GABAB receptor modulation of membrane excitability in human pluripotent stem cell-derived sensory neurons by baclofen and α-conotoxin Vc1.1
Ontology highlight
ABSTRACT: 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.
Project description:Electrical excitability—the ability to fire and propagate action potentials—is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons or requires signaling from glial cells remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage gated sodium channels, and to fire action potential trains. In this RNA-Seq study, we discovered that treating cultured DRG neurons with Schwann cell-conditioned media or PGE2 increased the expression of several genes required for neuronal maturation and excitability, including voltage-gated sodium channels.
Project description:Voltage-gated potassium channels (VGKCs) comprise one of the largest, most diverse and complex families of ion channels. Approximately 70 genes encode the alpha subunits that form homomeric VGKC channels. In addition, these subunits can form functional heteromeric channels, thus exponentially increasing the diversity of VGKCs. The functional expression and physiological role of heteromeric K-channels have remained largely unexplored due to the lack of tools to probe their functions. Conotoxins have high affinity and specificity for heteromeric combinations of K-channels and show great promise for elucidating their functions. In this work, using conotoxin kM-RIIIJ as a pharmacological probe, we explore the expression and physiological functions of heteromeric Kv1.2 channels using constellation pharmacology. We report that heteromers of Kv1.2/1.1 are highly expressed in proprioceptive neurons found in the dorsal root ganglion (DRG). Inhibition of Kv1.2/1.1 heteromers leads to an influx of calcium ions, suggesting that these channels regulate neuronal excitability. We also present evidence that Kv1.2/1.1 heteromers counteract persistent sodium currents, and that inhibiting these channels leads to tonic firing of action potentials. Additionally, kM-RIIIJ induces impaired proprioception in mice, uncovering a previously unrecognized physiological role of heteromeric Kv1.2/1.1 channels in proprioceptive sensory neurons of the DRG.
Project description:Pain is the leading cause of disability in the developed world but remains a poorly treated condition. Specifically, post-surgical pain continues to be a frequent and undermanaged condition. Here, we investigate the analgesic potential of pharmacological NaV1.7 inhibition in a mouse model of acute post-surgical pain, based on incision of the plantar skin and underlying muscle of the hind paw. We demonstrate that local and systemic treatment with the selective NaV1.7 inhibitor μ-theraphotoxin-Pn3a is effectively anti-allodynic in this model and completely reverses mechanical hypersensitivity in the absence of motor adverse effects. In addition, the selective NaV1.7 inhibitors ProTx-II and PF-04856264 as well as the clinical candidate CNV1014802 also reduced mechanical allodynia. Interestingly, co-administration of the opioid receptor antagonist naloxone completely reversed analgesic effects of Pn3a, indicating an involvement of endogenous opioids in the analgesic activity of Pn3a. Additionally, we found super-additive antinociceptive effects of sub-therapeutic Pn3a doses not only with the opioid oxycodone but also with the GABAB receptor agonist baclofen. Transcriptomic analysis of gene expression changes in dorsal root ganglia of mice post-surgery revealed decreased expression of several pro-nociceptive genes including N- and P/Q-type voltage-gated calcium channels important for neurotransmitter release, which suggest a reactive compensatory mechanism to reduce excessive pain similar to the endogenous opioid system. In summary, these findings suggest that pain after surgery can be successfully treated with NaV1.7 inhibitors alone or in combination with baclofen or opioids, which may present a novel and safe treatment strategy for this frequent and poorly managed condition.
Project description:To explore the molecular basis of the distinct intrinsic membrane properties and other dstinguishing features of functionally defined DRG neuron subtypes, we bulk-sequenced RNA at high depth of genetically-labeled DRG neurons to generate transcriptome profiles of eight major DRG neuron subtypes. The trancriptome profiles revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by the transcriptome pofiles, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns of the DRG neuron subtypes. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties.
Project description:Using whole-cell patch clamp recording and unbiased gene expression profiling in rat dissociated hippocampal neurons cultured at high density, we demonstrate here that chronic activity blockade induced by the sodium channel blocker tetrodotoxin leads to a homeostatic increase in action potential firing and down-regulation of potassium channel genes. In addition, chronic activity blockade reduces total potassium current, as well as protein expression and current of voltage-gated Kv1 and Kv7 potassium channels, which are critical regulators of action potential firing. Importantly, inhibition of N-Methyl-D-Aspartate receptors alone mimics the effects of tetrodotoxin, including the elevation in firing frequency and reduction of potassium channel gene expression and current driven by activity blockade, whereas inhibition of L-type voltage-gated calcium channels has no effect.
Project description:GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic GBR function is the gating of ion channels. However, where stable GBR-effector channel signaling units are formed in the biosynthetic pathway has remained unclear. Here we show that the vesicular protein synaptotagmin-11 (Syt11) binds the auxiliary GBR subunit KCTD16 and Cav2.2 channels. This enables Syt11 to recruit GBR/Cav2.2 channel complexes to post-Golgi vesicles that transport the pre-assembled signaling complexes in axons and dendrites. Bimolecular fluorescence complementation experiments reveal that GBR/Syt11 complexes are delivered to synaptic sites. Furthermore, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Syt11-deficient neurons exhibit reduced glutamatergic synaptic transmission and impaired GBR-mediated presynaptic inhibition, thus highlighting a key role for Syt11 in the transport and stable expression of functional GBR/Cav2.2 complexes at synapses.
Project description:Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of Kv7 channels is regulated by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signalling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP2), Ca2+/Calmodulin and phosphorylation. Recent studies have reported that PIP2 sensitivity of Kv7.2 channels is affected by two PTMs, phosphorylation and methylation, harboured in the PIP2 binding domains. Here this project aimed to update phosphorylation and methylation sites on Kv7.2 from heterologous cells, the rat brain and GST-fusion Kv7.2 N- and C-terminal ends proteins.
Project description:In the present work, we gain insight into the impact of oligodendrocyte secreted factors on hippocampal GABAergic neuron physiology, and performed patch-clamp recordings followed by single-cell RNA sequencing of neurons. Our results demonstrate that the GABAergic neuron excitability is influenced by glial cells and oligodendrocyte-secreted factors. Moreover, we show specific ion channels alterations and find a correlation with electrophysiological parameters suggesting possible mechanisms for the OCM-induced regulation of neuronal function.
Project description:Electrical excitability—the ability to fire and propagate action potentials—is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons or requires signaling from glial cells remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage gated sodium channels, and to fire action potential trains. In this scRNAseq study, we found that inactivating PGE2 synthesis in Schwann cells, in vivo, impaired somatosensory neuron maturation, with the most dramatic effects on nociceptor and proprioceptor somatosensory neuron subtypes.
Project description:Electrical excitability—the ability to fire and propagate action potentials—is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons or requires signaling from glial cells remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage gated sodium channels, and to fire action potential trains. In this scRNAseq study, we found that inactivating PGE2 synthesis in Schwann cells in vivo impaired somatosensory neuron maturation, with the most dramatic effects on nociceptor and proprioceptor somatosensory neuron subtypes.