Nalcn Is a "Leak" Sodium Channel That Regulates Excitability of Brainstem Chemosensory Neurons and Breathing.

UNLABELLED: The activity of background potassium and sodium channels determines neuronal excitability, but physiological roles for "leak" Na(+) channels in specific mammalian neurons have not been established. Here, we show that a leak Na(+) channel, Nalcn, is expressed in the CO2/H(+)-sensitive neurons of the mouse retrotrapezoid nucleus (RTN) that regulate breathing. In RTN neurons, Nalcn expression correlated with higher action potential discharge over a more alkalized range of activity; shRNA-mediated depletion of Nalcn hyperpolarized RTN neurons, and reduced leak Na(+) current and firing rate. Nalcn depletion also decreased RTN neuron activation by the neuropeptide, substance P, without affecting pH-sensitive background K(+) currents or activation by a cotransmitter, serotonin. In vivo, RTN-specific knockdown of Nalcn reduced CO2-evoked neuronal activation and breathing; hypoxic hyperventilation was unchanged. Thus, Nalcn regulates RTN neuronal excitability and stimulation by CO2, independent of direct pH sensing, potentially contributing to respiratory effects of Nalcn mutations; transmitter modulation of Nalcn may underlie state-dependent changes in breathing and respiratory chemosensitivity. SIGNIFICANCE STATEMENT: Breathing is an essential, enduring rhythmic motor activity orchestrated by dedicated brainstem circuits that require tonic excitatory drive for their persistent function. A major source of drive is from a group of CO2/H(+)-sensitive neurons in the retrotrapezoid nucleus (RTN), whose ongoing activity is critical for breathing. The ionic mechanisms that support spontaneous activity of RTN neurons are unknown. We show here that Nalcn, a unique channel that generates "leak" sodium currents, regulates excitability and neuromodulation of RTN neurons and CO2-stimulated breathing. Thus, this work defines a specific function for this enigmatic channel in an important physiological context.

Silencing the α2 subunit of γ-aminobutyric acid type A receptors in rat dorsal root ganglia reveals its major role in antinociception posttraumatic nerve injury.

BACKGROUND: Neuropathic pain (NPP) is likely the result of repetitive high-frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn. Drugs that promote γ-aminobutyric acid (GABA) activity in the dorsal horn provide partial relief of neuropathic symptoms. The authors examined how in vivo silencing of the GABA receptor type A (GABAA) α2 gene in dorsal root ganglia (DRG) controls NPP. METHODS: After crush injury to the right sciatic nerve of female rats, the α2 GABAA antisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG. In vivo behavioral assessment of nociception was conducted before the injury and ensuing 10 days (n = 4 to 10). In vitro quantification of α2 GABAA protein and electrophysiological studies of GABAA currents were performed on acutely dissociated L5 DRG neurons at relevant time points (n = 6 to 14). RESULTS: NPP postcrush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α2 subunit expression in the ipsilateral DRG (approximately 30%). Selective down-regulation of α2 expression in DRGs significantly worsens mechanical (2.55 ± 0.75 to 5.16 ± 1.16) and thermal (7.97 ± 0.96 to 5.51 ± 0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33 ± 0.40 to 5.00 ± 0.33) and thermal (10.80 ± 0.29 to 7.34 ± 0.81) hypersensitivity in sham animals (data shown as mean ± SD). Conversely, up-regulation of endogenous GABA via blockade of its uptake in DRG alleviates NPP. CONCLUSION: The GABAA receptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represents promising target for novel pain therapies.

S-nitrosylation inhibits pannexin 1 channel function.

S-nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1-1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1(C40A), Panx1(C346A), and Panx1(C426A)). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1(C40A/C346A)) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.

Hematopoietic pannexin 1 function is critical for neuropathic pain.

Neuropathic pain symptoms respond poorly to available therapeutics, with most treated patients reporting unrelieved pain and significant impairment in daily life. Here, we show that Pannexin 1 (Panx1) in hematopoietic cells is required for pain-like responses following nerve injury in mice, and a potential therapeutic target. Panx1 knockout mice (Panx1-/-) were protected from hypersensitivity in two sciatic nerve injury models. Bone marrow transplantation studies show that expression of functional Panx1 in hematopoietic cells is necessary for mechanical hypersensitivity following nerve injury. Reconstitution of irradiated Panx1 knockout mice with hematopoietic Panx1-/- cells engineered to re-express Panx1 was sufficient to recover hypersensitivity after nerve injury; this rescue required expression of a Panx1 variant that can be activated by G protein-coupled receptors (GPCRs). Finally, chemically distinct Panx1 inhibitors blocked development of nerve injury-induced hypersensitivity and partially relieved this hypersensitivity after it was established. These studies indicate that Panx1 expressed in immune cells is critical for pain-like effects following nerve injury in mice, perhaps via a GPCR-mediated activation mechanism, and suggest that inhibition of Panx1 may be useful in treating neuropathic pain.

PHYSIOLOGY. Regulation of breathing by CO2 requires the proton-activated receptor GPR4 in retrotrapezoid nucleus neurons.

Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H(+) and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency, and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2 (K(2P)5), a pH-sensitive K(+) channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel, and essential central mediators of respiratory chemosensitivity.

Ion channel profile of TRPM8 cold receptors reveals a role of TASK-3 potassium channels in thermosensation.

Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold-sensitive neurons, combining bacterial artificial chromosome (BAC) transgenesis with a molecular-profiling approach in fluorescence-activated cell sorting (FACS)-purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3, and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity.

Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane.

Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.