Exploring conformational states of the bacterial voltage-gated sodium channel NavAb via molecular dynamics simulations

The X-ray structure of the bacterial voltage-gated sodium channel NavAb has been reported in a conformation with a closed conduction pore. Comparison between this structure and the activated-open and resting-closed structures of the voltage-gated Kv1.2 potassium channel suggests that the voltage-sensor domains (VSDs) of the reported structure are not fully activated. Using the aforementioned structures of Kv1.2 as templates, molecular dynamics simulations are used to identify analogous functional conformations of NavAb. Specifically, starting from the NavAb crystal structure, conformations of the membrane-bound channel are sampled along likely pathways for activation of the VSD and opening of the pore domain. Gating charge computations suggest that a structural rearrangement comparable to that occurring between activated-open and resting-closed states is required to explain experimental values of the gating charge, thereby confirming that the reported VSD structure is likely an intermediate along the channel activation pathway. Our observation that the X-ray structure exhibits a low pore domain-opening propensity further supports this notion. The present molecular dynamics study also identifies conformations of NavAb that are seemingly related to the resting-closed and activated-open states. Our findings are consistent with recent structural and functional studies of the orthologous channels NavRh, NaChBac, and NavMs and offer possible structures for the functionally relevant conformations of NavAb.     

Targeting voltage-gated sodium channels for treatment for chronic visceral pain

Voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability, and their abnormal activity is related to several pathological processes, including cardiac arrhythmias, epilepsy, neurodegenerative diseases, spasticity and chronic pain. In particular, chronic visceral pain, the central symptom of functional gastrointestinal disorders such as irritable bowel syndrome, is a serious clinical problem that affects a high percentage of the world population. In spite of intense research efforts and after the dedicated decade of pain control and research, there are not many options to treat chronic pain conditions. However, there is a wealth of evidence emerging to give hope that a more refined approach may be achievable. By using electronic databases, available data on structural and functional properties of VGSCs in chronic pain, particularly functional gastrointestinal hypersensitivity, were reviewed. We summarize the involvement and molecular bases of action of VGSCs in the pathophysiology of several organic and functional gastrointestinal disorders. We also describe the efficacy of VGSC blockers in the treatment of these neurological diseases, and outline future developments that may extend the therapeutic use of compounds that target VGSCs. Overall, clinical and experimental data indicate that isoform-specific blockers of these channels or targeting of their modulators may provide effective and novel approaches for visceral pain therapy.     

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel

Voltage-gated sodium (Na_v) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the “hinged lid”-based fast inactivation, seen in eukaryotic Na_v channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of Na_vAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water–protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Na_v inhibitors.Voltage-gated sodium (Na_v) channel inhibitors can modulate sensory or motor activity without threatening vital bodily functions, enabling a wide range of therapeutic applications. In particular, the ability to control Na_v channel current allows for effective use as local anesthetic (LA) and antiepileptic (AE) drugs, normalizing function in conditions of hyperexcitability, such as epilepsy, cardiac arrhythmias, hyperalgesia, and myotonia (1, 2). Recent breakthroughs in the solution of atomic structures of the bacterial Na_vBac family (3–6) provide an excellent opportunity to describe drug binding at the molecular level.Pharmacological Na_v channel modulation is a complex phenomenon involving voltage- and state-dependent block of current, as well as prolongation of recovery from inactivated states, with interrelated observations that have implicated different active sites, pathways and inactivated channel states (7). Despite historical focus on fast inactivation (8), it has now been discovered that slow inactivation is affected by LA/AE drugs (9) and that mutation of key residues involved in inhibition impairs slow inactivation (10). The Na_vBac family lacks the Domain (D) III–DIV linker responsible for fast inactivation (3), enabling molecular-level investigation into this common modulation process. Moreover, key binding regions are well conserved in bacterial and mammalian channels (3, 11), and it has been shown that bacterial channels interact with common inhibitory drugs (11–14). However, questions remain as to how structurally disparate inhibitor molecules can cause qualitatively similar modulation of different mammalian and bacterial Na_v channels. Here, we explore the binding of LA benzocaine (BZC) and AE phenytoin (PHT) to the bacterial Na_vAb channel (3) (Fig. 1), providing detailed insight into Na_v modulation mechanisms.Open in a separate windowFig. 1.(A) Simulation system showing Na_vAb (two of four subunits as ribbons, with voltage sensor domain (VSD) S1 and S2 in blue and S3 and S4 in green, pore domain (PD) S5 in yellow and S6 in red, P-loop P1, P2, and selectivity filter (SF; orange) in a hydrated lipid bilayer (chains as gray lines; water as red/white sticks; NaCl as yellow/cyan balls) and PHT (dark gray sticks). (B and C) Stick representations of BZC (B) and PHT (C).Functional studies have suggested at least two major LA/AE binding sites: a high-affinity, state- and voltage-dependent site; and a low-affinity, less state-dependent site (15). The high-affinity site has been the focus of considerable investigation (16) and appears to be shared by many drugs (2). Studies of mammalian Na_v have suggested this site is on helix S6 of DIV, involving residue FS6 [F1764 in Nav1.2 (16), F1579 in Nav1.4, F1759 in Nav1.5 (17)], as well as a Tyr (Y1771 in Nav1.2, Y1586 in Nav1.4, Y1766 in Nav1.5). Binding at this site is use-dependent, such that under brief repetitive stimuli that open the channel, the channel enters a stable nonconducting state, with a cumulative reduction of current (18), signifying preferential binding to an inactivated state (10). Binding to FS6 appears to involve cation–π interaction for charged lidocaine (19), but π-stacking for neutral agents such as BZC and PHT. In contrast, little information exists to pinpoint the low-affinity site(s), whose binding may result from a “combination of hydrophobic interactions” occurring when the channel is maintained at hyperpolarized voltages (15). Complete sampling of the drug-channel binding distribution is needed to define these binding modes.Residue FS6 on domain IV of the mammalian channels is a critical feature in drug modulation, but does not exist at that position in the bacterial channels (Fig. 2A; with full comparison in SI Appendix, Fig. S1). The absence of this important residue may contribute to low LA affinities observed for NaChBac (12). However, the recently solved Na_vAb (3) possesses a nonconserved F203, which appears to mimic FS6. Fig. 2B shows a simple overlay of the hNa_v1.2 sequence on the Na_vAb structural template, with FS6 location and orientation similar to F203 (shifted only slightly along the S6 helix), and thus could play a similar role in drug binding. Furthermore, mammalian Na_v channels possess a second Phe in domain I that is equivalent to F203 (SI Appendix, Fig. S1). In contrast, the other bacterial Na_v channels only possess two conserved Phe (F201 and F207 in Na_vAb), with orientations inconsistent with an FS6 site (participating in interactions away from the pore; Fig. 2B). Na_vAb may therefore offer unique similarity to mammalian Na_v for the investigation of LA/AE binding (we note, however, that binding may still occur in the absence of an FS6-like residue, as seen for brominated drug-like compounds to more distant F214 on S6 in Na_vMs; ref. 11).Open in a separate windowFig. 2.(A) Sequence alignment of segment S6 of Na_vAb to other Na_v channels (showing only FS6-containing domain IV; see SI Appendix, Fig. S1, for full comparison). F201, F203, F207, and N211 in Na_vAb and F1764 in Na_v1.2 are indicated. Amino acids were colored with Jalview (46, 47) using the Zappo scheme. (B) Aligned structures (showing three subunits) of Na_vAb and model Na_v1.2 (based on Na_vAb) comparing Na_vAb F201, F203, and F207 to Na_v1.2 F1764. Conserved N211 (Na_vAb numbering) is also indicated.In the 1970s, Hille proposed two separate pathways for inhibitor binding, with charged drugs such as titratable amines and quaternary ammonium derivatives requiring an open channel, and neutral drugs such as BZC and PHT entering via a lipophilic pathway somewhere in the membrane (allowing for binding and dissociation, even when the pore is nonconducting) (20). Our previous simulations revealed dynamic interplay between the protein and lipids, with fenestrations allowing lipid tails to enter the pore (21), representing a potential drug pathway (20, 22). However, access to these openings, and subsequent binding, have yet to be described. Here we use the Anton supercomputer (23) to carry out extensive unbiased fully atomistic simulations of BZC and PHT binding to the Na_vAb channel to reveal the distribution of binding sites and observe their pathways to shed light on inhibition mechanisms and aid future Na_v-drug development.     

Frequency of Val1016Ile mutation in the voltage-gated sodium channel gene of Aedes aegypti Brazilian populations

One of the major insect pyrethroid resistance mechanisms affects its target site, the voltage-gated sodium channel (Na_v). In Aedes aegypti , the Val1016Ile substitution of the AaNa _v gene is associated to resistance in several Latin American countries. Genotyping of susceptible and resistant mosquitoes from seven Brazilian localities detected the 1016Ile mutation in five populations with a higher frequency of this substitution in resistant specimens in all cases. Furthermore, analysis of nine additional field populations revealed that five also presented the 1016Ile mutation. Our data suggest a recent dissemination and involvement of this substitution with pyrethroid resistance in Brazil.     

Targeted ubiquitination of sensory neuron calcium channels reduces the development of neuropathic pain

Neuropathic pain caused by lesions to somatosensory neurons due to injury or disease is a widespread public health problem that is inadequately managed by small-molecule therapeutics due to incomplete pain relief and devastating side effects. Genetically encoded molecules capable of interrupting nociception have the potential to confer long-lasting analgesia with minimal off-target effects. Here, we utilize a targeted ubiquitination approach to achieve a unique posttranslational functional knockdown of high-voltage-activated calcium channels (HVACCs) that are obligatory for neurotransmission in dorsal root ganglion (DRG) neurons. Ca_V-aβlator comprises a nanobody targeted to Ca_V channel cytosolic auxiliary β subunits fused to the catalytic HECT domain of the Nedd4-2 E3 ubiquitin ligase. Subcutaneous injection of adeno-associated virus serotype 9 encoding Ca_V-aβlator in the hind paw of mice resulted in the expression of the protein in a subset of DRG neurons that displayed a concomitant ablation of Ca_V currents and also led to an increase in the frequency of spontaneous inhibitory postsynaptic currents in the dorsal horn of the spinal cord. Mice subjected to spare nerve injury displayed a characteristic long-lasting mechanical, thermal, and cold hyperalgesia underlain by a dramatic increase in coordinated phasic firing of DRG neurons as reported by in vivo Ca²⁺ spike recordings. Ca_V-aβlator significantly dampened the integrated Ca²⁺ spike activity and the hyperalgesia in response to nerve injury. The results advance the principle of targeting HVACCs as a gene therapy for neuropathic pain and demonstrate the therapeutic potential of posttranslational functional knockdown of ion channels achieved by exploiting the ubiquitin-proteasome system.

Neuropathic pain is a debilitating ailment caused by lesions in the somatosensory system that can arise from diverse conditions including nerve injuries, diabetes, thoracic surgery, cancer, and chemotherapy-induced neuropathy. The condition is widely prevalent, affecting ∼10% of the general population and is the most difficult type of chronic pain to treat (1, 2). First-line treatments for neuropathic pain commonly include gabapentinoids (gabapentin or pregabalin) or antidepressants, with opioid agonists such as tramadol frequently prescribed as second-line treatments (3–5). Unfortunately, up to 40% of patients remain refractory to these treatments, and serious side effects of these medications further limit their clinical utility. For example, gabapentin only reduces chronic neuropathic pain by at least 50% in fewer than 20% of patients (6), and pregabalin is not effective in controlling chronic pain after traumatic nerve injury (7). Adverse side effects of gabapentinoids due to central actions include sedation, dizziness, drowsiness, fatigue, and blurred vision (8, 9). Opiate agonists also have well-recognized limitations—dose-limiting adverse effects, inadequate pain relief, requirement for multiple daily dosing, liver/kidney toxicity, high potential for abuse and addiction, and risk of overdose—that largely reduce their efficacy for treating neuropathic pain (10). Overall, neuropathic pain is a significant public health problem lacking adequate therapeutic options. As such, there is a salient need to develop treatment modalities that provide strong, long-lasting, and nonaddictive pain relief, with few side effects.Gene therapy has emerged as an attractive alternative approach to treat chronic pain, with the notion that sustained but locally restricted expression of a therapeutic transgene can potentially enable long-lasting alleviation of nociception after a single dose, with minimal side effects (11, 12). A critical decision is the choice of the gene or protein to be targeted and its role in the pain processing pathway. High-voltage-activated calcium channels (HVACCs) are attractive potential targets for a gene therapy approach because 1) presynaptic Ca_V2 channels in nociceptive somatosensory neurons mediate the central release of a neurotransmitter in the dorsal horn of the spinal cord, which is an obligatory step in nociception, and 2) several analgesics including gabapentinoids (13, 14), ziconotide (15), and opiate agonists acting on μ-opioid receptors to release G_βγ subunits (16) inhibit Ca_V2 channels as a mechanism for alleviating pain. Thus, we hypothesized that targeted down-regulation of HVACCs in somatosensory neurons in vivo would be efficacious as a long-lasting treatment for neuropathic pain. Previous studies seeking to downregulate the expression of proteins involved in nociception have used RNA interference or antisense approaches (17, 18). More recently, epigenetic suppression of Nav1.7 in mice was achieved using catalytically dead CRISPR-Cas9 and zinc finger protein approaches, respectively, to achieve analgesia in rodent models (19). By comparison with these methods, posttranslational knockdown of relevant proteins offers an alternative approach with potential advantages that include quicker onset of pain relief, easier titration of effects, and fewer obstacles to clinical development.We recently developed a genetically encoded potent HVACC blocker comprised of an auxiliary Ca_Vβ-subunit-targeted nanobody (nb.F3) fused to the catalytic HECT domain of the E3 ubiquitin ligase Nedd4L (neural precursor cell expressed developmentally downregulated gene 4-like) (20). Here, we investigated whether the expression of nb.F3-Nedd4L_HECT (also known as Ca_V-aβlator) in sensory neurons in mice is able to inhibit Ca_V channels in vivo and result in the alleviation of experimentally induced nocifensive responses. We show that Ca_V-aβlator can be targeted to sensory neurons via hind paw injection of adeno-associated virus serotype 9 (AAV9) in vivo. The expressed Ca_V-aβlator posttranslationally inhibits HVACCs in DRG neurons, results in increased spontaneous inhibitory postsynaptic currents (sIPSCs) in the spinal cord dorsal horn, and reduces the development of nocifensive responses following nerve injury with no apparent adverse effects. The results not only provide a paradigm of genetically encoded calcium channel blocker development and use for pre-emptive analgesia but also demonstrate the efficacy of harnessing a ubiquitin-dependent posttranslational knockdown approach to achieve treatment of chronic pain.     

PIP2 controls voltage-sensor movement and pore opening of Kv channels through the S4-S5 linker

Voltage-gated K(+) (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4-S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP(2) depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP(2). Gating current measurements revealed that PIP(2) constrains the movement of the sensor through interactions with the S4-S5 linker. Thus, PIP(2) controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.     

In vivo examination of membrane protein localization and degradation with green fluorescent protein.

To test the utility of green fluorescent protein (GFP) as an in vivo reporter protein when fused to a membrane domain, we made a fusion protein between yeast hydroxymethylglutaryl-CoA reductase and GFP. Fusion proteins displayed spatial localization and regulated degradation consistent with the native hydroxymethylglutaryl-CoA reductase proteins. Thus, GFP should be useful in the study of both membrane protein localization and protein degradation in vivo.     

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R in vitro. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent K_d of ≈15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.     

A common SCN5A variant alters the responsiveness of human sodium channels to class I antiarrhythmic agents

Background: The potential pathophysiological role of common SCN5A polymorphisms in cardiac arrhythmias has been increasingly recognized. However, little is known about the impact of those polymorphisms on the pharmocological response of hNav1.5 to various antiarrhythmic agents.
Methods and Results: The known SCN5A polymorphism, S524Y, was studied in comparison with the wild type (WT) define the SCN5A-Q1077del variant. The ion channel gating kinetics and pharmacology were evaluated using whole-cell patch-clamp methods in HEK-293 cells. Consistent with a previous report, the basal ion channel gating kinetics of S524Y were indistinguishable from the WT. Quinidine (20 μM) caused similar extent of tonic block reduction of sodium currents at –120 mV in WT and S524Y. Surprisingly, quinidine (20 μM) exerted a more use-dependent block by a 10 Hz pulse train in S524Y than in WT at 22°C ( Ki : WT, 51.3 μM; S524Y, 20.3 μM). S524Y significantly delayed recovery from the use-dependent block, compared with the WT (τ= 88.6 ± 7.9 s vs 41.9 ± 6.6 s, P < 0.005). Under more physiological conditions using a 2 Hz pulse train at 37°C, S524Y similarly enhanced the use-dependent block by quinidine. In addition, S524Y enhanced the use-dependent block by flecainide (12.5 μM), but not by mexiletine (100 μM).
Conclusion: A common SCN5A polymorphism, S524Y, can enhance a use-dependent block by class Ia and Ic antiarrhythmic agents. Our findings may have clinical implications in pharmacological management of cardiac arrhythmias since this common SCN5A polymorphism might be a contributing factor to the variable antiarrhythmic response.     

Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier

The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K⁺ channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.     

替米沙坦对电压依赖性的Kv1.3和Kv1.5通道电流阻断作用的实验研究

目的 通过观察替米沙坦对电压依赖性的Kv1.3和Kv1.5的阻断作用,探讨替米沙坦对此类通道的阻断可能具有的临床作用.方法 使用双电极电压钳技术记录表达于非洲爪蟾卵母细胞的Kv1.3和Kv1.5钾通道电流,不同浓度灌流观察其对电流影响.结果 (1)替米沙坦浓度依赖性的阻断Kv1.3通道,其阻断的IC50是2.05 μmol/L.替米沙坦对Kv1.3电流的阻断具有电压依赖性.(2)替米沙坦浓度依赖件的阻断Kv1.5通道,其阻断的IC50是2.37 μmol/L.替米沙坦对Kv1.5电流的阻断具有更显著的电压依赖性.结论 替米沙坦阻断开放状态的Kv1.3可能是其发挥免疫调节和抗动脉粥样硬化作用的机制之一.替米沙坦对开放状态的Kv1.5钾通道的阻断可能是其减少心房颤动发生率的作用机制之一.     

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.     

Glucocorticoids differentially regulate degradation of MyoD and Id1 by N-terminal ubiquitination to promote muscle protein catabolism

Accelerated protein degradation via the ubiquitin-proteasome pathway is the principal cause of skeletal muscle wasting associated with common human disease states and pharmacological treatment with glucocorticoids. Although many protein regulatory factors essential for muscle development and regeneration are degraded via the ubiquitin system, little is known about the mechanisms and regulation of this pathway that promote wasting muscle. Here, we demonstrate that, in differentiated myotubes, glucocorticoid, via the glucocorticoid receptor, selectively induces a decrease in protein abundance of MyoD, a master switch for muscle development and regeneration, but not that of its negative regulator Id1. This decrease in MyoD protein results from accelerated degradation after glucocorticoid exposure. Using MyoD and Id1 mutants deficient in either N terminus-dependent or internal lysine-dependent ubiquitination, we further show that these ubiquitination pathways of MyoD degradation are regulated differently from those of Id1 degradation. Specifically, glucocorticoid activates the N-terminal ubiquitination pathway in MyoD degradation in myotubes, without concomitant effects on Id1 degradation. This effect of glucocorticoid on MyoD and Id1 protein degradation is associated with the distinct cellular compartments in which their degradation occurs. Taken together, these results support a key role for the N terminus-dependent ubiquitination pathway in the physiology of muscle protein degradation.     

Developmental changes in the expression of voltage-gated potassium channels in the ductus arteriosus of the fetal rat

Oxygen-sensitive, voltage-gated potassium channels (Kv) may contribute to the determination of the membrane potential in smooth muscle cells of the ductus arteriosus (DA), and thus to regulation of contractile tone in response to oxygen. Developmental changes in Kv during gestation may be related to closure of the DA after birth. This study investigated developmental changes in the expression of Kv in the DA and compared it with that of the pulmonary artery (PA) and the aorta (Ao). The DA, PA, and Ao were isolated from fetal rats at days 19 and 21 of gestation (term: 21.5 days). The expression of Kv1.2, Kv1.5, Kv2.1, and Kv3.1, putative oxygen-sensitive Kv channels that open in response to oxygen, was evaluated at both the mRNA and protein levels, using quantitative real-time polymerase chain reaction and immunohistochemistry. In the Kv family studied, Kv1.5 mRNA was expressed most abundantly in the DA, PA, and Ao in both day-19 and day-21 fetuses. Although the expression levels of Kv1.2, Kv1.5, Kv2.1, and Kv3.1 did not change much with development in the PA and Ao, in the DA they decreased with development. The decrease in the expression of Kv channels may enhance DA closure after birth by eliminating the opening of Kv channels when oxygen increases.     

Role of sodium channels in propagation in heart muscle: how subtle genetic alterations result in major arrhythmic disorders

Sodium channels play a crucial role in initiation, propagation, and maintenance of cardiac excitation throughout the heart. Indeed, dysfunctional sodium channels have been shown to be responsible for several inherited cardiac electrical disorders, such as Long QT and Brugada syndromes (BrS), potentially leading to fatal arrhythmic events. Genetic approaches and functional experiments using heterologous systems have enabled the characterization of the molecular determinants involved in these disorders and their consequences on ion channel function. The improved understanding of the mechanisms leading to these cardiac arrhythmic events represents a first step in the development of therapeutic treatments.     

Alterations of sodium channel kinetics and gene expression in the postinfarction remodeled myocardium

INTRODUCTION: After a myocardial infarction (MI), the heart undergoes a remodeling process that includes hypertrophy of noninfarcted left ventricular myocytes. Alterations in the genetic expression, including reexpression of fetal isogene patterns, can result in electrophysiologic changes that contribute to the arrhythmogenicity of post-MI heart. The present study investigated possible alterations in gene expression of Na+ channel subtypes, as well as the kinetics of the Na+ current (I(Na)), in 3- to 4-week-old post-MI rat remodeled left ventricular myocardium. METHODS AND RESULTS: Using a macropatch technique, we showed increased Na+ channel bursting activity during sustained depolarization in post-MI remodeled myocytes resulting in a large slow component of the I(Na) decay. A tetrodotoxin-sensitive current contributed 18% to the prolonged APD90 of isolated post-MI myocytes compared with 6% in control myocytes. Our molecular studies revealed that, in addition to the rat heart I (rH I) subtype, thought to be the predominant subtype that encodes a tetrodotoxin-resistant isoform, the brain subtypes NaCh I and NaCh Ia also are expressed in the rat myocytes. Post-MI remodeled myocardium showed increased expression of NaCh I protein with reversion of the NaCh Ia/NaCh I isoform ratio toward the fetal phenotype. CONCLUSION: Our findings raise the possibility that the increase in the slow component of I(Na) in post-MI remodeled myocytes is secondary to the increased expression of NaCh I. Additional studies are required to address these questions and to characterize the functional role of the NaCh I subtypes in cardiac myocytes.     

Na(V)1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms

Na(V)1.1 is the primary voltage-gated Na(+) channel in several classes of GABAergic interneurons, and its reduced activity leads to reduced excitability and decreased GABAergic tone. Here, we show that Na(V)1.1 channels are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. Mice carrying a heterozygous loss of function mutation in the Scn1a gene (Scn1a(+/-)), which encodes the pore-forming α-subunit of the Na(V)1.1 channel, have longer circadian period than WT mice and lack light-induced phase shifts. In contrast, Scn1a(+/-) mice have exaggerated light-induced negative-masking behavior and normal electroretinogram, suggesting an intact retina light response. Scn1a(+/-) mice show normal light induction of c-Fos and mPer1 mRNA in ventral SCN but impaired gene expression responses in dorsal SCN. Electrical stimulation of the optic chiasm elicits reduced calcium transients and impaired ventro-dorsal communication in SCN neurons from Scn1a(+/-) mice, and this communication is barely detectable in the homozygous gene KO (Scn1a(-/-)). Enhancement of GABAergic transmission with tiagabine plus clonazepam partially rescues the effects of deletion of Na(V)1.1 on circadian period and phase shifting. Our report demonstrates that a specific voltage-gated Na(+) channel and its associated impairment of SCN interneuronal communication lead to major deficits in the function of the master circadian pacemaker. Heterozygous loss of Na(V)1.1 channels is the underlying cause for severe myoclonic epilepsy of infancy; the circadian deficits that we report may contribute to sleep disorders in severe myoclonic epilepsy of infancy patients.     

Structure and function of splice variants of the cardiac voltage-gated sodium channel Nav1.5

Voltage-gated sodium channels mediate the rapid upstroke of the action potential in excitable tissues. The tetrodotoxin (TTX) resistant isoform Na_v1.5, encoded by the SCN5A gene, is the predominant isoform in the heart. This channel plays a key role for excitability of atrial and ventricular cardiomyocytes and for rapid impulse propagation through the specific conduction system. During recent years, strong evidence has been accumulated in support of the expression of several Na_v1.5 splice variants in the heart, and in various other tissues and cell lines including brain, dorsal root ganglia, breast cancer cells and neuronal stem cell lines. This review summarizes our knowledge on the structure and putative function of nine Na_v1.5 splice variants detected so far. Attention will be paid to the distinct biophysical properties of the four functional splice variants, to the pronounced tissue- and species-specific expression, and to the developmental regulation of Na_v1.5 splicing. The implications of alternative splicing for SCN5A channelopathies, and for a better understanding of genotype-phenotype correlations, are discussed.     

STIM protein coupling in the activation of Orai channels

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca²⁺ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca²⁺ entry through the Orai family of highly Ca²⁺-selective “store-operated” channels (SOCs). Dissecting the STIM–Orai coupling process is restricted by the abstruse nature of the ER–PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca²⁺ release-activated Ca²⁺ (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct–Orai1 coupling occurred in STIM1/STIM2^−/− DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct–Orai1 and S2-ct–Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB''s action, the locked STIMct–Orai complex provides a potentially useful probe to structurally examine coupling.