scholarly journals Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Alejandra Leo-Macias ◽  
Esperanza Agullo-Pascual ◽  
Jose L. Sanchez-Alonso ◽  
Sarah Keegan ◽  
Xianming Lin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Sodium Current ◽  
Intercellular Adhesion ◽  
Intercalated Disc ◽  
Voltage Gated Sodium Channels ◽  
Mathematical Simulations ◽  
Nanoscale Imaging ◽  
Key Sites ◽  
Myelinated Fibres ◽  
Macromolecular Organization

Abstract Intercellular adhesion and electrical excitability are considered separate cellular properties. Studies of myelinated fibres, however, show that voltage-gated sodium channels (VGSCs) aggregate with cell adhesion molecules at discrete subcellular locations, such as the nodes of Ranvier. Demonstration of similar macromolecular organization in cardiac muscle is missing. Here we combine nanoscale-imaging (single-molecule localization microscopy; electron microscopy; and ‘angle view’ scanning patch clamp) with mathematical simulations to demonstrate distinct hubs at the cardiac intercalated disc, populated by clusters of the adhesion molecule N-cadherin and the VGSC NaV1.5. We show that the N-cadherin-NaV1.5 association is not random, that NaV1.5 molecules in these clusters are major contributors to cardiac sodium current, and that loss of NaV1.5 expression reduces intercellular adhesion strength. We speculate that adhesion/excitability nodes are key sites for crosstalk of the contractile and electrical molecular apparatus and may represent the structural substrate of cardiomyopathies in patients with mutations in molecules of the VGSC complex.

Control of neuronal excitability by phosphorylation and dephosphorylation of sodium channels

2006 ◽  
Vol 34 (6) ◽  
pp. 1299-1302 ◽  
Author(s):  
T. Scheuer ◽  
W.A. Catterall
Keyword(s):  
Protein Kinases ◽  
Sodium Channels ◽  
Neuronal Excitability ◽  
Sodium Current ◽  
Molecular Complexes ◽  
Neuronal Firing ◽  
Signal Integration ◽  
Excitable Cells ◽  
Signalling Molecules ◽  
Voltage Gated Sodium Channels

Currents through voltage-gated sodium channels drive action potential depolarization in neurons and other excitable cells. Smaller currents through these channels are key components of currents that control neuronal firing and signal integration. Changes in sodium current have profound effects on neuronal firing. Sodium channels are controlled by neuromodulators acting through phosphorylation of the channel by serine/threonine and tyrosine protein kinases. That phosphorylation requires specific molecular interaction of kinases and phosphatases with the channel molecule to form localized signalling complexes. Such localization is required for effective neurotransmitter-mediated regulation of sodium channels by protein kinase A. Analogous molecular complexes between sodium channels, kinases and other signalling molecules are expected to be necessary for specific and localized transmitter-mediated modulation of sodium channels by other protein kinases.

Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution

2015 ◽  
Vol 43 (3) ◽  
pp. 309-314 ◽  
Author(s):  
Sarah R. Needham ◽  
Laura C. Zanetti-Domingues ◽  
Kathrin M. Scherer ◽  
Michael Hirsch ◽  
Daniel J. Rolfe ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Spatial Resolution ◽  
Single Molecule ◽  
Egf Receptor ◽  
Limited Range ◽  
Length Scales ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth ◽  
Macromolecular Organization

There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5–50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.

scholarly journals Rapid single-wavelength lightsheet localization microscopy for clarified tissue

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Li-An Chu ◽  
Chieh-Han Lu ◽  
Shun-Min Yang ◽  
Yen-Ting Liu ◽  
Kuan-Lin Feng ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Biological Tissues ◽  
Adult Brain ◽  
Imaging Data ◽  
Transporter Protein ◽  
Index Matching ◽  
Refractive Index Matching ◽  
Nanoscale Imaging ◽  
Resolution Imaging

Abstract Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 107 µm3) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 µm3 per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.

Potentiation of rat brain sodium channel currents by PKA inXenopus oocytes involves the I-II linker

2000 ◽  
Vol 278 (4) ◽  
pp. C638-C645 ◽  
Author(s):  
Raymond D. Smith ◽  
Alan L. Goldin
Keyword(s):  
Sodium Channel ◽  
Xenopus Oocytes ◽  
Sodium Current ◽  
Sodium Currents ◽  
Voltage Gated Sodium Channels ◽  
Single Region ◽  
Functional Modulation ◽  
Excitability Of Neurons ◽  
Channel Currents ◽  
The Brain

Functional modulation of voltage-gated sodium channels affects the electrical excitability of neurons. Protein kinase A (PKA) can decrease sodium currents by phosphorylation at consensus sites in the cytoplasmic I-II linker. Once the sites are phosphorylated, however, additional PKA activity can increase sodium currents by an unknown mechanism. When the PKA sites were eliminated by substitutions of alanine for serine, peak sodium current amplitudes were increased by 20–80% when PKA was activated in Xenopus oocytes either by stimulation of a coexpressed β2-adrenergic receptor or by perfusion with reagents that increase cAMP. Potentiation required the I-II linker of the brain channel, in that a chimeric channel in which the brain linker was replaced with the comparable linker from the skeletal muscle channel did not demonstrate potentiation. Using a series of chimeric and deleted channels, we demonstrate that potentiation is not dependent on any single region of the linker and that the extent of potentiation varies depending on the total length and the residues throughout the linker. These data are consistent with the hypothesis that potentiation by PKA is an indirect process involving phosphorylation of an accessory protein that interacts with the I-II linker of the sodium channel.

scholarly journals Effect of Oxaliplatin on Voltage-Gated Sodium Channels in Peripheral Neuropathic Pain

Processes ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 680 ◽  
Author(s):  
Woojin Kim
Keyword(s):  
Neuropathic Pain ◽  
Action Potential ◽  
Sodium Channels ◽  
Sodium Current ◽  
Voltage Response ◽  
Treatment Schedule ◽  
Voltage Gated ◽  
Peripheral Neurons ◽  
Voltage Gated Sodium Channels ◽  
Current Voltage

Oxaliplatin is a chemotherapeutic drug widely used to treat various types of tumors. However, it can induce a serious peripheral neuropathy characterized by cold and mechanical allodynia that can even disrupt the treatment schedule. Since the approval of the agent, many laboratories, including ours, have focused their research on finding a drug or method to decrease this side effect. However, to date no drug that can effectively reduce the pain without causing any adverse events has been developed, and the mechanism of the action of oxaliplatin is not clearly understood. On the dorsal root ganglia (DRG) sensory neurons, oxaliplatin is reported to modify their functions, such as the propagation of the action potential and induction of neuropathic pain. Voltage-gated sodium channels in the DRG neurons are important, as they play a major role in the excitability of the cell by initiating the action potential. Thus, in this small review, eight studies that investigated the effect of oxaliplatin on sodium channels of peripheral neurons have been included. Its effects on the duration of the action potential, peak of the sodium current, voltage–response relationship, inactivation current, and sensitivity to tetrodotoxin (TTX) are discussed.

scholarly journals Chemometric Models of Differential Amino Acids at the Navα and Navβ Interface of Mammalian Sodium Channel Isoforms

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3551
Author(s):  
Fernando Villa-Diaz ◽  
Susana Lopez-Nunez ◽  
Jordan E. Ruiz-Castelan ◽  
Eduardo Marcos Salinas-Stefanon ◽  
Thomas Scior
Keyword(s):  
Sodium Channel ◽  
Sodium Current ◽  
Schematic Model ◽  
Excitable Cells ◽  
Protein Protein Interaction ◽  
Voltage Gated Sodium Channels ◽  
Gating Mechanism ◽  
In Silico Modeling ◽  
Variable Extent ◽  
Domain I

(1) Background: voltage-gated sodium channels (Navs) are integral membrane proteins that allow the sodium ion flux into the excitable cells and initiate the action potential. They comprise an α (Navα) subunit that forms the channel pore and are coupled to one or more auxiliary β (Navβ) subunits that modulate the gating to a variable extent. (2) Methods: after performing homology in silico modeling for all nine isoforms (Nav1.1α to Nav1.9α), the Navα and Navβ protein-protein interaction (PPI) was analyzed chemometrically based on the primary and secondary structures as well as topological or spatial mapping. (3) Results: our findings reveal a unique isoform-specific correspondence between certain segments of the extracellular loops of the Navα subunits. Precisely, loop S5 in domain I forms part of the PPI and assists Navβ1 or Navβ3 on all nine mammalian isoforms. The implied molecular movements resemble macroscopic springs, all of which explains published voltage sensor effects on sodium channel fast inactivation in gating. (4) Conclusions: currently, the specific functions exerted by the Navβ1 or Navβ3 subunits on the modulation of Navα gating remain unknown. Our work determined functional interaction in the extracellular domains on theoretical grounds and we propose a schematic model of the gating mechanism of fast channel sodium current inactivation by educated guessing.

Effects of lactate on the voltage-gated sodium channels of rat skeletal muscle: modulating current opinion

2012 ◽  
Vol 112 (9) ◽  
pp. 1454-1465 ◽  
Author(s):  
F. Rannou ◽  
R. Leschiera ◽  
M. A. Giroux-Metges ◽  
J. P. Pennec
Keyword(s):  
Skeletal Muscle ◽  
Sodium Current ◽  
Lactate Production ◽  
Petri Dish ◽  
Potential Effect ◽  
Muscle Physiology ◽  
Membrane Area ◽  
Electrophysiological Properties ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

During muscle contraction, lactate production and translocation across the membrane increase. While it has recently been shown that lactate anion acts on chloride channel, less is known regarding a potential effect on the voltage-gated sodium channel (Nav) of skeletal muscle. The electrophysiological properties of muscle Nav were studied in the absence and presence of lactate (10 mM) by using the macropatch-clamp method in dissociated fibers from rat peroneus longus (PL). Lactate in the external medium (petri dish + pipette) increases the maximal sodium current, while the voltage dependence of activation and fast inactivation are shifted toward the hyperpolarized potentials. Lactate induces a leftward shift in the relationship between the kinetic parameters and the imposed potentials, resulting in an earlier recruitment of muscle Nav. In addition, lactate significantly decreases the time constant of activation at voltages more negative than −10 mV, corresponding to an acceleration of Nav activation. The slow inactivation process is decreased by lactate, corresponding to an enhancement in the number of excitable Nav. In an additional series of experiments, lactate (10 mM) was only added to the petri dish, while the pipette remained sealed on the membrane area. With this approach, the electrophysiological properties of Nav were unaffected by lactate compared with the control condition. Altogether, these data indicate that lactate modulates muscle Nav properties by an extracellular pathway. These effects are consistent with an enhancement in excitability, providing new insights into the role of lactate in muscle physiology.

scholarly journals Mutant D96V calmodulin induces unexpected remodeling of cardiac nanostructure and physiology

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Heather L. Struckman ◽  
Mikhail Tarasov ◽  
Yusuf Olgar ◽  
Alec Miller ◽  
Jonathan P. Davis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Sodium Current ◽  
Close Association ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Super Resolution ◽  
Sodium Calcium Exchanger ◽  
Patch Clamp Recording ◽  
Specific Expression

Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized &lt;100 nm from RYR2. However, NaV1.6 density within these regions was 67% greater in cD96V relative to WT. Consistent with this result, SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings revealed more frequent late-burst openings involving larger NaV clusters in cD96V myocytes relative to WT. Previous work identifies the sodium-calcium exchanger (NCX) as a key link between aberrant late NaV1.6 activity and proarrhythmic Ca2+ mishandling. Therefore, we explored the spatial organization of NaV1.6 and NCX using STORM. Consistent with their close association, 89% of NaV1.6 clusters localized &lt;100 nm from NCX in cD96V hearts, compared with 77% in WT. Notably, density of both NaV1.6 and NCX was increased at these sites by 48% and 31%, respectively, in cD96V relative to WT. Consistent with these data, cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. These proarrhythmic functional effects were abrogated by cardiac-specific knockout of NaV1.6. To our knowledge, this is the first demonstration of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, offering novel mechanistic insight into calmodulinopathy.

Abstract 16722: Perinexal Width Modulates Sodium Channel Recruitment During Extracellular Stimulation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Katrina Colucci Chang ◽  
Xiaobo Wu ◽  
Grace Blair ◽  
Alicia Lozano ◽  
Alexandra Hanlon ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Voltage Divider ◽  
Extracellular Calcium ◽  
Extracellular Potassium ◽  
Intercalated Disc ◽  
Activation Threshold ◽  
Voltage Gated Sodium Channels ◽  
Cardiac Sodium Channels ◽  
Ephaptic Coupling

Excitability in cardiomyocytes is dependent on the subthreshold current required to raise transmembrane potential to the activation threshold of voltage gated sodium channels and sodium channel recruitment to trigger an action potential. Cardiac sodium channels are densely expressed in the intercalated disc within the perinexal nanodomain, which is 2 orders of magnitude narrower than bulk extracellular interstitium. We hypothesized that perinexal narrowing reduces extracellular induced excitability because the perinexus functions as a voltage divider. Methods: Excitability with an extracellular stimulus was quantified in isolated Langendorff perfused male retired breeder guinea pig hearts by strength duration curves using the Lapicque method. Interventions included changing extracellular potassium (K+: 3, 4.5, and 10 mM), inhibiting sodium channels (90-uM Flecainide), and narrowing the perinexus by increasing extracellular calcium (Ca2+: 1.25 to 2.5 mM). Results: Consistent with previous studies, decreasing K+ from 4.56 to 3 mM depressed excitability with 2.5 mM Ca2+ but not 1.25 mM Ca2+, and conduction velocity (CV) decreased by 10.5 % with both 1.25 and 2.5 mM Ca2+. When K+ was raised from 4.56 to 10 mM, no change was seen in excitability with both Ca2+ concentrations. However, CV decreased by 16% with both Ca2+ concentrations. Flecainide depressed excitability only with 2.5 but not 1.25 mM Ca2+. Meanwhile CV decreased by 13% with 1.25 but CV did not change with 2.5 mM Ca2+. Finally, raising Ca2+ alone at baseline decreased excitability, without substantially changing conduction. Conclusions: Elevating extracellular calcium to narrow perinexi reduces excitability measured by extracellular stimulation consistent with a hypothesis that sodium channels in the intercalated disc are electrically isolated from the bulk interstitium. Furthermore, excitability and conduction do not correlate in response to similar K+ changes when Ca2+ also varies, suggesting cardiac excitability and propagation are independent mechanisms when the excitatory current occurs through regenerative propagation as occurs through gap junctions or arrives via an extracellular field as occurs with pacing and ephaptic coupling.

Tumor necrosis factor-α downregulates sodium current in skeletal muscle by protein kinase C activation: involvement in critical illness polyneuromyopathy

2011 ◽  
Vol 301 (5) ◽  
pp. C1057-C1063 ◽  
Author(s):  
Maité Guillouet ◽  
Gildas Gueret ◽  
Fabrice Rannou ◽  
Marie-Agnès Giroux-Metges ◽  
Maxime Gioux ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Sodium Current ◽  
Dose Effect ◽  
Membrane Excitability ◽  
Voltage Gated Sodium Channels ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

Sepsis is involved in the decrease of membrane excitability of skeletal muscle, leading to polyneuromyopathy. This effect is mediated by alterations of the properties of voltage-gated sodium channels (NaV), but the exact mechanism is still unknown. The aim of the present study was to check whether tumor necrosis factor (TNF-α), a cytokine released during sepsis, exerts a rapid effect on NaV. Sodium current ( INa) was recorded by macropatch clamp in skeletal muscle fibers isolated from rat peroneus longus muscle, in control conditions and after TNF-α addition. Analyses of dose-effect and time-effect relationships were carried out. Effect of chelerythrine, a PKC inhibitor, was also studied to determine the way of action of TNF-α. TNF-α induced a reversible dose- and time-dependent inhibition of INa. A maximum inhibition of 75% of the control current was observed. A shift toward more negative potentials of activation and inactivation curves of INa was also noticed. These effects were prevented by chelerythrine pretreatment. TNF-α is a cytokine released in the early stages of sepsis. Besides a possible transcriptional role, i.e., modification of the channel type and/or number, we demonstrated the existence of a rapid, posttranscriptional inhibition of NaV by TNF-α. The downregulation of the sodium current could be mediated by a PKC-induced phosphorylation of the sodium channel, thus leading to a significant decrease in muscle excitability.

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