A novel O2-sensing mechanism in rat glossopharyngeal neurones mediated by a halothane-inhibitable background K+ conductance

Modulation of K+ channels by hypoxia is a common O2-sensing mechanism in specialised cells. More recently, acid-sensitive TASK-like background K+ channels, which play a key role in setting the resting membrane potential, have been implicated in O2-sensing in certain cell types. Here, we report a novel O2 sensitivity mediated by a weakly pH-sensitive background K+ conductance in nitric oxide synthase (NOS)-positive neurones of the glossopharyngeal nerve (GPN). This conductance was insensitive to 30 mm TEA, 5 mm 4-aminopyridine (4-AP) and 200 μm Cd2+, but was reversibly inhibited by hypoxia (O2 tension (PO2) = 15 mmHg), 2–5 mm halothane, 10 mm barium and 1 mm quinidine. Notably, the presence of halothane occluded the inhibitory effect of hypoxia. Under current clamp, these agents depolarised GPN neurones. In contrast, arachidonic acid (5–10 μm) caused membrane hyperpolarisation and potentiation of the background K+ current. This pharmacological profile suggests the O2-sensitive conductance in GPN neurones is mediated by a class of background K+ channels different from the TASK family; it appears more closely related to the THIK (tandem pore domain halothane-inhibited K+) subfamily, or may represent a new member of the background K+ family. Since GPN neurones are thought to provide NO-mediated efferent inhibition of the carotid body (CB), these channels may contribute to the regulation of breathing during hypoxia via negative feedback control of CB function, as well as to the inhibitory effect of volatile anaesthetics (e.g. halothane) on respiration

Identification of a single amino acid in GluN1 that is critical for glycine-primed internalization of NMDA receptors

Abstract Background NMDA receptors are ligand-gated ion channels with essential roles in glutamatergic synaptic transmission and plasticity in the CNS. As co-receptors for glutamate and glycine, gating of the NMDA receptor/channel pore requires agonist binding to the glycine sites, as well as to the glutamate sites, on the ligand-binding domains of the receptor. In addition to channel gating, glycine has been found to prime NMDA receptors for internalization upon subsequent stimulation of glutamate and glycine sites. Results Here we address the key issue of identifying molecular determinants in the glycine-binding subunit, GluN1, that are essential for priming of NMDA receptors. We found that glycine treatment of wild-type NMDA receptors led to recruitment of the adaptor protein 2 (AP-2), and subsequent internalization after activating the receptors by NMDA plus glycine. However, with a glycine-binding mutant of GluN1 – N710R/Y711R/E712A/A714L – we found that treating with glycine did not promote recruitment of AP-2 nor were glycine-treated receptors internalized when subsequently activated with NMDA plus glycine. Likewise, GluN1 carrying a single point mutation – A714L – did not prime upon glycine treatment. Importantly, both of the mutant receptors were functional, as stimulating with NMDA plus glycine evoked inward currents. Conclusions Thus, we have identified a single amino acid in GluN1 that is critical for priming of NMDA receptors by glycine. Moreover, we have demonstrated the principle that while NMDA receptor gating and priming share a common requirement for glycine binding, the molecular constraints in GluN1 for gating are distinct from those for priming

RAGE-dependent potentiation of TRPV1 currents in sensory neurons exposed to high glucose.

Diabetes mellitus is associated with sensory abnormalities, including exacerbated responses to painful (hyperalgesia) or non-painful (allodynia) stimuli. These abnormalities are symptoms of diabetic peripheral neuropathy (DPN), which is the most common complication that affects approximately 50% of diabetic patients. Yet, the underlying mechanisms linking hyperglycemia and symptoms of DPN remain poorly understood. The transient receptor potential vanilloid 1 (TRPV1) channel plays a central role in such sensory abnormalities and shows elevated expression levels in animal models of diabetes. Here, we investigated the function of TRPV1 channels in sensory neurons cultured from the dorsal root ganglion (DRG) of neonatal mice, under control (5mM) and high glucose (25mM) conditions. After maintaining DRG neurons in high glucose for 1 week, we observed a significant increase in capsaicin (CAP)-evoked currents and CAP-evoked depolarizations, independent of TRPV1 channel expression. These functional changes were largely dependent on the expression of the receptor for Advanced Glycation End-products (RAGE), calcium influx, cytoplasmic ROS accumulation, PKC, and Src kinase activity. Like cultured neurons from neonates, mature neurons from adult mice also displayed a similar potentiation of CAP-evoked currents in the high glucose condition. Taken together, our data demonstrate that under the diabetic condition, DRG neurons are directly affected by elevated levels of glucose, independent of vascular or glial signals, and dependent on RAGE expression. These early cellular and molecular changes to sensory neurons in vitro are potential mechanisms that might contribute to sensory abnormalities that can occur in the very early stages of diabetes

High glucose did not increase expression of TRPV1 channels.

<p>A, representative images immunocytochemistry for the detection of TRPV1 protein in cultured DRG maintained in either control (CTL) or high glucose (HG) conditions. B, bar graph summarizes mean pixel intensity for CTL (n = 71; HG, n = 51). C, western blots showing TRPV1 and β-actin expression in 4 independent samples (per treatment) of cultured DRG neurons exposed to either CTL or HG for 10 days. B, bar graph summarizes the ratio of TRPV1/β-actin per treatment. All data are represented as mean ± SEM. Scale bar in A represents 30 μm. Statistical analysis by Mann<i>–</i>Whitney test; no significant differences were found between treatments (B-D). AU, arbitrary units.</p

Passive membrane properties for cultured DRG neurons from RAGE KO mice.

<p>Passive membrane properties for cultured DRG neurons from RAGE KO mice.</p

High glucose induces intracellular ROS accumulation.

<p>A, representative images of ROS detection by CM-H2DCFDA fluorescence, in cultured DRG neurons from WT and RAGE KO mice, maintained in control (CTL) and high glucose (HG) conditions for 1 week. B, the bar graph summarizes mean ± SEM pixel intensity for HG/CTL ratio for neurons from WT (n = 110) and RAGE KO (n = 156) mice, respectively. C, representative traces showing CAP-evoked currents recorded in the presence of ALA+CAT in CTL and HG conditions. D-F, bar graphs summarize maximal current (I_max) density (D), maximal current charge (Q_max; E), and current rundown calculated as the ratio I₁₅/ I_max (F). All data are represented as mean ± SEM, ALA + CAT (CTL, n = 10; HG, n = 12). Scale bar in A represent 30 μm. Statistical analysis by one sample <i>t</i>-test hypothetical mean = 1 (B), unpaired <i>t</i>-test (D-E), and Mann-Whitney test (F). ***, p < 0.001.</p

High glucose potentiated CAP-evoked currents in cultured DRG neurons.

<p>A-B, representative traces of CAP-evoked currents recorded in whole-cell voltage-clamp mode (holding potential set at -60 mV). Agonist was applied for 1 s and repeated every 30 s, in control (CTL), and high glucose (HG) in Ca²⁺-ECF (A), and in the same glucose conditions but in Ba²⁺-ECF (B), respectively. C-E, bar graphs summarizing maximal current (I_max) density (C), maximal current charge (Q_max; D), and maximal current rundown calculated as the ratio of I_max relative to the 15^th application (I₁₅) in a series (E), for CTL (n = 24) and HG (n = 24) in Ca²⁺-ECF; and for CTL (n = 8) and HG (n = 11) in Ba²⁺-ECF, respectively. All data are represented as mean ± SEM. The absolute current density and charge were used in C and D for simplicity. Statistical analysis by two-way ANOVA followed by Sidak's post-hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Passive membrane properties for cultured DRG neurons from adult wild type mice.

<p>Passive membrane properties for cultured DRG neurons from adult wild type mice.</p

High glucose failed to potentiate CAP-evoked currents in cultured DRG neurons from RAGE KO mice.

<p>A-B, representative traces of CAP-evoked currents recorded during whole-cell voltage-clamp recording (holding potential set at -60 mV) from DRG neurons maintained in either control (CTL) or high glucose (HG) conditions. C-E, bar graphs summarize maximal current (I_max) density (C), maximal current charge (Q_max; D), and current rundown calculated as the ratio I₁₅/ I_max (E). All data are represented as mean ± SEM, n = 14 for each condition. The absolute current density and charge were used in C and D for simplicity. Statistical analysis by unpaired <i>t</i>-test (C-D), and Mann-Whitney test (E); no significant differences between treatments were found.</p