapse as a Potential Mechanism of Partial Loss of Pain in Nav1.7 M899I

Complete loss of function of the voltage-gated sodium channel subtype Nav1.7, encoded by SCN9A, results in congenital insensitivity to pain. Here, we investigate a previously identified variant, M899I, in which methionine at position 899 is substituted by isoleucine. This variant was originally described in a Chinese patient with loss of pain. We confirmed membrane expression of the mutant channel in HEK cells using extracellular HA-tagging; however, no sodium currents were detectable from the variant in patch-clamp recordings. The M899I substitution is located within a tightly packed hydrophobic region of the pore module. Introducing the corresponding variant into Nav1.2 and Nav1.5 similarly abolished channel function, underscoring the high conservation and functional importance of this residue. To further investigate the underlying mechanism, we combined in-silico coarse-grained molecular dynamics simulations with in-vitro electrophysiological analysis. Our simulations predicted that the M899I substitution induces collapse of the outer pore, substantially reducing both pore radius and volume. Substitution with other hydrophobic residues was likewise predicted to alter pore geometry and, consequently, ion permeation to varying degrees. Whole-cell voltage-clamp recordings validated these predictions, with observed current densities closely correlating with the extent of pore collapse predicted in silico. Together, our findings establish pore collapse as a mechanism underlying disease-relevant loss-of-function variants in Nav1.7 and suggest that this principle may extend to other sodium channel subtypes. Moreover, our results demonstrate that in-silico molecular dynamics approaches can reliably predict structural and functional consequences of channel mutations, as confirmed by in-vitro electrophysiological data.

biophysics↗

bioRxiv2019

Uncoupling sodium channel dimers rescues phenotype of pain-linked Nav1.7 mutation

The voltage-gated sodium channel Nav1.7 is essential for an adequate perception of painful stimuli. Its mutations cause various pain syndromes in human patients. The hNav1.7/A1632E mutation induces symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD), and its main gating change is a strongly enhanced persistent current.\n\nUsing molecular simulations, we demonstrate that the disease causing persistent current of hNav1.7/A1632E is due to impaired binding of the IFM motif, thus affecting proper function of the recently proposed allosteric fast inactivation mechanism. By using native polyacrylamide gel electrophoresis (PAGE) gels, we show that hNav1.7 dimerizes. The disease-linked persistent current depends on the channels functional dimerization status: Using difopein, a 14-3-3 inhibitor known to uncouple dimerization of hNav1.5, we detect a significant decrease in hNav1.7/A1632E induced persistent currents.\n\nOur work identifies that functional uncoupling of hNav1.7/A1632E dimers rescues the pain-causing molecular phenotype by interferes with an allosteric fast inactivation mechanism, which we link for the first time to channel dimerization. Our work supports the concept of sodium channel dimerization and reveals its relevance to human pain syndromes.

neuroscience↗