Ion channel regulation and function (15)
Cardiac electrophysiology and arrhythmias (10)
Pain Mechanisms and Treatments (7)
Neuroscience and Neural Engineering (7)
Ion Channels and Receptors (5)
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.
Meents JE, Bressan E, Sontag S, Foerster A, Hautvast P , et al.
Pain •
The chronic pain syndrome inherited erythromelalgia (IEM) is attributed to mutations in the voltage-gated sodium channel (NaV) 1.7. Still, recent studies targeting NaV1.7 in clinical trials have provided conflicting results. Here, we differentiated induced pluripotent stem cells from IEM patients with the NaV1.7/I848T mutation into sensory nociceptors. Action potentials in these IEM nociceptors displayed a decreased firing threshold, an enhanced upstroke, and afterhyperpolarization, all of which may explain the increased pain experienced by patients. Subsequently, we investigated the voltage dependence of the tetrodotoxin-sensitive NaV activation in these human sensory neurons using a specific prepulse voltage protocol. The IEM mutation induced a hyperpolarizing shift of NaV activation, which leads to activation of NaV1.7 at more negative potentials. Our results indicate that NaV1.7 is not active during subthreshold depolarizations, but that its activity defines the action potential threshold and contributes significantly to the action potential upstroke. Thus, our model system with induced pluripotent stem cell-derived sensory neurons provides a new rationale for NaV1.7 function and promises to be valuable as a translational tool to profile and develop more efficacious clinical analgesics.
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.