Single-Molecule Imaging of Na v 1.6 on the Surface of Hippocampal Neurons Reveals Somatic Nanoclusters

Akin, Elizabeth J.; Solé, Laura; Johnson, Ben; Beheiry, Mohamed El; Masson, Jean-Baptiste; Krapf, Diego; Tamkun, Michael M.

doi:10.1016/j.bpj.2016.08.016

Cited by 51 publications

(76 citation statements)

References 61 publications

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“…Most trajectories exhibit sub-diffusive behavior, i.e., the MSD of individual trajectories is not linear in lag time but it scales as a power law = with < 1, where is the anomalous exponent and is the generalized diffusion coefficient [20][21][22] . Furthermore, a substantial number of trajectories have a flat MSD, indicating that these particles are either immobile or confined to small domains 23 . In all cases, only particles that were observed for at least 3 s were analyzed.…”

Section: Resultsmentioning

confidence: 99%

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking

Metz

Pennock

Krapf

et al. 2018

Preprint

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Agonist binding to the mu opioid receptor (MOR) results in conformational changes that allow recruitment of G-proteins, activation of downstream effectors and eventual desensitization and internalization, all of which could affect receptor mobility. The present study employed single particle tracking (SPT) of quantum dot labeled FLAG-tagged MORs to examine shifts in MOR mobility after agonist binding. FLAG-MORs on the plasma membrane were in both mobile and immobile states under basal conditions. Activation of FLAG-MORs with DAMGO caused an acute increase in the fraction of mobile MORs, and free portions of mobile tracks were partially dependent on interactions with G-proteins. In contrast, 10-minute exposure to DAMGO or morphine increased the fraction of immobile FLAG-MORs. While the decrease in mobility with prolonged DAMGO exposure corresponded to an increase in colocalization with clathrin, the increase in colocalization was present in both mobile and immobile FLAG-MORs. Thus, no single mobility state of the receptor accounted for colocalization with clathrin. These findings demonstrate that SPT can be used to track agonist-dependent changes in MOR mobility over time, but that the mobility states observed likely arise from a diverse set of interactions and will be most informative when examined in concert with particular downstream effectors.

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Section: Resultsmentioning

confidence: 99%

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking

Metz

Pennock

Krapf

et al. 2018

Preprint

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“…Plasmids constructs. hNa v 1.6-GFP, hNa v 1.6-LoopBAD-GFP, hNa v 1.6ABM-GFP, and hNa v 1.6ABM-LoopBAD-GFP were previously described (Akin et al, , 2016. The 77/80 VAVP-AAAA mutation was incorporated by site-directed mutagenesis into hNa v 1.6-GFP and hNa v 1.6-LoopBAD-GFP.…”

Section: Methodsmentioning

confidence: 99%

“…Cell culture and transfections. Rat hippocampal neuronal cultures (rHNs) were used as previously described (Akin et al, , 2016Fox et al, 2015;Johnson et al, 2018). Briefly, dissociated rat E18 hippocampal neurons from both sexes were seeded onto poly-L-lysine (Sigma-Aldrich, catalog #RNBG0769) (dilution 1:2 in 0.15 M borate buffer, pH 8.4) coated glassbottom 35 mm dishes (Matsunami, catalog #D11130H) and cultured in Neurobasal media (Invitrogen, catalog #21103-049) supplemented with Glutamax (Sigma-Aldrich, catalog #350350-061), penicillin/streptomycin (Cellgro, catalog #SV30010), and SM1 neuronal supplement (Stemcell Technologies, catalog #05711), per the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…The ion-conducting Na v ␣ sub-units are encoded by nine genes, with Na v 1.1, Na v 1.2, Na v 1.3, and Na v 1.6 predominant in the CNS (Catterall, 2012). Na v 1.6, encoded by SCN8A, is one of the most abundant Na v channels and localizes to specific nanodomains on the neuronal soma (Akin et al, 2016), axon initial segment (AIS) and nodes of Ranvier (Schaller et al, 1995;Whitaker et al, 1999;Tzoumaka et al, 2000). Maintenance of the structure and composition of the AIS is essential for proper neuronal activity, with the AIS demonstrating plasticity by changing both its composition and location in response to neuronal activity and injury (Grubb and Burrone, 2010;Kuba et al, 2010;Evans et al, 2013;Hatch et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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The MAP1B Binding Domain of Na_v1.6 Is Required for Stable Expression at the Axon Initial Segment

et al. 2019

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Na v 1.6 (SCN8A) is a major voltage-gated sodium channel in the mammalian CNS, and is highly concentrated at the axon initial segment (AIS). As previously demonstrated, the microtubule associated protein MAP1B binds the cytoplasmic N terminus of Na v 1.6, and this interaction is disrupted by the mutation p.VAVP(77-80)AAAA. We now demonstrate that this mutation results in WT expression levels on the somatic surface but reduced surface expression at the AIS of cultured rat embryonic hippocampal neurons from both sexes. The mutation of the MAP1B binding domain did not impair vesicular trafficking and preferential delivery of Na v 1.6 to the AIS; nor was the diffusion of AIS inserted channels altered relative to WT. However, the reduced AIS surface expression of the MAP1B mutant was restored to WT levels by inhibiting endocytosis with Dynasore, indicating that compartment-specific endocytosis was responsible for the lack of AIS accumulation. Interestingly, the lack of AIS targeting resulted in an elevated percentage of persistent current, suggesting that this late current originates predominantly in the soma. No differences in the voltage dependence of activation or inactivation were detected in the MAP1B binding mutant relative to WT channel. We hypothesize that MAP1B binding to the WT Na v 1.6 masks an endocytic motif, thus allowing long-term stability on the AIS surface. This work identifies a critical and important new role for MAP1B in the regulation of neuronal excitability and adds to our understanding of AIS maintenance and plasticity, in addition to identifying new target residues for pathogenic mutations of SCN8A. Significance StatementNa v 1.6 is a major voltage-gated sodium channel in human brain, where it regulates neuronal activity due to its localization at the axon initial segment (AIS). Na v 1.6 mutations cause epilepsy, intellectual disability, and movement disorders. In the present work, we show that loss of interaction with MAP1B within the Na v 1.6 N terminus reduces the steady-state abundance of Na v 1.6 at the AIS. The effect is due to increased Na v 1.6 endocytosis at this neuronal compartment rather than a failure of forward trafficking to the AIS. This work confirms a new biological role of MAP1B in the regulation of sodium channel localization and will contribute to future analysis of patient mutations in the cytoplasmic N terminus of Na v 1.6.

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“…Because the pipette solution used in our whole-cell recordings contained a constant level of pH and ATP, it seems unlikely that Rot-induced suppression of peak I Na is linked to changes in either intracellular pH or ATP content. It is also tempting to speculate that the α-subunit of the Na V 1.1 or Na V 1.6 channels, which are respectively encoded by the SCN1A or SCN8A gene, is functionally expressed in mHippoE-14 cells [16, 28]. …”

Section: Discussionmentioning

confidence: 99%

Multiple Actions of Rotenone, an Inhibitor of Mitochondrial Respiratory Chain, on Ionic Currents and Miniature End-Plate Potential in Mouse Hippocampal (mHippoE-14) Neurons

Huang

Lin

Hung

et al. 2018

Cell Physiol Biochem

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Background/Aims: Rotenone (Rot) is known to suppress the activity of complex I in the mitochondrial chain reaction; however, whether this compound has effects on ion currents in neurons remains largely unexplored. Methods: With the aid of patch-clamp technology and simulation modeling, the effects of Rot on membrane ion currents present in mHippoE-14 cells were investigated. Results: Addition of Rot produced an inhibitory action on the peak amplitude of I_Na with an IC₅₀ value of 39.3 µM; however, neither activation nor inactivation kinetics of I_Na was changed during cell exposure to this compound. Addition of Rot produced little or no modifications in the steady-state inactivation curve of I_Na. Rot increased the amplitude of Ca²⁺-activated Cl^- current in response to membrane depolarization with an EC₅₀ value of 35.4 µM; further addition of niflumic acid reversed Rot-mediated stimulation of this current. Moreover, when these cells were exposed to 10 µM Rot, a specific population of ATP-sensitive K⁺ channels with a single-channel conductance of 18.1 pS was measured, despite its inability to alter single-channel conductance. Under current clamp condition, the frequency of miniature end-plate potentials in mHippoE-14 cells was significantly raised in the presence of Rot (10 µM) with no changes in their amplitude and time course of rise and decay. In simulated model of hippocampal neurons incorporated with chemical autaptic connection, increased autaptic strength to mimic the action of Rot was noted to change the bursting pattern with emergence of subthreshold potentials. Conclusions: The Rot effects presented herein might exert a significant action on functional activities of hippocampal neurons occurring in vivo.

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Single-Molecule Imaging of Na v 1.6 on the Surface of Hippocampal Neurons Reveals Somatic Nanoclusters

Cited by 51 publications

References 61 publications

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking

The MAP1B Binding Domain of Na_v1.6 Is Required for Stable Expression at the Axon Initial Segment

Multiple Actions of Rotenone, an Inhibitor of Mitochondrial Respiratory Chain, on Ionic Currents and Miniature End-Plate Potential in Mouse Hippocampal (mHippoE-14) Neurons

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Single-Molecule Imaging of Na v 1.6 on the Surface of Hippocampal Neurons Reveals Somatic Nanoclusters

Cited by 51 publications

References 61 publications

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking

Temporal dependence of shifts in mu opioid receptor mobility at the cell surface after agonist binding observed by single-particle tracking

The MAP1B Binding Domain of Nav1.6 Is Required for Stable Expression at the Axon Initial Segment

Multiple Actions of Rotenone, an Inhibitor of Mitochondrial Respiratory Chain, on Ionic Currents and Miniature End-Plate Potential in Mouse Hippocampal (mHippoE-14) Neurons

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The MAP1B Binding Domain of Na_v1.6 Is Required for Stable Expression at the Axon Initial Segment