Ion channel regulation and function (9)
Pain Mechanisms and Treatments (8)
Hereditary Neurological Disorders (4)
Neuroscience and Neuropharmacology Research (3)
Ion Channels and Receptors (3)
Shim J, Tanaka B, Taub DG, Mis MA, Schulman BR , et al.
Brain : a journal of neurology •
Inherited erythromelalgia, small fibre neuropathy and paroxysmal extreme pain disorder are caused by gain-of-function mutations in the voltage-gated sodium channel Nav1.7. It remains unknown how different mutations in the same channel enhancing electrogenesis in sensory neurons results in such distinct disease presentations. Most of the work analysing the impact of these mutations on electrophysiological properties has used overexpression systems in cell lines and rodent sensory neurons, which might differ from the natural context. We have differentiated sensory neurons from induced pluripotent stem cells derived from patient samples that have the Nav1.7 A1632G mutation. This strategy reveals changes in electrophysiological properties, not previously observed in cell lines, that might be important for disease presentation. Furthermore, using CRISPR/Cas9, we corrected this mutation, which reduced the underlying hyperexcitability, providing a path for personalized medicine to treat these disorders, and we introduced the mutation into control induced pluripotent stem cells, which generated hyperexcitability, providing causality. Induced pluripotent stem cell sensory neurons are a robust, scalable and relevant model to study the effects of gain-of-function mutations in ion channels in pain-related disorders.
Mis MA, Tyagi S, Akin EJ, Ghovanloo MR, Zhao P , et al.
Neurobiology of pain (Cambridge, Mass.) •
Gain-of-function mutations which enhance activation of Na1.7, a widely expressed sodium channel in nociceptors, cause human pain disorders including inherited erythromelalgia (IEM). IEM is characterized by attacks of burning pain in distal extremities triggered by warmth, with cooling of affected limbs providing temporary relief. We investigated the behaviour of the IEM-linked L858F mutant Na1.7 channel at physiological normal skin temperature (NST, 33-35 °C) in IB4-negative DRG sensory neurons known to include thermosensors. Using voltage-clamp recordings at NST we found that the Na1.7-L858F mutant channel shows the characteristic hyperpolarizing shift in activation as has been previously found in recordings at room temperature, and that the current density of the L858F channels is significantly larger than that of WT channels. Using a live-cell optical pulse-chase imaging methodology at NST we observed that accelerated forward-trafficking significantly increases membrane insertion of mutant channels in IB4 neurons. Current-clamp recordings at NST show increased firing of IB4 neurons that express the L858F mutant channel, consistent with increased trafficking of the channel at this physiological temperature. Our findings identify enhanced trafficking and membrane insertion of the L858F mutant channels at normal skin temperature in IB4 neurons as an additional mechanism underlying IEM-related neuronal hyperexcitability.
Yuan JH, Estacion M, Mis MA, Tanaka BS, Schulman BR , et al.
Brain communications •
There is a pressing need for understanding of factors that confer resilience to pain. Gain-of-function mutations in sodium channel Nav1.7 produce hyperexcitability of dorsal root ganglion neurons underlying inherited erythromelalgia, a human genetic model of neuropathic pain. While most individuals with erythromelalgia experience excruciating pain, occasional outliers report more moderate pain. These differences in pain profiles in blood-related erythromelalgia subjects carrying the same pain-causative Nav1.7 mutation and markedly different pain experience provide a unique opportunity to investigate potential genetic factors that contribute to inter-individual variability in pain. We studied a patient with inherited erythromelalgia and a Nav1.7 mutation (c.4345T>G, p. F1449V) with severe pain as is characteristic of most inherited erythromelalgia patients, and her mother who carries the same Nav1.7 mutation with a milder pain phenotype. Detailed six-week daily pain diaries of pain episodes confirmed their distinct pain profiles. Electrophysiological studies on subject-specific induced pluripotent stem cell-derived sensory neurons from each of these patients showed that the excitability of these cells paralleled their pain phenotype. Whole-exome sequencing identified a missense variant (c.2263C>T, p. D755N) in (Kv7.3) in the pain resilient mother. Voltage-clamp recordings showed that co-expression of Kv7.2-wild type (WT)/Kv7.3-D755N channels produced larger M-currents than that of Kv7.2-WT/Kv7.3-WT. The difference in excitability of the patient-specific induced pluripotent stem cell-derived sensory neurons was mimicked by modulating M-current levels using the dynamic clamp and a model of the mutant Kv7.2-WT/Kv7.3-D755N channels. These results show that a 'pain-in-a-dish' model can be used to explicate genetic contributors to pain, and confirm that variants can confer pain resilience via an effect on peripheral sensory neurons.
Mis MA, Yang Y, Tanaka BS, Gomis-Perez C, Liu S , et al.
The Journal of neuroscience : the official journal of the Society for Neuroscience •
Pain is a complex process that involves both detection in the peripheral nervous system and perception in the CNS. Individual-to-individual differences in pain are well documented, but not well understood. Here we capitalized on inherited erythromelalgia (IEM), a well characterized human genetic model of chronic pain, and studied a unique family containing related IEM subjects with the same disease-causing Na1.7 mutation, which is known to make dorsal root ganglion (DRG) neurons hyperexcitable, but different pain profiles (affected son with severe pain, affected mother with moderate pain, and an unaffected father). We show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell (iPSC)-derived sensory neurons ; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing (WES) and dynamic clamp, we show that it is possible to pinpoint a specific variant of another gene, in this particular kindred, that modulates the excitability of iPSC-derived sensory neurons in this family. While different gene variants may modulate DRG neuron excitability and thereby contribute to interindividual differences in pain in other families, this study shows that subject-specific iPSCs can be used to model interindividual differences in pain. We further provide proof-of-principle that iPSCs, WES, and dynamic clamp can be used to investigate peripheral mechanisms and pinpoint specific gene variants that modulate pain signaling and contribute to interindividual differences in pain. Individual-to-individual differences in pain are well documented, but not well understood. In this study, we show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell-derived sensory neurons ; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing and dynamic clamp, we show that it is possible to pinpoint a specific gene variant that modulates pain signaling and contributes to interindividual differences in pain.
Yang Y, Huang J, Mis MA, Estacion M, Macala L , et al.
The Journal of neuroscience : the official journal of the Society for Neuroscience •
Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM. Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients.
Cao L, McDonnell A, Nitzsche A, Alexandrou A, Saintot PP , et al.
Science translational medicine •
In common with other chronic pain conditions, there is an unmet clinical need in the treatment of inherited erythromelalgia (IEM). TheSCN9Agene encoding the sodium channel Nav1.7 expressed in the peripheral nervous system plays a critical role in IEM. A gain-of-function mutation in this sodium channel leads to aberrant sensory neuronal activity and extreme pain, particularly in response to heat. Five patients with IEM were treated with a new potent and selective compound that blocked the Nav1.7 sodium channel resulting in a decrease in heat-induced pain in most of the patients. We derived induced pluripotent stem cell (iPSC) lines from four of five subjects and produced sensory neurons that emulated the clinical phenotype of hyperexcitability and aberrant responses to heat stimuli. When we compared the severity of the clinical phenotype with the hyperexcitability of the iPSC-derived sensory neurons, we saw a trend toward 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, our approach may have broader utility for a wide range of pain and sensory conditions.