Reductions in External Divalent Cations Evoke Novel Voltage-Gated Currents in Sensory Neurons

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg2+ and Ca2+ from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na+ influx that then causes depolarization-mediated activation of voltage-gated Ca2+ channels (CaV), which allows Ca2+ influx upon divalent re-introduction. Inhibition of CaV (ω-conotoxin, nifedipine) or NaV (tetrodotoxin, lidocaine) fails to reduce the Na+ influx. The Ca2+ influx is inhibited by CaV inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg2+ or Ca2+ alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg2+ and Ca2+ from external solutions evokes a large slowly-inactivating voltage-gated current (IDF) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca2+ alone fails to evoke IDF. Evidence suggests IDF is a non-selective cation current. The IDF is not reduced by inhibition of NaV (lidocaine, riluzole), CaV (cilnidipine, nifedipine), KV (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca2+ (IC50∼0.5 µM) or Mg2+ (IC50∼3 µM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons

A study of small and intermediate conductance calcium-activated potassium channels in sensory neurones

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Functional evidence of distinct electrophile-induced activation states of the ion channel TRPA1

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, nonselective cation channel expressed on nociceptive sensory nerves whose activation elicits nocifensive responses (e.g. pain). TRPA1 is activated by electrophiles found in foods and pollution, or produced during inflammation and oxidative stress, via covalent modification of reactive cysteines, but the mechanism underlying electrophilic activation of TRPA1 is poorly understood. Here we studied TRPA1 activation by the irreversible electrophiles iodoacetamide and N-ethylmaleimide (NEM) following transient expression in HEK293 cells. We found that in Ca2+ imaging studies C621 is critical for electrophile-induced TRPA1 activation, but the role of C665 in TRPA1 activation is dependent on the size of the electrophile. We identified slower TRPA1 activation in whole-cell recordings compared to studies with intact cells, which is rescued by pipette solution supplementation with the antioxidant glutathione. Single-channel recordings identified two distinct electrophilic-induced TRPA1 activation phases: a partial activation that, in some channels, switched to full activation with continued electrophile exposure. Full activation but not the initial activation was regulated by C665. Fitting of open time distributions suggests that full activation correlated with an additional (and long) exponential component, thus suggesting the phases are manifestations of distinct activation states. Our results suggest that distinct NEM-induced TRPA1 activation states are evoked by sequential modification of C621 then C665

Increasing external [Mg²⁺] inhibits <i>I_DF</i> activation.

<p>Mean ± S.E.M. Vagal neurons were held at −120 mV and stepped to −70 mV to record the inward current (A) or stepped to +60 mV to record the resulting outward current (B). Neurons were perfused with normal bath solution containing 2.5 mM Ca²⁺ and 1.2 mM Mg²⁺ (Control; white bar), or solution containing 5 mM EGTA and different [Mg²⁺] (0 µM, 1 µM, 10 µM, 100 µM and 1 mM; solid black bars). Statistical analysis was carried out between control conditions and increasing [Mg²⁺] using the unpaired Student's t-test (*** p<0.005).</p

Re-introduction of divalent cations following EDTA evokes Ca²⁺ influx in vagal neurons.

<p>Mean ± S.E.M. Ca²⁺ responses of vagal neurons in response to brief treatment with EDTA (5 mM; 0 Ca²⁺, 0 Mg²⁺) followed by re-introduction of Ca²⁺ (2.2 mM) and Mg²⁺ (1.2 mM) as measured by Fura 2AM. Response to capsaicin (1 µM) and KCl (75 mM) also shown. Data comprised of capsaicin-sensitive (black squares; n = 41) and capsaicin-insensitive neurons (grey squares; n = 11) from C57BL/6 mice. Blocked line denotes application of drugs.</p

The <i>I_DF</i> is a Na⁺-permeable non-selective cation current.

<p>Mean ± S.E.M. Vagal neurons were held at −120 mV, prepulsed to −20 mV for 200 ms and then stepped from −120 to +40 mV in 20 mV increments for 25 ms (current measured at 1 ms). The current-voltage relationship from this Na_V inactivation protocol is shown for cells bathed in “Normal Na⁺” 154 mM NaCl-containing solutions: control external solution (solid line, black squares) and solution containing 5 mM EDTA (0 Ca²⁺, 0 Mg²⁺) (solid line, grey squares; n = 15). Also depicted are the current-voltage relationships for cells bathed in “low” 100 mM NaCl-containing solutions: control external solution (dotted line, empty black circles) and solution containing 5 mM EDTA (0 Ca²⁺, 0 Mg²⁺; dotted line, empty grey circles; n = 6).</p

Chelation of external divalent cations activates a voltage-gated current in vagal neurons.

<p>Mean ± S.E.M. A, voltage steps of 10 mV increments were applied to vagal neurons held at −120 mV. Neurons were perfused with normal bath solution (Control and Recovery) or bath solution containing 5 mM EDTA with nominally 0 mM Ca²⁺ and 0 mM Mg²⁺ (EDTA). The resulting currents are depicted. B, the current-voltage relationship of the peak amplitude of the fast component of the current (n = 8 and 14 for Control (black squares) and EDTA (grey triangles) respectively). C, the current-voltage relationship of the persistent component of the current recorded at 24.5 ms after the onset of the voltage step (indicated by dotted line in inset; n = 24).</p

Chelation of external divalent cations evokes Na⁺ influx in sensory neurons.

<p>Mean ± S.E.M. Na⁺ responses of vagal neurons as measured by SBFI. Blocked line denotes the chelation of divalent cations, at all other times Ca²⁺ and Mg²⁺ are at 2.2 mM and 1.2 mM, respectively. A, the response to divalent chelation (5 mM EDTA; 0 Ca²⁺, 0 Mg²⁺) in the presence of external Na⁺ (black squares) or external NMDG⁺ (grey squares). B, the response to chelation of Ca²⁺ alone (5 mM EGTA; 0 Ca²⁺, 1.2 mM Mg²⁺; grey squares) or the response to 5 mM EDTA saturated with 7.2 mM Ca²⁺ and 1.2 mM Mg²⁺ (black outlined boxes). C, the effect of lidocaine (1 mM; grey squares) or TTX (1 µM; black outlined boxes) on the response to 5 mM EDTA (0 Ca²⁺, 0 Mg²⁺). D, the effect of a combination of ω-conotoxin (1 µM) and nifedipine (10 µM; grey squares) or ruthenium red (RR, 30 µM, black outlined boxes) on the response to 5 mM EDTA (0 Ca²⁺, 0 Mg²⁺). E, the response to 5 mM EDTA (0 Ca²⁺, 0 Mg²⁺) in vagal neurons (black squares) and HEK293 cells (grey squares).</p

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K) – Akt signalling pathways retains FoxO3a in the cytoplasm thereby inhibiting the transcriptional activation of death promoting genes. We hypothesised that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localisation of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and upregulated Fas ligand expression. In contrast the phenolic antioxidant tBHQ caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a prevented NMDA-induced upregulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration