Deletion Of Channel Research Articles

Molecular mechanism of GIRK2 channel gating modulated by cholesteryl hemisuccinate.

Cholesterol, an essential lipid of cell membranes, regulates G protein-gated inwardly rectifying potassium (GIRK) channel activity. Previous studies have shown that cholesterol activates GIRK2 homotetrameric channels, which are expressed in dopaminergic neurons of the brain. Deletion of GIRK2 channels affects both GIRK2 homo- and heterotetrames and can lead to abnormal neuronal excitability, including conditions such as epilepsy and addiction. A 3.5Å cryo-EM structure of GIRK2 in complex with CHS (cholesteryl hemisuccinate) and PIP2 (phosphatidylinositol 4,5-bisphosphate) has been solved. This structure provides the opportunity to study GIRK2 channel gating dynamics regulated by cholesterol using gating molecular dynamics (GMD) simulations. In the present study, we conducted microsecond-long GMD simulations on the GIRK2 channel in its APO, PIP2, and PIP2/CHS bound states, followed by systematic analysis to gain molecular insights into how CHS modulates GIRK2 channel gating. We found that CHS binding facilitates GIRK2 channel opening, with 43 K+ ion permeation events observed, compared to 0 and 2 K+ ion permeation events for GIRK2-APO and GIRK2/PIP2, respectively. Binding of CHS to the GIRK2 channel enhances PIP2 and channel interactions, which is consistent with previous experimental results. The negatively charged PIP2 alters the internal electrostatic potential field in the channel and lowers the negative free energy barrier for K+ ion permeation.

Nociceptive TRP channels function as molecular target for several antifungal drugs.

Topically applied antifungal agents can induce adverse effects, such as pain and irritation. The transient receptor potential (TRP) channels-TRPA1 and TRPV1-mainly expressed in sensory neurons, act as sensors for detecting irritants. This study aims to evaluate the involvement of nociceptive channels in topical antifungal-induced pain and irritation. We tested nine topical antifungals belonging five classes: isoconazole, econazole, miconazole, clotrimazole, and ketoconazole as imidazoles; liranaftate as a thiocarbamate; terbinafine as an allylamine; amorolfine as a morpholine; and butenafine as a benzylamine. Intracellular calcium concentrations ([Ca2+]i) and membrane currents in response to antifungals were measured to estimate channel activity using heterologously expressing cells and isolated mouse sensory neurons. In mouse TRPA1-expressing cells, all the tested drugs induced an increase in [Ca2+]i, which was abrogated or reduced by a TRPA1 blocker. Although many drugs evoked the TRPA1-nonspecific [Ca2+]i response at high concentrations, responses to clotrimazole, ketoconazole, and liranaftate were TRPA1 specific and elicited current responses in TRPA1-expressing cells. In mouse TRPV1-expressing cells, clotrimazole and ketoconazole elicited [Ca2+]i and current responses. In mouse sensory neurons, liranaftate-induced increase in [Ca2+]i was abrogated by a TRPA1 blocker and Trpa1 deletion. Responses to ketoconazole were inhibited by TRPA1 and TRPV1 blockers and by the genetic deletion of either channel. These results suggest that topical antifungal-induced pain and irritation are attributable to the activation of nociceptive TRPA1 and/or TRPV1 channel/s. Consequently, caution should be exercised in the use of topical antifungals with symptoms of pain.

Mouse brain elastography changes with sleep/wake cycles, aging, and Alzheimer's disease

Understanding the physiological processes in aging and how neurodegenerative disorders affect cognitive function is a high priority for advancing human health. One specific area of recently enabled research is the in vivo biomechanical state of the brain. This study utilized reverberant optical coherence elastography, a high-resolution elasticity imaging method, to investigate stiffness changes during the sleep/wake cycle, aging, and Alzheimer's disease in murine models. Four-dimensional scans of 44 wildtype mice, 13 mice with deletion of aquaporin-4 water channel, and 12 mice with Alzheimer-related pathology (APP/PS1) demonstrated that (1) cortical tissue became softer (on the order of a 10% decrease in shear wave speed) when young wildtype mice transitioned from wake to anesthetized, yet this effect was lost in aging and with mice overexpressing amyloid-β or lacking the water channel AQP4. (2) Cortical stiffness increased with age in all mice lines, but wildtype mice exhibited the most prominent changes as a function of aging. The study provides novel insight into the brain's biomechanics, the constraints of fluid flow, and how the state of brain activity affects basic properties of cortical tissues.

Schwann cell TRPA1 elicits reserpine-induced fibromyalgia pain in mice.

Fibromyalgia is a complex clinical disorder with an unknown aetiology, characterized by generalized pain and co-morbid symptoms such as anxiety and depression. An imbalance of oxidants and antioxidants is proposed to play a pivotal role in the pathogenesis of fibromyalgia symptoms. However, the precise mechanisms by which oxidative stress contributes to fibromyalgia-induced pain remain unclear. The transient receptor potential ankyrin 1 (TRPA1) channel, known as both a pain sensor and an oxidative stress sensor, has been implicated in various painful conditions. The feed-forward mechanism that implicates reactive oxygen species (ROS) driven by TRPA1 was investigated in a reserpine-induced fibromyalgia model in C57BL/6J mice employing pharmacological interventions and genetic approaches. Reserpine-treated mice developed pain-like behaviours (mechanical/cold hypersensitivity) and early anxiety-depressive-like disorders, accompanied by increased levels of oxidative stress markers in the sciatic nerve tissues. These effects were not observed upon pharmacological blockade or global genetic deletion of the TRPA1 channel and macrophage depletion. Furthermore, we demonstrated that selective silencing of TRPA1 in Schwann cells reduced reserpine-induced neuroinflammation (NADPH oxidase 1-dependent ROS generation and macrophage increase in the sciatic nerve) and attenuated fibromyalgia-like behaviours. Activated Schwann cells expressing TRPA1 promote an intracellular pathway culminating in the release of ROS and recruitment of macrophages in the mouse sciatic nerve. These cellular and molecular events sustain mechanical and cold hypersensitivity in the reserpine-evoked fibromyalgia model. Targeting TRPA1 channels on Schwann cells could offer a novel therapeutic approach for managing fibromyalgia-related behaviours.

Opioid-Induced Hyperalgesia and Tolerance Are Driven by HCN Ion Channels.

Prolonged exposure to opioids causes an enhanced sensitivity to painful stimuli (opioid-induced hyperalgesia, OIH) and a need for increased opioid doses to maintain analgesia (opioid-induced tolerance, OIT), but the mechanisms underlying both processes remain obscure. We found that pharmacological block or genetic deletion of HCN2 ion channels in primary nociceptive neurons of male mice completely abolished OIH but had no effect on OIT. Conversely, pharmacological inhibition of central HCN channels alleviated OIT but had no effect on OIH. Expression of C-FOS, a marker of neuronal activity, was increased in second-order neurons of the dorsal spinal cord by induction of OIH, and the increase was prevented by peripheral block or genetic deletion of HCN2, but block of OIT by spinal block of HCN channels had no impact on C-FOS expression in dorsal horn neurons. Collectively, these observations show that OIH is driven by HCN2 ion channels in peripheral nociceptors, while OIT is driven by a member of the HCN family located in the CNS. Induction of OIH increased cAMP in nociceptive neurons, and a consequent shift in the activation curve of HCN2 caused an increase in nociceptor firing. The shift in HCN2 was caused by expression of a constitutively active μ-opioid receptor (MOR) and was reversed by MOR antagonists. We identified the opioid-induced MOR as a six-transmembrane splice variant, and we show that it increases cAMP by coupling constitutively to Gs HCN2 ion channels therefore drive OIH, and likely OIT, and may be a novel therapeutic target for the treatment of addiction.

Deletion of voltage-gated calcium channels in astrocytes decreases neuroinflammation and demyelination in a murine model of multiple sclerosis

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.

BK Channel Depletion Promotes Adipocyte Differentiation by Activating the MAPK/ERK Pathway.

The expression of large conductance calcium-activated potassium channels (BK channels) in adipose tissue has been identified for years. BK channel deletion can improve metabolism in vivo, but the relative mechanisms remain unclear. Here, we examined the effects of BK channels on the differentiation of adipose-derived stem cells (ADSCs) and the related mechanisms. BKα and β1 subunits were expressed on adipocytes. We found that both deletion of the KCNMA1 gene, encoding the pore forming α subunit of BK channels, and the BK channel inhibitor paxilline increased the expression of key genes in the peroxisome proliferator activated receptor (PPAR) pathway and promoted adipogenetic differentiation of ADSCs. We also observed that the MAPK-ERK pathway participates in BK channel deficiency-promoted adipogenic differentiation of ADSCs and that ERK inhibitors blocked the differentiation-promoting effect of BK channel deficiency. Hyperplasia of adipocytes is considered beneficial for metabolic health. These results indicate that BK channels play an important role in adipose hyperplasia by regulating the differentiation of ADSCs and may become an important target for studying the pathogenesis and treatment strategies of metabolic disorder-related diseases.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wntless (Wls), a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulates the expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and β-catenin-deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/β-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.NEW & NOTEWORTHY Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

Heterogeneous off-target impact of ion-channel deletion on intrinsic properties of hippocampal model neurons that self-regulate calcium.

How do neurons that implement cell-autonomous self-regulation of calcium react to knockout of individual ion-channel conductances? To address this question, we used a heterogeneous population of 78 conductance-based models of hippocampal pyramidal neurons that maintained cell-autonomous calcium homeostasis while receiving theta-frequency inputs. At calcium steady-state, we individually deleted each of the 11 active ion-channel conductances from each model. We measured the acute impact of deleting each conductance (one at a time) by comparing intrinsic electrophysiological properties before and immediately after channel deletion. The acute impact of deleting individual conductances on physiological properties (including calcium homeostasis) was heterogeneous, depending on the property, the specific model, and the deleted channel. The underlying many-to-many mapping between ion channels and properties pointed to ion-channel degeneracy. Next, we allowed the other conductances (barring the deleted conductance) to evolve towards achieving calcium homeostasis during theta-frequency activity. When calcium homeostasis was perturbed by ion-channel deletion, post-knockout plasticity in other conductances ensured resilience of calcium homeostasis to ion-channel deletion. These results demonstrate degeneracy in calcium homeostasis, as calcium homeostasis in knockout models was implemented in the absence of a channel that was earlier involved in the homeostatic process. Importantly, in reacquiring homeostasis, ion-channel conductances and physiological properties underwent heterogenous plasticity (dependent on the model, the property, and the deleted channel), even introducing changes in properties that were not directly connected to the deleted channel. Together, post-knockout plasticity geared towards maintaining homeostasis introduced heterogenous off-target effects on several channels and properties, suggesting that extreme caution be exercised in interpreting experimental outcomes involving channel knockouts.

The impact of HCN4 channels on CNS brain networks as a new target in pain development.

While it is well established that the isoform 2 of the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN2) plays an important role in the development and maintenance of pain, the role of the closely related HCN4 isoform in the processing of nociceptive signals is not known. HCN4 channels are highly expressed in the thalamus, a region important for stimulus transmission and information processing. We used a brain-specific HCN4-knockout mouse line (HCN4-KO) to explore the role of HCN4 channels in acute nociceptive processing using several behavioral tests as well as a multimodal magnetic resonance imaging (MRI) approach. Functional MRI (fMRI) brain responses were measured during acute peripheral thermal stimulation complemented by resting state (RS) before and after stimulation. The data were analyzed by conventional and graph-theoretical approaches. Finally, high-resolution anatomical brain data were acquired. HCN4-KO animals showed a central thermal, but not a mechanical hypersensitivity in behavioral experiments. The open field analysis showed no significant differences in motor readouts between HCN4-KO and controls but uncovered increased anxiety in the HCN4-KO mice. Thermal stimulus-driven fMRI (s-fMRI) data revealed increased response volumes and response amplitudes for HCN4-KO, most pronounced at lower stimulation temperatures in the subcortical input, the amygdala as well as in limbic/hippocampal regions, and in the cerebellum. These findings could be cross-validated by graph-theoretical analyses. Assessment of short-term RS before and after thermal stimulation revealed that stimulation-related modulations of the functional connectivity only occurred in control animals. This was consistent with the finding that the hippocampus was found to be smaller in HCN4-KO. In summary, the deletion of HCN4 channels impacts on processing of acute nociception, which is remarkably manifested as a thermal hypersensitive phenotype. This was mediated by the key regions hypothalamus, somatosensory cortex, cerebellum and the amygdala. As consequence, HCN4-KO mice were more anxious, and their brain-wide RS functional connectivity could not be modulated by thermal nociceptive stimulation.

Abstract WP240: TRPM2 Channel Deletion In Neurons Reduces Injury Following Ischemic Stroke And Improves Post-stroke Cognitive Function

Background: Emerging evidence implicates post-stroke cognitive impairment as a major contributor to long-term disability. Therefore, optimal therapeutic targets reduce acute ischemic injury and enhance post-stroke brain function. Strong data demonstrates that a novel TRPM2 channel antagonist (tat-M2NX) provides neuroprotection and improves synaptic function, thereby reducing post-stroke cognitive impairment (PSCI). Hypothesis: Knockout of neuron-specific TRPM2 channel expression reduces infarct volume and enhances functional recovery following MCAO Methods: Transient MCAO (60 min) was performed on adult (8-10 week) male and female TRPM2 neuron-specific KO (TRPM2fl/fl, CaMKII Cre) and TRPM2 floxed controls (TRPM2fl/fl)Hemispheric infarct volume analyzed from MRI (T2) images 3 days post-injury by a blinded investigator. Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 7 days after recovery from MCAO to analyze synaptic placticity (LTP). Results: We observed that neuronal-specific TRPM2 channel knockout reduces acute ischemic injury (infarct volume) in male animals, while having minimal effect on female mice. Consistent with the hypothesis that neuronal TRPM2 channels contribute to ischemia-induced synaptic dysfunction, recordings obtained in brain slices from neuronal TRPM2 channel KO mice (TRPM2fl/fl-CaMKII CRE) mice 7 days after recovery from 60 min MCAo exhibited intact hippocampal plasticity compared to control TRPM2fl/fl mice not expressing CRE. Male sham control mice exhibit robust LTP of 163±10.4% (n=3) compared to 115±5.6% (n=4) in TRPM2fl/fl mice after MCAO. Neuronal KO exhibited 175±9.8% (n=2). Similarly, female mice had control LTP of 170±6.8% (n=4) compared to 125±6.8% (n=3) in TRPM2fl/fl mice after MCAO. Neuronal KO exhibited 208±7.8% (n=5, p<0.05 ANOVA compared to TRPM2fl/fl MCAO). Conclusion: Our data highlight that TRPM2 channels expressed in neurons contribute to both acute injury following transient ischemic stroke and subacute/chronic functional recovery.

Kidney and blood pressure regulation-latest evidence for molecular mechanisms.

Hypertension is one of the major health problems leading to the development of cardiovascular diseases. Despite a rapid expansion in global hypertension prevalence, molecular mechanisms leading to hypertension are not fully understood largely due to the complexity of pathogenesis involving several factors. Salt intake is recognized as a leading determinant of blood pressure, since reduced dietary salt intake is related to lower morbidity and mortality, and hypertension in relation to cardiovascular events. Compared with salt-resistant populations, salt-sensitive individuals exhibit high sensitivity in blood pressure responses according to changes in salt intake. In this setting, the kidney plays a major role in the maintenance of blood pressure under the hormonal control of the renin-angiotensin-aldosterone system. In the present review, we summarize the current overview on the molecular mechanisms for modulation of blood pressure associated with renal ion channels/transporters including sodium-hydrogen exchanger isoform 3 (NHE3), Na+-K+-2Cl- cotransporter (NKCC2), sodium-chloride cotransporter (NCC), epithelial sodium channel (ENaC) and pendrin expressed in different nephron segments. In particular, recent studies on experimental animal models with deletion of renal ion channels led to the identification of several crucial physiological mechanisms and molecules involved in hypertension. These findings could further provide a potential for novel therapeutic approaches applicable on human patients with hypertension.

Macrophages and glia are the dominant P2X7-expressing cell types in the gut nervous system—No evidence for the role of neuronal P2X7 receptors in colitis

The blockade or deletion of the pro-inflammatory P2X7 receptor channel has been shown to reduce tissue damage and symptoms in models of inflammatory bowel disease, and P2X7 receptors on enteric neurons were suggested to mediate neuronal death and associated motility changes. Here, we used P2X7-specific antibodies and nanobodies, as well as a bacterial artificial chromosome transgenic P2X7-EGFP reporter mouse model and P2rx7-/- controls to perform a detailed analysis of cell type-specific P2X7 expression and possible overexpression effects in the enteric nervous system of the distal colon. In contrast to previous studies, we did not detect P2X7 in neurons but found dominant expression in glia and macrophages, which closely interact with the neurons. The overexpression of P2X7 per se did not induce significant pathological effects. Our data indicate that macrophages and/or glia account for P2X7-mediated neuronal damage in inflammatory bowel disease and provide a refined basis for the exploration of P2X7-based therapeutic strategies.

The absence of AQP4/TRPV4 complex substantially reduces acute cytotoxic edema following ischemic injury.

Astrocytic Aquaporin 4 (AQP4) and Transient receptor potential vanilloid 4 (TRPV4) channels form a functional complex that likely influences cell volume regulation, the development of brain edema, and the severity of the ischemic injury. However, it remains to be fully elucidated whether blocking these channels can serve as a therapeutic approach to alleviate the consequences of having a stroke. In this study, we used in vivo magnetic resonance imaging (MRI) to quantify the extent of brain lesions one day (D1) and seven days (D7) after permanent middle cerebral artery occlusion (pMCAO) in AQP4 or TRPV4 knockouts and mice with simultaneous deletion of both channels. Our results showed that deletion of AQP4 or TRPV4 channels alone leads to a significant worsening of ischemic brain injury at both time points, whereas their simultaneous deletion results in a smaller brain lesion at D1 but equal tissue damage at D7 when compared with controls. Immunohistochemical analysis 7 days after pMCAO confirmed the MRI data, as the brain lesion was significantly greater in AQP4 or TRPV4 knockouts than in controls and double knockouts. For a closer inspection of the TRPV4 and AQP4 channel complex in the development of brain edema, we applied a real-time iontophoretic method in situ to determine ECS diffusion parameters, namely volume fraction (α) and tortuosity (λ). Changes in these parameters reflect alterations in cell volume, and tissue structure during exposure of acute brain slices to models of ischemic conditions in situ, such as oxygen-glucose deprivation (OGD), hypoosmotic stress, or hyperkalemia. The decrease in α was comparable in double knockouts and controls when exposed to hypoosmotic stress or hyperkalemia. However, during OGD, there was no decrease in α in the double knockouts as observed in the controls, which suggests less swelling of the cellular components of the brain. Although simultaneous deletion of AQP4 and TRPV4 did not improve the overall outcome of ischemic brain injury, our data indicate that the interplay between AQP4 and TRPV4 channels plays a critical role during neuronal and non-neuronal swelling in the acute phase of ischemic injury.

Deletion of large-conductance calcium-activated potassium channels promotes vascular remodelling through the CTRP7-mediated PI3K/Akt signaling pathway.

The large-conductance calcium-activated potassium (BK) channel is a critical regulator and potential therapeutic target of vascular tone and architecture, and abnormal expression or dysfunction of this channel is linked to many vascular diseases. Vascular remodelling is the early pathological basis of severe vascular diseases. Delaying the progression of vascular remodelling can reduce cardiovascular events, but the pathogenesis remains unclear. To clarify the role of BK channels in vascular remodelling, we use rats with BK channel α subunit knockout (BK α ‒/‒). The results show that BK α ‒/‒ rats have smaller inner and outer diameters, thickened aortic walls, increased fibrosis, and disordered elastic fibers of the aortas compared with WT rats. When the expression and function of BK α are inhibited in human umbilical arterial smooth muscle cells (HUASMCs), the expressions of matrix metalloproteinase 2 (MMP2), MMP9, and interleukin-6 are enhanced, while the expressions of smooth muscle cell contractile phenotype proteins are reduced. RNA sequencing, bioinformatics analysis and qPCR verification show that C1q/tumor necrosis factor-related protein 7 ( CTRP7) is the downstream target gene. Furthermore, except for that of MMPs, a similar pattern of IL-6, smooth muscle cell contractile phenotype proteins expression trend is observed after CTRP7 knockdown. Moreover, knockdown of both BK α and CTRP7 in HUASMCs activates PI3K/Akt signaling. Additionally, CTRP7 is expressed in vascular smooth muscle cells (VSMCs), and BK α deficiency activates the PI3K/Akt pathway by reducing CTRP7 level. Therefore, we first show that BK channel deficiency leads to vascular remodelling. The BK channel and CTRP7 may serve as potential targets for the treatment of cardiovascular diseases.

Intrinsic Adrenal TWIK-Related Acid-Sensitive TASK Channel Dysfunction Produces Spontaneous Calcium Oscillations Sufficient to Drive AngII (Angiotensin II)-Unresponsive Hyperaldosteronism.

Ion channel mutations in calcium regulating genes strongly associate with AngII (angiotensin II)-independent aldosterone production. Here, we used an established mouse model of in vivo aldosterone autonomy, Cyp11b2-driven deletion of TWIK-related acid-sensitive potassium channels (TASK-1 and TASK-3, termed zona glomerulosa [zG]-TASK-loss-of-function), and selective pharmacological TASK channel inhibition to determine whether channel dysfunction in native, electrically excitable zG cell rosette-assemblies: (1) produces spontaneous calcium oscillatory activity and (2) is sufficient to drive substantial aldosterone autonomy. We imaged calcium activity in adrenal slices expressing a zG-specific calcium reporter (GCaMP3), an in vitro experimental approach that preserves the native rosette assembly and removes potentially confounding extra-adrenal contributions. In parallel experiments, we measured acute aldosterone production from adrenal slice cultures. Absent from untreated WT slices, we find that either adrenal-specific genetic deletion or acute pharmacological TASK channel inhibition produces spontaneous oscillatory bursting behavior and steroidogenic activity (2.4-fold) that are robust, sustained, and equivalent to activities evoked by 3 nM AngII in WT slices. Moreover, spontaneous activity in zG-TASK-loss-of-function slices and inhibitor-evoked activity in WT slices are unresponsive to AngII regulation over a wide range of concentrations (50 pM to 3 µM). We provide proof of principle that spontaneous activity of zG cells within classic rosette assemblies evoked solely by a change in an intrinsic, dominant resting-state conductance can be a significant source of AngII-independent aldosterone production from native tissue.

The Slack Channel Deletion Causes Mechanical Pain Hypersensitivity in Mice

The role of the Slack (also known as Slo2.2, KNa1.1, or KCNT1) channel in pain-sensing is still in debate on which kind of pain it regulates. In the present study, we found that the Slack–/– mice exhibited decreased mechanical pain threshold but normal heat and cold pain sensitivity. Subsequently, X-gal staining, in situ hybridization, and immunofluorescence staining revealed high expression of the Slack channel in Isolectin B4 positive (IB4+) neurons in the dorsal root ganglion (DRG) and somatostatin-positive (SOM+) neurons in the spinal cord. Patch-clamp recordings indicated the firing frequency was increased in both small neurons in DRG and spinal SOM+ neurons in the Slack–/– mice whereas no obvious slow afterhyperpolarization was observed in both WT mice and Slack–/– mice. Furthermore, we found Kcnt1 gene expression in spinal SOM+ neurons in Slack–/– mice partially relieved the mechanical pain hypersensitivity of Slack–/– mice and decreased AP firing rates of the spinal SOM+ neurons. Finally, deletion of the Slack channel in spinal SOM+ neurons is sufficient to result in mechanical pain hypersensitivity in mice. In summary, our results suggest the important role of the Slack channel in the regulation of mechanical pain-sensing both in small neurons in DRG and SOM+ neurons in the spinal dorsal horn.

The Slack Channel Regulates Anxiety-Like Behaviors via Basolateral Amygdala Glutamatergic Projections to Ventral Hippocampus.

Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. In the present study, we find that the global Slack KO male mice exhibit anxious behaviors, whereas the Slack Y777H male mice manifest anxiolytic behaviors. The expression of Slack channels is rich in basolateral amygdala (BLA) glutamatergic neurons and downregulated in chronic corticosterone-treated mice. In addition, electrophysiological data show enhanced excitability of BLA glutamatergic neurons in the Slack KO mice and decreased excitability of these neurons in the Slack Y777H mice. Furthermore, the Slack channel deletion in BLA glutamatergic neurons is sufficient to result in enhanced avoidance behaviors, whereas Kcnt1 gene expression in the BLA or BLA-ventral hippocampus (vHPC) glutamatergic projections reverses anxious behaviors of the Slack KO mice. Our study identifies the role of the Slack channel in controlling anxious behaviors by decreasing the excitability of BLA-vHPC glutamatergic projections, providing a potential target for anxiolytic therapies.SIGNIFICANCE STATEMENT Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. Here, we examined the behaviors of loss- and gain-of-function of Slack channel mice in elevated plus maze and open field tests and found the anxiolytic role of the Slack channel. By altering the Slack channel expression in the specific neuronal circuit, we demonstrated that the Slack channel played its anxiolytic role by decreasing the excitability of BLA-vHPC glutamatergic projections. Our data reveal the role of the Slack channel in the regulation of anxiety, which may provide a potential molecular target for anxiolytic therapies.

20-hydroxyeicosatetraenoic acid (20-HETE) is a pivotal endogenous ligand for TRPV1-mediated neurogenic inflammation in the skin.

Transient receptor potential cation channel subfamily V member 1 (TRPV1) is localized to sensory C-fibres and its opening leads to membrane depolarization, resulting in neuropeptide release and neurogenic inflammation. However, the identity of the endogenous activator of TRPV1 in this setting is unknown. The arachidonic acid metabolites 12-hydroperoxyeicosatetraenoyl acid (12-HpETE) and 20-hydroxyeicosatetraenoic acid (20-HETE) have emerged as potential endogenous activators of TRPV1. However, whether these lipids underlie TRPV1-mediated neurogenic inflammation remains unknown. We analysed human cantharidin-induced blister samples and inflammatory responses in TRPV1 transgenic mice. In a human cantharidin-blister model, the potent TRPV1 activators 20-HETE but not 12-HETE (stable metabolite of 12-HpETE) correlated with arachidonic acid levels. Similarly, in mice, levels of 20-HETE (but not 12-HETE) and arachidonic acid were strongly positively correlated within the inflammatory milieu. Furthermore, LPS-induced oedema formation and neutrophil recruitment were substantially and significantly attenuated by pharmacological block or genetic deletion of TRPV1 channels, inhibition of 20-HETE formation or SP receptor neurokinin 1 (NK1 ) blockade. LPS treatment also increased cytochrome P450 ω-hydroxylase gene expression, the enzyme responsible for 20-HETE production. Taken together, our findings suggest that endogenously generated 20-HETE activates TRPV1 causing C-fibre activation and consequent oedema formation. These findings identify a novel pathway that may be useful in the therapeutics of diseases/conditions characterized by a prominent neurogenic inflammation, as in several skin diseases.

Hyperpolarization-activated cyclic nucleotide-gated channels working as pacemaker channels in colonic interstitial cells of Cajal.

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels function as pacemaker channels in spontaneously active cells. We studied the existence of HCN channels and their functional roles in the interstitial cells of Cajal (ICC) from the mouse colon using electrophysiological, immunohistochemical and molecular techniques. HCN1 and HCN3 channels were detected in anoctamin‐1 (Ca2+‐activated Cl− channel; ANO1)‐positive cells within the muscular and myenteric layers in colonic tissues. The mRNA transcripts of HCN1 and HCN3 channels were expressed in ANO1‐positive ICC. In the deletion of HCN1 and HCN3 channels in colonic ICC, the pacemaking potential frequency was reduced. Basal cellular adenylate cyclase activity was decreased by adenylate cyclase inhibitor in colonic ICC, whereas cAMP‐specific phosphodiesterase inhibitors increased it. 8‐Bromo‐cyclic AMP and rolipram increased spontaneous intracellular Ca2+ oscillations. In addition, Ca2+‐dependent adenylate cyclase 1 (AC1) mRNA was detected in colonic ICC. Sulprostone, a PGE2‐EP3 agonist, increased the pacemaking potential frequency, maximum rate of rise of resting membrane in pacemaker potentials and basal cellular adenylate cyclase activity in colonic ICC. These results indicate that HCN channels exist in colonic ICC and participate in generating pacemaking potentials. Thus, HCN channels may be therapeutic targets in disturbed colonic motility disorders.