Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na(v)1.7-specific inhibitor, ProTxII, allowed the isolation of Na(v)1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na(v)1.7 and Na(v)1.8 currents. The redistribution of Na(v)1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L(-/-)). SNS-Nedd4L(-/-) mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na(v)1.7 and Na(v)1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na(v)s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Chloride channels in myotonia congenita assessed by velocity recovery cycles

Introduction: Myotonia congenita (MC) is caused by congenital defects in the muscle chloride channel CLC-1. This study used muscle velocity recovery cycles (MVRCs) to investigate how membrane function is affected. Methods: MVRCs and responses to repetitive stimulation were compared between 18 patients with genetically confirmed MC (13 recessive, 7 dominant) and 30 age-matched normal controls. Results: MC patients exhibited increased early supernormality, but treatment with sodium channel blockers prevented this. After multiple conditioning stimuli, late supernormality was enhanced in all MC patients, indicating delayed repolarization. These abnormalities were similar between the MC subtypes, but recessive patients showed a greater drop in amplitude during repetitive stimulation. Discussion: MVRCs indicate that chloride conductance only becomes important when muscle fibers are depolarized. The differential responses to repetitive stimulation suggest that in dominant MC the affected chloride channels are activated by strong depolarization, consistent with a positive shift of the CLC-1 activation curve. © 2013 Wiley Periodicals, Inc.

Geomorphic coupling between hillslopes and channels in the Swiss Alps

The coupling relationships between hillslope and channel network are fundamental for the understanding of mountainous landscapes' evolution. Here, we applied dendrogeomorphic methods to identify the hillslope–channel relationship and the sediment transfer dynamics within an alpine catchment, at the highest possible resolution. The Schimbrig catchment is located in the central Swiss Alps and can be divided into two distinct geomorphic sectors. To the east, the Schimbrig earth flow is the largest sediment source of the basin, while to the west, the Rossloch channel network is affected by numerous shallow landslides responsible for the supply of sediment from hillslopes to channels. To understand the connectivity between hillslopes and channels and between sources and sink, trees were sampled along the main Rossloch stream, on the Schimbrig earth flow and on the Rossloch depositional area. Geomorphic observations and dendrogeomophic results indicate different mechanisms of sediment production, transfer and deposition between upper and lower segments of the channel network. In the source areas (upper part of the Rossloch channel system), sediment is delivered to the channel network through slow movements of the ground, typical of earth flow, shallow landslides and soil creep. Contrariwise, in the depositional area (lower part of the channel network), the mechanisms of sediment transfer are mainly due to torrential activity, floods and debris flows. Tree analysis allowed the reconstruction of periods of high activity during the last century for the entire catchment. The collected dataset presents a very high temporal resolution but we encountered some limitations in establishing the source-to-sink connectivity at the catchment-wide scale. Despite these uncertainties, for decennial timescales the results suggest a direct coupling between hillslopes and neighbouring channels in the Rossloch channel network, and a de-coupling between sediment sources and sink farther downstream, with connections possible only during extraordinary events.

Inhibition of dendritic Ca2+ spikes by GABAB receptors in cortical pyramidal neurons is mediated by a direct Gi/o- -subunit interaction with Cav1 channels

Voltage-dependent calcium channels (VDCCs) serve a wide range of physiological functions and their activity is modulated by different neurotransmitter systems. GABAergic inhibition of VDCCs in neurons has an important impact in controlling transmitter release, neuronal plasticity, gene expression and neuronal excitability. We investigated the molecular signalling mechanisms by which GABAB receptors inhibit calcium-mediated electrogenesis (Ca2+ spikes) in the distal apical dendrite of cortical layer 5 pyramidal neurons. Ca2+ spikes are the basis of coincidence detection and signal amplification of distal tuft synaptic inputs characteristic for the computational function of cortical pyramidal neurons. By combining dendritic whole-cell recordings with two-photon fluorescence Ca2+ imaging we found that all subtypes of VDCCs were present in the Ca2+ spike initiation zone, but that they contribute differently to the initiation and sustaining of dendritic Ca2+ spikes. Particularly, Cav1 VDCCs are the most abundant VDCC present in this dendritic compartment and they generated the sustained plateau potential characteristic for the Ca2+ spike. Activation of GABAB receptors specifically inhibited Cav1 channels. This inhibition of L-type Ca2+ currents was transiently relieved by strong depolarization but did not depend on protein kinase activity. Therefore, our findings suggest a novel membrane-delimited interaction of the Gi/o-βγ-subunit with Cav1 channels identifying this mechanism as the general pathway of GABAB receptor-mediated inhibition of VDCCs. Furthermore, the characterization of the contribution of the different VDCCs to the generation of the Ca2+ spike provides new insights into the molecular mechanism of dendritic computation.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Search for resonant diboson production in the lvjj decay channels with the ATLAS detector at 7 TeV

A search for resonant diboson production using a data sample corresponding to 4.7 fb(-1) of integrated luminosity collected by the ATLAS experiment at the Large Hadron Collider in pp collisions at root s = 7 TeV is presented. The search for a narrow resonance in the WW or WZ mass distribution is conducted in a final state with an electron or a muon, missing transverse momentum, and at least two jets. No significant excess is observed and limits are set using three benchmark models: WW resonance masses below 940 and 710 GeV are excluded at 95% confidence level for spin-2 Randall-Sundrum and bulk Randall-Sundrum gravitons, respectively; WZ resonance masses below 950 GeV are excluded at 95% confidence level for a spin-1 extended gauge model W' boson.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

A pentasymmetric open channel blocker for Cys-loop receptor channels.

γ-Aminobutyric acid type A receptors (GABAA receptors) are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. These receptors show near five-fold symmetry that is most pronounced in the second trans-membrane domain M2 lining the Cl- ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclopentdienyl anion (PCCP-) that exhibited all characteristics of an open channel blocker. Inhibition was strongly dependent on the membrane potential. Through mutagenesis and covalent modification, we identified the region α1V256-α1T261 in the rat recombinant GABAA receptor to be important for PCCP- action. Introduction of positive charges into M2 increased the affinity for PCCP- while PCCP- prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP-, among them the Drosophila RDL GABAA receptor carrying an insecticide resistance mutation, suggesting that PCCP- could serve as an insecticide.

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Ca v1.2 channels

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.