GABAB receptor modulation of membrane excitability in human pluripotent stem cell-derived sensory neurons by baclofen and α-conotoxin Vc1.1

GABAB receptor (GABABR) activation is known to alleviate pain by reducing neuronal excitability, primarily through inhibition of high voltage-activated (HVA) calcium (CaV2.2) channels and potentiating G protein-coupled inwardly rectifying potassium (GIRK) channels. While the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons, emerging strategies to develop, study, and characterise human pluripotent stem cell (hPSC)-derived sensory neurons present a promising alternative. In this study, hPSCs were efficiently differentiated into peripheral DRG-induced sensory neurons (iSNs) using a combined chemical and transcription factor-driven approach, via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A, ISLET1, and PRPH, in addition to GABABR and ion channels including CaV2.2 and GIRK1 in iSNs. Functional characterisation of GABABR was conducted using whole-cell patch clamp electrophysiology, assessing neuronal excitability under current clamp conditions in the absence and presence of GABABR agonists baclofen and α-conotoxin Vc1.1. Both baclofen (100 mM) and Vc1.1 (1 mM) significantly reduced membrane excitability by hyperpolarizing the resting membrane potential, and increasing the rheobase for action potential firing. In voltage-clamp mode, baclofen and Vc1.1 inhibited HVA Ca2+ channel currents, which were attenuated by the selective GABABR antagonist CGP 55845. However, modulation of GIRK channels by GABABRs was not observed in the presence of baclofen or Vc1.1, suggesting that functional GIRK1/2 channels were not coupled to GABABRs in hPSC-derived iSNs. This study reports is the first to report GABABR modulation of membrane excitability in iSNs by baclofen and Vc1.1, highlighting their potential as a future model for studying analgesic compounds. In preparation for iSN differentiation, cell culture plasticware was first coated with 10 μg/ml Poly-D-Lysine (Sigma–Aldrich) for 30 min at room temperature, then coated with Matrigel overnight at 37 °C. CD271+ NCCs were then seeded at a density of approximately 8.0 ´ 103 cells/cm2 on 12 mm circular glass coverslips for patch-clamp studies, 2.6 ´ 104 cells/cm2 on 13 mm circular glass coverslips for immunocytochemistry, or 2.6 ´ 104 cells/cm2 directly on plasticware for transcriptomic analyses. iSN differentiation was completed following previously published methods with some modifications (Hulme et al., 2020) St Clair-Glover et al, 2024). Briefly, cells were cultured in neuronal medium containing Neurobasal™ medium containing 1% N2, 1% B-27 minus vitamin A, 1% Insulin-transferrin-Selenium-A, and 1% GlutaMAX™ (all from Gibco, Waltham, MA, USA). Throughout iSN differentiation, the media was supplemented with 10 μM Y-27632, 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml NT-3, and 10 ng/ml b-NGF (all from STEMCELL Technologies), with media changes conducted every 2-3 days. For the initial 96 h after plating (day 6-10), NGN2 expression was induced by the application of doxycycline at 2 µg/mL (Sigma-Aldrich, D9891). From day 14, cells were phased into BrainPhys™ neuronal medium (BrainPhys™ Neuronal Medium, 0.02% NeuroCult™ SM1 without Vitamin A, 0.01% N2 Supplement-A [all from STEMCELL Technologies]) for maturation, with a ratio of 25:75, 50:50, 75:25, 100:0 (BrainPhys™: neuronal media) at each media change. Where required, proliferating cells were removed by adding anti-mitotic agent, cytosine β-D-arabinofuranoside at 2.5 μM (AraC, Sigma-Aldrich), for 48 h between days 16-18