Changes In Nav1 Research Articles

Depletion of membrane cholesterol modifies structure, dynamic and activation of Nav1.7

Cholesterol is a major component of plasma membranes and plays a significant role in actively regulating the functioning of several membrane proteins in humans. In this study, we focus on the role of cholesterol depletion on the voltage-gated sodium channel Nav1.7, which is primarily expressed in the peripheral sensory neurons and linked to various chronic inherited pain syndromes. Coarse-grained molecular dynamics simulations revealed key dynamic changes of Nav1.7 upon membrane cholesterol depletion: A loss of rigidity in the structural motifs linked to activation and fast-inactivation is observed, suggesting an easier transition of the channel between different gating states. In-vitro whole-cell patch clamp experiments on HEK293t cells expressing Nav1.7 validated these predictions at the functional level: Hyperpolarizing shifts in the voltage-dependence of activation and fast-inactivation were observed along with an acceleration of the time to peak and onset kinetics of fast inactivation. These results underline the critical role of membrane composition, and of cholesterol in particular, in influencing Nav1.7 gating characteristics. Furthermore, our results also point to cholesterol-driven changes of the geometry of drug-binding regions, hinting to a key role of the membrane environment in the regulation of drug effects.

Computational modeling to study the impact of changes in Nav1.8 sodium channel on neuropathic pain.

Nav1.8 expression is restricted to sensory neurons; it was hypothesized that aberrant expression and function of this channel at the site of injury contributed to pathological pain. However, the specific contributions of Nav1.8 to neuropathic pain are not as clear as its role in inflammatory pain. The aim of this study is to understand how Nav1.8 present in peripheral sensory neurons regulate neuronal excitability and induce various electrophysiological features on neuropathic pain. To study the effect of changes in sodium channel Nav1.8 kinetics, Hodgkin-Huxley type conductance-based models of spiking neurons were constructed using the NEURON v8.2 simulation software. We constructed a single-compartment model of neuronal soma that contained Nav1.8 channels with the ionic mechanisms adapted from some existing small DRG neuron models. We then validated and compared the model with our experimental data from in vivo recordings on soma of small dorsal root ganglion (DRG) sensory neurons in animal models of neuropathic pain (NEP). We show that Nav1.8 is an important parameter for the generation and maintenance of abnormal neuronal electrogenesis and hyperexcitability. The typical increased excitability seen is dominated by a left shift in the steady state of activation of this channel and is further modulated by this channel's maximum conductance and steady state of inactivation. Therefore, modified action potential shape, decreased threshold, and increased repetitive firing of sensory neurons in our neuropathic animal models may be orchestrated by these modulations on Nav1.8. Computational modeling is a novel strategy to understand the generation of chronic pain. In this study, we highlight that changes to the channel functions of Nav1.8 within the small DRG neuron may contribute to neuropathic pain.

Periodic injections of Relaxin 2, its pharmacokinetics and remodeling of rat hearts

Relaxin-2 (RLX), a critical hormone in pregnancy, has been investigated as a therapy for heart failure. In most studies, the peptide was delivered continuously, subcutaneously for 2 weeks in animals or intravenously for 2-days in human subjects, for stable circulating [RLX]. However, pulsatile hormone levels may better uncover the normal physiology.This premise was tested by subcutaneously injecting Sprague Dawley rats (250 g, N = 2 males, 2 females/group) with human RLX (0, 30, 100, or 500 µg/kg), every 12 h for 1 day, then measuring changes in Nav1.5, connexin43, and β-catenin, 24 h later. Pulsatile RLX was measured by taking serial blood draws, post-injection.After an injection, RLX reached a peak in ∼ 60 min, fell to 50 % in 5–6 h; injections of 0, 30, 100 or 500 µg/kg yielded peak levels of 0, 11.26 ± 3.52, 58.33 ± 16.10, and 209.42 ± 29.04 ng/ml and residual levels after 24-hrs of 0, 4.9, 45.1 and 156 pg/ml, respectively. The 30 µg/kg injections had no effect and 100 µg/kg injections increased Nav1.5 (25 %), Cx43 (30 %) and β-catenin (90 %). The 500 µg/kg injections also increased Nav1.5 and Cx43 but were less effective at upregulating β-catenin (up by 25 % vs. 90 %).Periodic injections of 100 µg/kg were highly effective at increasing the expression of Nav1.5 and Cx43 which are key determinants of conduction velocity in the heart and the suppression of arrhythmias. Periodic RLX is effective at eliciting changes in cardiac protein expression and may be a better strategy for its longer-term delivery in the clinical setting.

Targeted transcriptional upregulation of SENP1 by CRISPR activation enhances deSUMOylation pathways to elicit antinociception in the spinal nerve ligation model of neuropathic pain.

The voltage-gated sodium channel Na V 1.7 is an essential component of human pain signaling. Changes in Na V 1.7 trafficking are considered critical in the development of neuropathic pain. SUMOylation of collapsin response mediator protein 2 (CRMP2) regulates the membrane trafficking and function of Na V 1.7. Enhanced CRMP2 SUMOylation in neuropathic pain correlates with increased Na V 1.7 activity. Pharmacological and genetic interventions that interfere with CRMP2 SUMOylation in rodents with neuropathic pain have been shown to reverse mechanical allodynia. Sentrin or SUMO-specific proteases (SENPs) are vital for balancing SUMOylation and deSUMOylation of substrates. Overexpression of SENP1 and/or SENP2 in CRMP2-expressing cells results in increased deSUMOylation and decreased membrane expression and currents of Na V 1.7. Although SENP1 is present in the spinal cord and dorsal root ganglia, its role in regulating Na V 1.7 function and pain is not known. We hypothesized that favoring SENP1 expression can enhance CRMP2 deSUMOylation to modulate Na V 1.7 channels. In this study, we used a clustered regularly interspaced short palindromic repeats activation (CRISPRa) SENP1 lentivirus to overexpress SENP1 in dorsal root ganglia neurons. We found that SENP1 lentivirus reduced CRMP2 SUMOylation, Na V 1.7-CRMP2 interaction, and Na V 1.7 membrane expression. SENP1 overexpression decreased Na V 1.7 currents through clathrin-mediated endocytosis, directly linked to CRMP2 deSUMOylation. Moreover, enhancing SENP1 expression did not affect the activity of TRPV1 channels or voltage-gated calcium and potassium channels. Intrathecal injection of CRISPRa SENP1 lentivirus reversed mechanical allodynia in male and female rats with spinal nerve injury. These results provide evidence that the pain-regulating effects of SENP1 overexpression involve, in part, the modulation of Na V 1.7 channels through the indirect mechanism of CRMP2 deSUMOylation.

Biophysical properties of Nav1.5 channel in ventricular-like and atrial-like chamber cardiomyocytes derived from human induced pluripotent stem cells.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) is a promising way modeling cardiac diseases and investigating their associated mechanisms. Nevertheless, it is still challenging to differentiate hiPSC-CMs into specific cardiac types to investigate differences in ion channel properties such as for the NaV1.5 channel. In this context, we investigated biophysical properties of NaV1.5 in ventricular-like and atrial-like hiPSC-CMs. Atrial-like cardiomyocytes were obtained from the differentiation of hiPSCs treated with retinoic acid (RA). The quality of atrial specification was assessed by immunocytofluorescence, qPCR and western blot. Calcium and sodium currents were recorded using the Patch-clamp technique and their electrophysiological properties were investigated using optical mapping technique. hiPSC-CMs differentiation with RA induced a higher expression of mlc2a protein, a marker of atrial cardiomyocytes, and a lower expression of mlc2v and Cx43 proteins, markers of ventricular cardiomyocytes. The amplitude, the duration, and the steady-state phase of action potentials (APs) were decreased in RA-differentiated cardiomyocytes, corresponding to the atrial AP shape. Interestingly, no difference in SCN5A gene expression was detected whereas changes in NaV1.5 currents were observed. Indeed, lower current densities and a shift of the activation and inactivation to more depolarized membrane potential were measured in RA-differentiated cardiomyocytes. Furthermore, the kinetics of the fast inactivation were slower in atrial-like cardiomyocytes. These results showed that RA treatment allowed to obtain atrial-like cardiomyocytes and permitted to investigate differences in NaV1.5 channel properties between ventricular and atrial compartments. This approach with RA could represent an interesting way to investigate specific-type channelopathies.

Inflammation provokes gating changes in the neuronal voltage-gated sodium channel, Nav1.6.

We recently showed that inflammation causes arrhythmogenic properties in cardiac voltage gated sodium channels (Nav). Here, we hypothesize that inflammation, via Nav, is a common trigger for other pathologies including neurological disorders. Based on our previous work, we further postulated that cannabidiol (CBD), the primary non-psychoactive constituent of Cannabis sativa, may protect against inflammation-induced neuropathy through its direct effects on neurological Nav1.6. To test these ideas, we used whole-cell patch clamp of human embryonic kidney (HEK 293) cells transiently transfected with SCN8A cDNA encoding neurological Nav1.6 α-subunit and incubated the cells under control conditions or in a cocktail of inflammatory mediators for 24 hours. Incubation in inflammatory mediators right-shifted the voltage dependence of both activation and steady-state fast inactivation, and increased late sodium current. Perfusion of CBD (IC50; 5 µM) during recording rescued all the gating changes in Nav1.6 provoked by incubation with inflammatory mediators. These findings suggest that inflammation, by affecting Nav, is a potential pathogenic trigger for neurological pathologies. Moreover, CBD, through its direct effects on Nav, could potentially mitigate the dysfunction induced by inflammation.

Clinical comparison and functional study of the L703P: a recurrent mutation in human SCN4A that causes sodium channel myotonia

The non-dystrophic myotonias are inherited skeletal muscle disorders characterized by skeletal muscle stiffness after voluntary contraction, without muscle atrophy. Based on their clinical features, non-dystrophic myotonias are classified into myotonia congenita, paramyotonia congenita, and sodium channel myotonia. Using whole-exome next-generation sequencing, we identified a L703P mutation (c.2108T>C, p.L703P) in SCN4A in a Chinese family diagnosed with non-dystrophic myotonias. The clinical findings of patients in this family included muscle stiffness and hypertrophy. The biophysical properties of wildtype and mutant channels were investigated using whole-cell patch clamp. L703P causes both gain-of-function and loss-of-function changes in Nav1.4 properties, including decreased current density, impaired recovery, enhanced activation and slow inactivation. Our study demonstrates that L703P is a pathogenic variant for myotonia, and provides additional electrophysiological information for understanding the pathogenic mechanism of SCN4A-associated channelopathies.

Molecular Determinants of Mechanical Itch Sensitization in Chronic Itch.

Chronic itch is associated with sensitization of the somatosensory nervous system. Recent studies have identified the neural circuits transmitting acute itch; however, the mechanisms by which itch transforms into a pathological state remain largely unknown. We have previously shown that Aβ low-threshold mechanoreceptors, together with spinal urocortin 3-positive (Ucn3+) excitatory interneurons and neuropeptide Y-positive (NPY+) inhibitory interneurons, form a microcircuit that transmits and gates acute mechanical itch. Here, using whole-cell patch-clamp recordings, we observed increased excitability in spinal Ucn3+ neurons under chronic itch conditions. In contrast to Ucn3+ neurons, the excitability of spinal NPY+ neurons was largely reduced under chronic itch conditions. To explore the molecular mechanisms underlying sensitization of this microcircuit, we examined the mRNA expression levels of voltage-gated ion channels in recorded spinal Ucn3+ and NPY+ neurons by single-cell quantitative real-time PCR (qRT-PCR). We found that the expression levels of Nav1.6 and Cav2.3 channels were increased in spinal Ucn3+ neurons in chronic itch mice, while the expression level of SK3 channels was decreased. By contrast, the expression levels of Nav1.6 and BK channels were decreased in spinal NPY+ neurons in chronic itch mice. To determine the contribution of different ion channels in chronic itch sensitization, we then used a Markov Chain Monte Carlo method to parameterize a large number of biophysically distinct multicompartment models of Ucn3+ and NPY+ neurons. These models included explicit representations of the ion channels that we found to be up- or down-regulated under chronic itch conditions. Our models demonstrated that changes in Nav1.6 conductance are predominantly responsible for the changes in excitability of both Ucn3+ and NPY+ neurons during chronic itch pathogenesis. Furthermore, when simulating microcircuits of our Ucn3+ and NPY+ models, we found that reduced Nav1.6 conductance in NPY+ models played a major role in opening the itch gate under chronic itch conditions. However, changing SK, BK, or R-type calcium channel conductance had negligible effects on the sensitization of this circuit. Therefore, our results suggest that Nav1.6 channels may play an essential role in mechanical itch sensitization. The findings presented here may open a new avenue for developing pharmaceutical strategies to treat chronic itch.

Lidocaine reduces pain behaviors by inhibiting the expression of Nav1.7 and Nav1.8 and diminishing sympathetic sprouting in SNI rats

Chronic neuropathic pain is a significant clinical challenge, and the mechanisms of neuropathic pain remain elusive. Previous studies have shown that spontaneous potential, which is triggered by Nav1.7 and Nav1.8 in the dorsal root ganglion (DRG), is crucial for the development of inflammatory and neuropathic pain. Functional coupling between the sympathetic nervous system and somatosensory nerves after a nerve injury has also been noted as an important factor in neuropathic pain. However, the relationship of sympathetic sprouting with Nav1.7 and Nav1.8 remains unclear. Therefore, we dynamically examined the mechanical withdrawal threshold (MWT), changes in Nav1.7 and Nav1.8, and sympathetic sprouting after lidocaine treatment in the spared nerve injury (SNI) model of rats. After lidocaine treatment, the MWT obviously increased, showing that hypersensitivity was significantly relieved and the abnormal expression of Nav1.7 and Nav1.8 caused by SNI was also significantly reduced. In addition, lidocaine distinctly inhibited sympathetic nerve sprouting and basket formation around the Nav1.7 and Nav1.8 neurons in the DRG. These results indicate that lidocaine may alleviate neuropathic pain by inhibiting the expression of Nav1.7 and Nav1.8, and diminishing sympathetic sprouting in DRG.

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels.

Background and purpose: Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug. Experimental Approach: Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Nav1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Nav1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Nav1.5 inactivation parameters and inhibition of different VGSC isoforms. Key results: Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Nav1.5), neuronal (Nav1.2), and skeletal muscle (Nav1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site. Conclusion and Implications: Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.

Distinctive Properties and Powerful Neuromodulation of Nav1.6 Sodium Channels Regulates Neuronal Excitability.

Voltage-gated sodium channels (Navs) are critical determinants of cellular excitability. These ion channels exist as large heteromultimeric structures and their activity is tightly controlled. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Changes in Nav1.6 expression and function profoundly impact the input-output properties of neurons in normal and pathological conditions. While mutations in Nav1.6 may cause channel dysfunction, aberrant changes may also be the result of complex modes of regulation, including various protein-protein interactions and post-translational modifications, which can alter membrane excitability and neuronal firing properties. Despite decades of research, the complexities of Nav1.6 modulation in health and disease are still being determined. While some modulatory mechanisms have similar effects on other Nav isoforms, others are isoform-specific. Additionally, considerable progress has been made toward understanding how individual protein interactions and/or modifications affect Nav1.6 function. However, there is still more to be learned about how these different modes of modulation interact. Here, we examine the role of Nav1.6 in neuronal function and provide a thorough review of this channel’s complex regulatory mechanisms and how they may contribute to neuromodulation.

Differential Functional Changes of Nav1.2 Channel Causing SCN2A-Related Epilepsy and Status Epilepticus During Slow Sleep.

Background: Nav1.2 encoded by the SCN2A gene is a brain-expressed voltage-gated sodium channel known to be associated with neurodevelopment disorders ranging from benign familial neonatal infantile seizures (BFIS) to developmental and epileptic encephalopathy (DEE) and autism spectrum disorder. Interestingly, status epilepticus during slow sleep (ESES), which aggravates cognitive impairment, has been found in SCN2A-related epilepsy. However, the functional features and the relationship between SCN2A and ESES have not been researched.Method: We herein investigated the functional consequences of an unpublished de novo V911A and the other two published variants in patients with SCN2A-related disorder and ESES by whole-cell patch-clamp studies in transfected HEK293T cells.Results: The unpublished V911A and published K1933M variants detected in patients with DEE exhibited a profound gain-of-functional (GOF) change. Another published BFIS variant S863F significantly reduced current density as a loss-of-functional (LOF) change. The refractory epilepsy in the patient with V911A was controlled by using the precise treatment of oxcarbazepine (OXC) since the age of 3 months. ESES was found at 18 months during the seizure-free period. We finally chose an aggressive treatment for eliminating ESES by using methylprednisolone combined with levetiracetam and nitrazepam instead of the precise treatment of OXC.Conclusion: Both GOF and LOF variants in the SCN2A gene can lead to ESES among the phenotypes of DEE and BFIS. We should monitor the electroencephalogram regularly in the patients with SCN2A-related epilepsy even during their seizure-free period.

Protein Kinases Mediate Anti-Inflammatory Effects of Cannabidiol and Estradiol Against High Glucose in Cardiac Sodium Channels.

Background: Cardiovascular anomalies are predisposing factors for diabetes-induced morbidity and mortality. Recently, we showed that high glucose induces changes in the biophysical properties of the cardiac voltage-gated sodium channel (Nav1.5) that could be strongly correlated to diabetes-induced arrhythmia. However, the mechanisms underlying hyperglycemia-induced inflammation, and how inflammation provokes cardiac arrhythmia, are not well understood. We hypothesized that inflammation could mediate the high glucose-induced biophyscial changes on Nav1.5 through protein phosphorylation by protein kinases A and C. We also hypothesized that this signaling pathway is, at least partly, involved in the cardiprotective effects of cannabidiol (CBD) and 17β-estradiol (E2). Methods and Results: To test these ideas, we used Chinese hamster ovarian (CHO) cells transiently co-transfected with cDNA encoding human Nav1.5 α-subunit under control, a cocktail of inflammatory mediators or 100 mM glucose conditions (for 24 h). We used electrophysiological experiments and action potential modeling. Inflammatory mediators, similar to 100 mM glucose, right shifted the voltage dependence of conductance and steady-state fast inactivation and increased persistent current leading to computational prolongation of action potential (hyperexcitability) which could result in long QT3 arrhythmia. We also used human iCell cardiomyocytes derived from inducible pluripotent stem cells (iPSC-CMs) as a physiologically relevant system, and they replicated the effects produced by inflammatory mediators observed in CHO cells. In addition, activators of PK-A or PK-C replicated the inflammation-induced gating changes of Nav1.5. Inhibitors of PK-A or PK-C, CBD or E2 mitigated all the potentially deleterious effects provoked by high glucose/inflammation. Conclusion: These findings suggest that PK-A and PK-C may mediate the anti-inflammatory effects of CBD and E2 against high glucose-induced arrhythmia. CBD, via Nav1.5, may be a cardioprotective therapeutic approach in diabetic postmenopausal population.

Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7

Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7

The nociceptive innervation of the normal and osteoarthritic mouse knee

To document the nociceptive innervation of the normal and osteoarthritic murine knee. Knees were collected from naïve male C57BL/6 NaV1.8-tdTomato reporter mice aged 10, 26, and 52 weeks (n=5/group). Destabilization of the medial meniscus (DMM) or sham surgeries (n=5/group) were performed in the right knee of 10-week old male NaV1.8-tdTomato mice, and knees were harvested 16 weeks later. Twenty 20-μm frozen sections from a 400-μm mid-joint region were collected for confocal microscopy. Integrated density of the tdTomato signal was calculated using Image J by two independent observers blinded to the groups. Consecutive sections were stained with hematoxylin & eosin. C57BL/6-Pirt-GCaMP3 mice (n=5/group) and protein gene product 9.5 (PGP9.5) immunostaining of C57BL/6 wild type (WT) mice (n=5/group) were used to confirm innervation patterns. In naive 10-week old mice, nociceptive innervation was most dense in bone marrow cavities, lateral synovium and at the insertions of the cruciate ligaments. By age 26 weeks, unoperated knees showed a marked decline in nociceptors in the lateral synovium and cruciate ligament insertions. No further decline was observed by age 1 year. Sixteen weeks after DMM, the medial compartment of OA knees exhibited striking changes in NaV1.8+innervation, including increased innervation of the medial synovium and meniscus, and nociceptors in subchondral bone channels. All results were confirmed through quantification, also in Pirt-GCaMP3 and PGP9.5-immunostained WT mice. Nociceptive innervation of the mouse knee markedly declines by age 26 weeks, before onset of spontaneous OA. Late-stage surgically induced OA is associated with striking plasticity of joint afferents in the medial compartment of the knee.

UBC9 regulates cardiac sodium channel Nav1.5 ubiquitination, degradation and sodium current density

Voltage-gated sodium channel Nav1.5 is critical for generation and conduction of cardiac action potentials. Mutations and expression level changes of Nav1.5 are associated with cardiac arrhythmias and sudden death. The ubiquitin (Ub) conjugation machinery utilizes three enzyme activities, E1, E2, and E3, to regulate protein degradation. Previous studies from us and others showed that Nedd4–2 acts as an E3 ubiquitin-protein ligase involved in ubiquitination and degradation of Nav1.5, however, more key regulators remain to be identified. In this study, we show that UBC9, a SUMO-conjugating enzyme, regulates ubiquitination and degradation of Nav1.5. Overexpression of UBC9 significantly decreased Nav1.5 expression and reduced sodium current densities, whereas knockdown of UBC9 expression significantly enhanced Nav1.5 expression and increased sodium current densities, in both HEK293 cells and primary neonatal cardiomyocytes. Overexpression of UBC9 increased ubiquitination of Nav1.5, and proteasome inhibitor MG132 blocked the effect of UBC9 overexpression on Nav1.5 degradation. Co-immunoprecipitation showed that UBC9 interacts with Nedd4–2. UBC9 with mutation C93S, which suppresses SUMO-conjugating activity of UBC9, was as active as wild type UBC9 in regulating Nav1.5 levels, suggesting that UBC9 regulates Nav1.5 expression levels in a SUMOylation-independent manner. Our findings thus identify a key structural element of the ubiquitin-conjugation machinery for Nav1.5 and provide important insights into the regulatory mechanism for ubiquitination and turnover of Nav1.5.

Nav1.7 is phosphorylated by Fyn tyrosine kinase which modulates channel expression and gating in a cell type-dependent manner.

Voltage-gated sodium channel Nav1.7 is a key molecule in nociception, and its dysfunction has been associated with various pain disorders. Here, we investigated the regulation of Nav1.7 biophysical properties by Fyn, an Src family tyrosine kinase. Nav1.7 was coexpressed with either constitutively active (FynCA) or dominant negative (FynDN) variants of Fyn kinase. FynCA elevated protein expression and tyrosine phosphorylation of Nav1.7 channels. Site-directed mutagenesis analysis identified two tyrosine residues (Y1470 and Y1471) located within the Nav1.7 DIII-DIV linker (L3) as phosphorylation sites of Fyn. Whole-cell recordings revealed that FynCA evoked larger changes in Nav1.7 biophysical properties when expressed in ND7/23 cells than in Human Embryonic Kidney (HEK) 293 cells, suggesting a cell type-specific modulation of Nav1.7 by Fyn kinase. In HEK 293 cells, substitution of both tyrosine residues with phenylalanine dramatically reduced current amplitude of mutant channels, which was partially rescued by expressing mutant channels in ND7/23 cells. Phenylalanine substitution showed little effect on FynCA-induced changes in Nav1.7 activation and inactivation, suggesting additional modifications in the channel or modulation by interaction with extrinsic factor(s). Our study demonstrates that Nav1.7 is a substrate for Fyn kinase, and the effect of the channel phosphorylation depends on the cell background. Fyn-mediated modulation of Nav1.7 may regulate DRG neuron excitability and contribute to pain perception. Whether this interaction could serve as a target for developing new pain therapeutics requires future study.

MiR-30b Attenuates Neuropathic Pain by Regulating Voltage-Gated Sodium Channel Nav1.3 in Rats.

Nav1.3 is a tetrodotoxin-sensitive isoform among voltage-gated sodium channels that are closely associated with neuropathic pain. It can be up-regulated following nerve injury, but its biological function remains uncertain. MicroRNAs (miRNAs) are endogenous non-coding RNAs that can regulate post-transcriptional gene expression by binding with their target mRNAs. Using Target Scan software, we discovered that SCN3A is the major target of miR-30b, and we then determined whether miR-30b regulated the expression of Nav1.3 by transfecting miR-30b agomir through the stimulation of TNF-α or by transfecting miR-30b antagomir in primary dorsal root ganglion (DRG) neurons. The spinal nerve ligation (SNL) model was used to determine the contribution of miR-30b to neuropathic pain, to evaluate changes in Nav1.3 mRNA and protein expression, and to understand the sensitivity of rats to mechanical and thermal stimuli. Our results showed that miR-30b agomir transfection down-regulated Nav1.3 mRNA stimulated with TNF-α in primary DRG neurons. Moreover, miR-30b overexpression significantly attenuated neuropathic pain induced by SNL, with decreases in the expression of Nav1.3 mRNA and protein both in DRG neurons and spinal cord. Activation of Nav1.3 caused by miR-30b antagomir was identified. These data suggest that miR-30b is involved in the development of neuropathic pain, probably by regulating the expression of Nav1.3, and might be a novel therapeutic target for neuropathic pain.Perspective: This study is the first to explore the important role of miR-30b and Nav1.3 in spinal nerve ligation-induced neuropathic pain, and our evidence may provide new insight for improving therapeutic approaches to pain.

Historical Contingency in a Multigene Family Facilitates Adaptive Evolution of Toxin Resistance

Novel adaptations must originate and function within an already established genome [1]. As a result, the ability of a species to adapt to new environmental challenges is predicted to be highly contingent on the evolutionary history of its lineage [2-6]. Despite a growing appreciation of the importance of historical contingency in the adaptive evolution of single proteins [7-11], we know surprisingly little about its role in shaping complex adaptations that require evolutionary change in multiple genes. One such adaptation, extreme resistance to tetrodotoxin (TTX), has arisen in several species of snakes through coevolutionary arms races with toxic amphibian prey, which select for TTX-resistant voltage-gated sodium channels (Nav) [12-16]. Here, we show that the relatively recent origins of extreme toxin resistance, which involve the skeletal muscle channel Nav1.4, were facilitated by ancient evolutionary changes in two other members of the same gene family. A substitution conferring TTX resistance to Nav1.7, a channel found in small peripheral neurons, arose in lizards ∼170 million years ago (mya) and was present in the common ancestor of all snakes. A second channel found in larger myelinated neurons, Nav1.6, subsequently evolved resistance in four different snake lineages beginning ∼38 mya. Extreme TTX resistance has evolved at least five times within the past 12 million years via changes in Nav1.4, but only within lineages that previously evolved resistant Nav1.6 and Nav1.7. Our results show that adaptive protein evolution may be contingent upon enabling substitutions elsewhere in the genome, in this case, in paralogs of the same gene family.

Abstract 17984: No Longer Silent: A Synonymous Coding SNP Modifying a Novel MicroRNA-24:SCN5A Interaction Associates With Non-Arrhythmic Death in Heart Failure

Mutations that disrupt the coding sequence of Nav1.5, the cardiac Na+ channel encoded by SCN5A, cause inherited arrhythmias and cardiomyopathy. Changes in Nav1.5 splice isoforms, localization and function have also been associated with prognosis in acquired heart disease. However, the role of genetic variants in SCN5A gene regulatory sequences (e.g. those modulating expression) remains poorly understood. MicroRNAs (miRs) have emerged as vital gene regulators in heart, having roles in cardiogenesis, contractile function, and disease. These small non-coding RNAs are loaded into Argonaute proteins to direct post-transcriptional gene suppression by base-pairing with target transcripts, and genetic variants altering miR binding sequences may influence disease. Here, we used high-throughput biochemical means (HITS-CLIP) to generate the first transcriptome-wide map of miR binding events in human heart (4000 sites across >2200 genes). Our query of the data uncovered >500 common SNPs residing in or nearby miR target sites. Among these, we identified a synonymous SNP (rs1805126, T/C) which alters a conserved miR-24 binding site in the terminal coding exon of SCN5A. We hypothesized that this SNP affects Nav1.5 expression and contributes to clinical outcomes in heart failure. In vitro experiments showed that miR-24 suppresses Na+ current on patch clamp analysis (~5-fold; p<0.001) and potently inhibits Nav1.5 expression, with rs1805126 allele C showing a greater decrease relative to T (66% vs. 52%, p<0.01). To assess the clinical relevance of this, we genotyped >1800 subjects with EFs≤30% and implantable cardioverter defibrillators (ICDs) from GRADE, a prospective multicenter study of genetic modulators of heart failure outcomes. We found that CC homozygotes had worse survival rates than T-allele carriers (HR=1.5, p=0.001), but no change in appropriate ICD shocks, a surrogate for sudden death. Together, these data reveal a novel miR-24:SCN5A:SNP regulatory axis that points to a surprising link between cardiac Na+ channel levels and non-arrhythmic death in heart failure. Overall, this work represents a key advance in exploring the interface of cardiac miRs with genetics and disease, perhaps signifying a broader functional relevance for “silent” SNPs.