Eberhardt E, Namer B, Neureiter A, Körner J, Jørum E , et al.
Pain •
Spontaneous activity of peripheral sensory nerve fibers is one of the main drivers of neuropathic pain. It can be assessed in microneurography recordings of patients' C fibers and in patch-clamp recordings of dissociated dorsal root ganglia from humans and rodents. In microneurography of human C fibers, a distinct subgroup of neurons, the so-called mechano-insensitive (CMi) or sleeping nociceptors, shows spontaneous activity during neuropathic pain. It was shown before that sensory neurons from patient-derived induced pluripotent stem cells (iSNs) can be used to model this increased spontaneous activity in vitro, suggesting that a disease relevant cell type is generated with this approach. The origin of the spontaneous activity in human C fibers is not fully understood. Derived sensory neurons offer the unique possibility to study patient-derived, single-cell function, allowing for identification of potential mechanisms underlying spontaneous C-fiber activity. Here, we identify 4 distinct functional subtypes of iSNs from healthy donors and a patient suffering from the neuropathic pain syndrome inherited erythromelalgia using patch-clamp recordings. Similar to microneurography recordings from the same patient, spontaneous activity is restricted to 1 functional subgroup that shows tonic firing behavior and seems to be especially prone to develop neuronal hyperexcitability. We demonstrate that spontaneous activity correlates with a reduced voltage threshold of action potential generation and increased spontaneous depolarizing fluctuations of the membrane potential. Our findings reveal that only the tonically firing functional subclass of iSNs shows spontaneous activity and suggest that these neurons may be related to the pathologically active CMi fibers identified during microneurography recordings in patients with pain.
Kesdoğan AB, Neureiter A, Gaebler AJ, Kalia AK, Körner J , et al.
Neuropharmacology •
Botulinum neurotoxin type A BoNT/A is used off-label as a third line therapy for neuropathic pain. However, the mechanism of action remains unclear. In recent years, the role of voltage-gated sodium channels (Nav) in neuropathic pain became evident and it was suggested that block of sodium channels by BoNT/A would contribute to its analgesic effect. We assessed sodium channel function in the presence of BoNT/A in heterologously expressed Nav1.7, Nav1.3, and the neuronal cell line ND7/23 by high throughput automated and manual patch-clamp. We used both the full protein and the isolated catalytic light chain LC/A for acute or long-term extracellular or intracellular exposure. To assess the toxin's effect in a human cellular system, we differentiated induced pluripotent stem cells (iPSC) into sensory neurons from a healthy control and a patient suffering from a hereditary neuropathic pain syndrome (inherited erythromelalgia) carrying the Nav1.7/p.Q875E-mutation and carried out multielectrode-array measurements. Both BoNT/A and the isolated catalytic light chain LC/A showed limited effects in heterologous expression systems and the neuronal cell line ND7/23. Spontaneous activity in iPSC derived sensory neurons remained unaltered upon BoNT/A exposure both in neurons from the healthy control and the mutation carrying patient. BoNT/A may not specifically be beneficial in pain syndromes linked to sodium channel variants. The favorable effects of BoNT/A in neuropathic pain are likely based on mechanisms other than sodium channel blockage and new approaches to understand BoNT/A's therapeutic effects are necessary.
Kriegeskorte S, Bott R, Hampl M, Korngreen A, Hausmann R , et al.
The Journal of general physiology •
Voltage-gated sodium channels (Nav) are key players in excitable tissues with the capability to generate and propagate action potentials. Mutations in the genes encoding Navs can lead to severe inherited diseases, and some of these so-called channelopathies show temperature-sensitive phenotypes, for example, paramyotonia congenita, Brugada syndrome, febrile seizure syndromes, and inherited pain syndromes like erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). Nevertheless, most investigations of mutation-induced gating effects have been conducted at room temperature, and thus the role of cooling or warming in channelopathies remains poorly understood. Here, we investigated the temperature sensitivity of four Nav subtypes: Nav1.3, Nav1.5, Nav1.6, and Nav1.7, and two mutations in Nav1.7 causing IEM (Nav1.7/L823R) and PEPD (Nav1.7/I1461T) expressed in cells of the human embryonic kidney cell line using an automated patch clamp system. Our experiments at 15°C, 25°C, and 35°C revealed a shift of the voltage dependence of activation to more hyperpolarized potentials with increasing temperature for all investigated subtypes. Nav1.3 exhibited strongly slowed inactivation kinetics compared with the other subtypes that resulted in enhanced persistent current, especially at 15°C, indicating a possible role in cold-induced hyperexcitability. Impaired fast inactivation of Nav1.7/I1461T was significantly enhanced by a cooling temperature of 15°C. The subtype-specific modulation as well as the intensified mutation-induced gating changes stress the importance to consider temperature as a regulator for channel gating and its impact on cellular excitability as well as disease phenotypes.
Le Cann K, Meents JE, Sudha Bhagavath Eswaran V, Dohrn MF, Bott R , et al.
Channels (Austin, Tex.) •
Mutations in the voltage-gated sodium channel Nav1.7 are linked to human pain. The Nav1.7/N1245S variant was described before in several patients suffering from primary erythromelalgia and/or olfactory hypersensitivity. We have identified this variant in a pain patient and a patient suffering from severe and life-threatening orthostatic hypotension. In addition, we report a female patient suffering from muscle pain and carrying the Nav1.7/E1139K variant. We tested both Nav1.7 variants by whole-cell voltage-clamp recordings in HEK293 cells, revealing a slightly enhanced current density for the N1245S variant when co-expressed with the β1 subunit. This effect was counteracted by an enhanced slow inactivation. Both variants showed similar voltage dependence of activation and steady-state fast inactivation, as well as kinetics of fast inactivation, deactivation, and use-dependency compared to WT Nav1.7. Finally, homology modeling revealed that the N1245S substitution results in different intramolecular interaction partners. Taken together, these experiments do not point to a clear pathogenic effect of either the N1245S or E1139K variant and suggest they may not be solely responsible for the patients' pain symptoms. As discussed previously for other variants, investigations in heterologous expression systems may not sufficiently mimic the pathophysiological situation in pain patients, and single nucleotide variants in other genes or modulatory proteins are necessary for these specific variants to show their effect. Our findings stress that biophysical investigations of ion channel mutations need to be evaluated with care and should preferably be supplemented with studies investigating the mutations in their context, ideally in human sensory neurons.
Kerth CM, Hautvast P, Körner J, Lampert A, Meents JE
The Journal of biological chemistry •
Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain.
Rühlmann AH, Körner J, Hausmann R, Bebrivenski N, Neuhof C , et al.
British journal of pharmacology •
The voltage-gated sodium channel Na 1.7 is essential for adequate perception of painful stimuli. Mutations in the encoding gene, SCN9A, cause various pain syndromes in humans. The hNa 1.7/A1632E channel mutant causes symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD), and its main gating change is a strongly enhanced persistent current. On the basis of recently published 3D structures of voltage-gated sodium channels, we investigated how the inactivation particle binds to the channel, how this mechanism is altered by the hNa 1.7/A1632E mutation, and how dimerization modifies function of the pain-linked mutation. We applied atomistic molecular simulations to demonstrate the effect of the mutation on channel fast inactivation. Native PAGE was used to demonstrate channel dimerization, and electrophysiological measurements in HEK cells and Xenopus laevis oocytes were used to analyze the links between functional channel dimerization and impairment of fast inactivation by the hNa 1.7/A1632E mutation. Enhanced persistent current through hNa 1.7/A1632E channels was caused by impaired binding of the inactivation particle, which inhibits proper functioning of the recently proposed allosteric fast inactivation mechanism. hNa 1.7 channels form dimers and the disease-associated persistent current through hNa 1.7/A1632E channels depends on their functional dimerization status: Expression of the synthetic peptide difopein, a 14-3-3 inhibitor known to functionally uncouple dimers, decreased hNa 1.7/A1632E channel-induced persistent currents. Functional uncoupling of mutant hNa 1.7/A1632E channel dimers restored their defective allosteric fast inactivation mechanism. Our findings support the concept of sodium channel dimerization and reveal its potential relevance for human pain syndromes.
The causes for neuropathic pain are manifold and remain unexplained in the majority of cases. In recent years a growing number of pain syndromes have been attributed to mutations in genes encoding voltage-gated sodium channels. Hence, this group of rare diseases should be considered in the differential diagnostics of neuropathic pain. Evaluation of topic-related literature and discussion of own experiences as well as consideration of current guidelines. Alterations in the electrical excitability of nociceptive neurons by pathogenic mutations in sodium channels lead to disease patterns, such as small fiber neuropathy and various pain syndromes. This article summarizes the knowledge on these genetic diseases and discusses the differential diagnosis of neuropathic pain. Current treatment concepts are presented and the predominantly experimental approaches to targeted modulation of sodium channels are discussed. The treatment of patients with chronic neuropathic pain requires interdisciplinary cooperation and is often difficult due to an unsatisfactory treatment response. Increasing knowledge on rare genetically determined channelopathies can contribute to the development of novel pharmaceuticals since ion channels are central players in the processing of pain.
Chronic pain patients are often left with insufficient treatment as the pathophysiology especially of neuropathic pain remains enigmatic. Recently, genetic variations in the genes of the voltage-gated sodium channels (Navs) were linked to inherited neuropathic pain syndromes, opening a research pathway to foster our understanding of the pathophysiology of neuropathic pain. More than 10 years ago, the rare, inherited pain syndrome erythromelalgia was linked to mutations in the subtype Nav1.7, and since then a plethora of mutations and genetic variations in this and other Nav genes were identified. Often the biophysical changes induced by the genetic alteration offer a straightforward explanation for the clinical symptoms, but mutations in some channels, especially Nav1.9, paint a more complex picture. Although efforts were undertaken to significantly advance our knowledge, translation from heterologous or animal model systems to humans remains a challenge. Here we present recent advances in translation using stem cell-derived human sensory neurons and their potential application for identification of better, effective, and more precise treatment for the individual pain patient.
Kist AM, Sagafos D, Rush AM, Neacsu C, Eberhardt E , et al.
PloS one •
Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences.