There has been considerable progress in identifying signaling pathways directing the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types including neurons. However, extrinsic factor-based differentiation of hPSCs is a slow, step-wise process mimicking the protracted timing of normal human development. Using a small molecule screen we identified a combination of five small molecule pathway inhibitors sufficient to yield hPSC-derived neurons at >75% efficiency within 10 days of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors including TTX-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli including ATP and capsaicin. Neuronal fate acquisition occurs three-fold faster than during in vivo1 development suggesting that use of small molecule pathway inhibitors could develop into a general strategy for accelerating developmental timing in vitro. The quick and high efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain.

The voltage-sensitive sodium channel confers electrical excitability on neurons, a fundamental property required for higher processes including cognition. The ion-conducting ␣-subunit of the channel is regulated by two known auxiliary subunits, ␤1 and ␤2. We have identified rat and human forms of an additional subunit, ␤3. It is most closely related to ␤1 and is the product of a separate gene localized to human chromosome 11q23.3. When expressed in Xenopus oocytes, ␤3 inactivates sodium channel opening more slowly than ␤1 does. Structural modeling has identified an amino acid residue in the putative ␣-subunit binding site of ␤3 that may play a role in this difference. The expression of ␤3 within the central nervous system differs significantly from ␤1. Our results strongly suggest that ␤3 performs a distinct neurophysiological function.T he voltage-sensitive sodium channel plays a fundamental role in excitable cells, transiently increasing the sodium permeability of the plasma membrane in response to changes in membrane potential and thus propagating the action potential (1, 2). Not surprisingly, mutations in sodium channel genes are implicated in several pathologies, including epilepsy and cardiac arrhythmias (3-5), and therapeutic drugs, including antiepileptics, local anesthetics, and anticonvulsants (6), act on the channel.In the central nervous system, the channel is conventionally described as a heterotrimer composed of a 260-kDa ␣-subunit, a noncovalently associated 36-kDa ␤1-subunit, and a disulfidelinked 33-kDa ␤2-subunit (2). The ␣-subunit forms the ion pore and is responsible for the voltage-sensitive characteristics of the complex. There are multiple isoforms of the ␣-subunit expressed in different regions of the brain and peripheral nervous system that differ in their kinetic properties (1). The ␤-subunits are auxiliary components acting in a regulatory capacity (7). ␤1 increases the fraction of ␣-subunits operating in a fast gating mode, thus accelerating the activation and inactivation kinetics of the channel and modulating the frequency with which neurons fire (8). The ␤2-subunit is required for the efficient assembly of the channel but has minor effects on gating kinetics. These two ␤-subunits are distantly related by sequence (9).We now report the cloning and analysis of the rat and human forms of a previously uncharacterized sequence that we call ␤3. It is homologous to ␤1, but differs from ␤1 both in its distribution within the brain and in some of its kinetic properties. The discovery of this subunit increases the complexity of the sodium channel and raises further questions about the role of these auxiliary subunits. Materials and MethodsCloning Methodology. We isolated a variant of the rat pheochromocytoma cell line PC12 (termed A35C), which lacks typical neuronal properties (10). To discover previously unidentified neuroendocrine-specific genes, subtractive cloning was used to identify transcripts expressed at a level in the variant cells lower than that in normal PC12 cells. Total RNA wa...

Ion channels are membrane proteins expressed in almost all living cells. The sequencing of the human genome has identified more than 400 putative ion channels, but only a fraction of these have been cloned and functionally tested. The widespread tissue distribution of ion channels, coupled with the plethora of physiological consequences of their opening and closing, makes ion-channel-targeted drug discovery highly compelling. However, despite some important drugs in clinical use today, as a class, ion channels remain underexploited in drug discovery and many existing drugs are poorly selective with significant toxicities or suboptimal efficacy. This Perspective seeks to review the ion channel family, its structural and functional features, and the diseases that are known to be modulated by members of the family. In particular, we will explore the structure and properties of known ligands and consider the future prospects for drug discovery in this challenging but high potential area.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7’s role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

One sentence summary: Bench to bedside translation using iPSC to characterise phenotype and pharmacology in primary erythromelalgia, an inherited chronic pain condition. ABSTRACTIn common with other chronic pain conditions, inherited erythromelalgia (IEM) represents a significant unmet medical need. The peripherally expressed SCN9A encoded sodium channel Nav1.7 plays a critical role in IEM with gain-of-function leading to aberrant sensory neuronal activity and extreme pain, particularly in response to heat. In five carefully phenotyped IEM patients, a novel highly potent and selective Nav1.7 blocker reduced heat-induced pain in the majority of subjects. In four of the five subjects we used induced pluripotent stem cell (iPSC) technology to create sensory neurons which uniquely emulated the clinical phenotype of hyperexcitability and aberrant responses to heat stimuli. When we compared the severity of the clinical phenotype with the iPSC-derived sensory neuron hyperexcitability we saw a trend towards a correlation for individual mutations. The in vitro IEM phenotype was sensitive to Nav1.7 blockers, including the clinical test agent. Given the importance of peripherally expressed sodium channels in many pain conditions, this translational approach is likely to have broader utility to a wide range of pain and sensory conditions. This emphasizes the use of iPSC approaches to bridge between clinical and preclinical studies, enabling greater understanding of a disease and the response to a therapeutic agent in defined patient populations.

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell–derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

1. We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Nab1.1 and Nab2.1 and the novel, recently cloned subunit, b3. 2.In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and b3 mRNA at embryonic stages whilst Nab1.1 and Nab2.1 mRNA was absent throughout this period.3. Nab1.1 and Nab2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. b3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum.4. Emulsion-dipped slides showed co-localisation of b3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction.5. Co-expression in Xenopus oocytes revealed that b3 could modify the properties of Nav1.3; b3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation.6. In conclusion, b3 is shown to be the predominant b subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage-gated sodium channels during development.

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