Danshensu reduces neuronal excitability by enhancing potassium currents in bushy cells in the mouse cochlear nucleus.

Danshensu, also known as salvianic acid A, is a primary active compound extracted from a traditional Chinese herb Danshen (Salvia miltiorrhiza). While its antioxidative and neuroprotective effects are well-documented, the underlying mechanisms are poorly understood. In this study, we sought out to investigate if and how Danshensu modulates neuronal excitability and voltage-gated ionic currents in the central nervous system. We prepared brain slices of the mouse brainstem and performed patch-clamp recording in bushy cells in the anteroventral cochlear nucleus, with or without Danshensu incubation for 1 h. QX-314 was used internally to block Na+ current, while tetraethylammonium and 4-aminopyridine were used to isolate different subtypes of K+ current. We found that Danshensu of 100 µm decreased the input resistance of bushy cells by approximately 60% and shifted the voltage threshold of spiking positively by approximately 7 mV, resulting in significantly reduced excitability. Furthermore, we found this reduced excitability by Danshensu was caused by enhanced voltage-gated K+ currents in these neurons, including both low voltage-activated IK,A, by approximately 100%, and high voltage-activated IK,dr, by approximately 30%. Lastly, we found that the effect of Danshensu on K+ currents was dose-dependent in that no enhancement was found for Danshensu of 50 µm and Danshensu of 200 µm failed to cause significantly more enhancement on K+ currents when compared to that of 100 µm. We found that Danshensu reduced neuronal excitability in the central nervous system by enhancing voltage-gated K+ currents, providing mechanistic support for its neuroprotective effect widely seen in vivo.

Cannabidiol potentiates hyperpolarization-activated cyclic nucleotide-gated (HCN4) channels.

Cannabidiol (CBD), the main non-psychotropic phytocannabinoid produced by the Cannabis sativa plant, blocks a variety of cardiac ion channels. We aimed to identify whether CBD regulated the cardiac pacemaker channel or the hyperpolarization-activated cyclic nucleotide-gated channel (HCN4). HCN4 channels are important for the generation of the action potential in the sinoatrial node of the heart and increased heart rate in response to ß-adrenergic stimulation. HCN4 channels were expressed in HEK 293T cells, and the effect of CBD application was examined using a whole-cell patch clamp. We found that CBD depolarized the V1/2 of activation in holo-HCN4 channels, with an EC50 of 1.6 µM, without changing the current density. CBD also sped activation kinetics by approximately threefold. CBD potentiation of HCN4 channels occurred via binding to the closed state of the channel. We found that CBD's mechanism of action was distinct from cAMP, as CBD also potentiated apo-HCN4 channels. The addition of an exogenous PIP2 analog did not alter the ability of CBD to potentiate HCN4 channels, suggesting that CBD also acts using a unique mechanism from the known HCN4 potentiator PIP2. Lastly, to gain insight into CBD's mechanism of action, computational modeling and targeted mutagenesis were used to predict that CBD binds to a lipid-binding pocket at the C-terminus of the voltage sensor. CBD represents the first FDA-approved drug to potentiate HCN4 channels, and our findings suggest a novel starting point for drug development targeting HCN4 channels.

Diazoxide attenuates DOX-induced cardiotoxicity in cultured rat myocytes.

Doxorubicin (DOX)-induced cardiotoxicity is a well known clinical problem, and many investigations have been made of its possible amelioration. We have investigated whether diazoxide (DIA), an agonist at mitochondrial ATP-sensitive potassium channels (mitoKATP), could reverse DOX-induced apoptotic myocardial cell loss, in cultured rat cardiomyocytes. The role of certain proteins in this pathway was also studied. The rat cardiomyocyte cell line (H9c2) was treated with DOX, and also co-treated with DOX and DIA, for 24 h. Distribution of actin filaments, mitochondrial membrane potential, superoxide dismutase (SOD) activity, total oxidant and antioxidant status (TOS and TAS, respectively), and some protein expressions, were assessed. DOX significantly decreased SOD activity, increased ERK1/2 protein levels, and depolarised the mitochondrial membrane, while DIA co-treatment inhibited such changes. DIA co-treatment ameliorated DOX-induced cytoskeletal changes via F-actin distribution and mitoKATP structure. Co-treatment also decreased ERK1/2 and cytochrome c protein levels. Cardiomyocyte loss due to oxidative stress-mediated apoptosis is a key event in DOX-induced cytotoxicity. DIA had protective effects on DOX-induced cardiotoxicity, via mitoKATP integrity, especially with elevated SUR2A levels; but also by a cascade including SOD/AMPK/ERK1/2. Therefore, DIA may be considered a candidate agent for protecting cardiomyocytes against DOX chemotherapy.

Lippia origanoides essential oil induces tocolytic effect in virgin rat uterus and inhibits writhing in a dysmenorrhea mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Lippia origanoides Kunth, popularly known as "salva-de-marajó", is used in Brazilian traditional "quilombola" communities to treat menstrual cramps and uterine inflammation. AIM OF THE STUDY: Evaluate the spasmolytic activity of Lippia origanoides essential oil (LOO) on experimental models of uterine conditions related to menstrual cramps and investigate its mechanism of action. MATERIALS AND METHODS: Virgin rat-isolated uterus was mounted in the organ bath apparatus to evaluate the spasmolytic effect of LOO on basal tonus and contractions induced by carbachol, KCl, or oxytocin. We used pharmacological agents to verify the relaxation mechanism of LOO. The evaluation of uterine contractility in virgin rats, after treatment with LOO for three consecutive days, was carried out by the construction of a concentration-response curve with oxytocin or carbachol. The primary dysmenorrhea animal model was replicated with an injection of estradiol cypionate in female mice for three consecutive days, followed by intraperitoneal application of oxytocin. RESULTS: LOO relaxed the rat uterus precontracted with 10-2 IU/mL oxytocin (logEC50 = 1.98 ± 0.07), 1 µM carbachol (logEC50 = 1.42 ± 0.07) or 60 mM KCl (logEC50 = 1.53 ± 0.05). It was also able relax uterus on spontaneous contractions (logEC50 = 0.41 ± 0.05). Preincubation with glibenclamide, propranolol, phentolamine or L-NAME in contractions induced by carbachol did not alter significantly the relaxing effect of LOO. However, in the presence of 4-aminopyridine, CsCl or tetraethylammonium there was a reduction of LOO potency, whereas the blockers methylene blue, ODQ, aminophylline and heparin potentiated the LOO relaxing effect. Preincubation with LOO in a Ca2+ free medium at concentrations of 27 µg/mL or 81 µg/mL reduced the contraction induced by carbachol. The administration of LOO for 3 days did not alter uterus contractility. The treatment with LOO at 30 or 100 mg/kg intraperitoneally, or 100 mg/kg orally, inhibited writhing in female mice. The association of LOO at 10 mg/kg with nifedipine or mefenamic acid potentiated writhing inhibition in mice. CONCLUSIONS: The essential oil of L. origanoides has tocolytic activity in rat isolated uterus pre-contracted with KCl, oxytocin, or carbachol. This effect is possibly related to the opening of potassium channels (Kir, KV, and KCa), cAMP increase, and diminution of intracellular Ca2+. This relaxant effect, probably, contributed to reduce the number of writhings in an animal model of dysmenorrhea being potentiated by nifedipine or mefenamic acid. Taken together, the results here presented indicate that this species has a pharmacological potential for the treatment of primary dysmenorrhea, supporting its use in folk medicine.

Pretreatment with the active fraction of Rhodiola tangutica (Maxim.) S.H. Fu rescues hypoxia-induced potassium channel inhibition in rat pulmonary artery smooth muscle cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Previous studies have shown that the active fraction of Rhodiola tangutica (Maxim.) S.H. Fu (ACRT) dilates pulmonary arteries and thwarts pulmonary artery remodelling. The dilatation effect of ACRT on pulmonary artery vascular rings could be reduced by potassium (K+) channel blockers. However the exact mechanisms of ACRT on ion channels are still unclear. AIM OF THE STUDY: This study aimed to investigate whether the effect of ACRT on K+ channels inhibits cell proliferation after pulmonary artery smooth muscle cells (PASMCs) are exposed to hypoxia. MATERIALS AND METHODS: The whole-cell patch-clamp method was used to clarify the effect of ACRT on the K+ current (IK) of rat PASMCs exposed to hypoxia. The mRNA and protein expression levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The intracellular calcium (Ca2+) concentration ([Ca2+]i) values in rat PASMCs were detected by laser scanning confocal microscopy. The cell cycle and cell proliferation were assessed using flow cytometry analysis and CCK-8 and EdU assays. RESULTS: ACRT pretreatment alleviated the inhibition of IK induced by hypoxia in rat PASMCs. Compared with hypoxia, ACRT upregulated voltage-dependent K+ channel (Kv) 1.5 and big-conductance calcium-activated K+ channel (BKCa) mRNA and protein expression and downregulated voltage-dependent Ca2+ channel (Cav) 1.2 mRNA and protein expression. ACRT decreased [Ca2+]i, inhibited the promotion of cyclin D1 and proliferating cell nuclear antigen (PCNA) expression, and prevented the proliferation of rat PASMCs exposed to hypoxia. CONCLUSION: In conclusion, the present study demonstrated that ACRT plays a key role in restoring ion channel function and then inhibiting the proliferation of PASMCs under hypoxia, ACRT has preventive and therapeutic potential in hypoxic pulmonary hypertension.

A Comparison of the Effect of Lead (Pb) on the Slow Vacuolar (SV) and Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles.

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.

Are ion channels potential therapeutic targets for Parkinson's disease?

Parkinson's disease (PD) is primarily associated with the progressive neurodegeneration of the dopaminergic neurons in the substantia nigra region of the brain. The resulting motor symptoms are managed with the help of dopamine replacement therapies. However, these therapeutics do not prevent the neurodegeneration underlying the disease and therefore lose their effectiveness in managing disease symptoms over time. Thus, there is an urgent need to develop newer therapeutics for the benefit of patients. The release of dopamine and the firing activity of substantia nigra neurons is regulated by several ion channels that act in concert. Dysregulations of these channels cause the aberrant movement of various ions in the intracellular milieu. This eventually leads to disruption of intracellular signalling cascades, alterations in cellular homeostasis, and bioenergetic deficits. Therefore, ion channels play a central role in driving the high vulnerability of dopaminergic neurons to degenerate during PD. Targeting ion channels offers an attractive mechanistic strategy to combat the process of neurodegeneration. In this review, we highlight the evidence pointing to the role of various ion channels in driving the PD processes. In addition, we also discuss the various drugs or compounds that target the ion channels and have shown neuroprotective potential in the in-vitro and in-vivo models of PD. We also discuss the current clinical status of various drugs targeting the ion channels in the context of PD.

Cardioprotective effect of anisodamine against ischemia/reperfusion injury through the mitochondrial ATP-sensitive potassium channel.

Previous clinical studies have shown that anisodamine could improve no-reflow phenomenon and prevent reperfusion arrhythmias, but whether this protective effect is related to the antagonism of the M-type cholinergic receptor or other potential mechanisms is uncertain. The aim of the present study was to investigate the role of the mitochondrial ATP-sensitive potassium channel (mitoK ATP ) in cardioprotective effect of anisodamine against ischemia/reperfusion injury. Anisodamine and 5- hydroxydecanoic acid were used to explore the relationship between anisodamine and mitoK ATP . Using a Langendorff isolated heart ischemia/reperfusion injury model, hemodynamic parameters and reperfusion ventricular arrhythmia were evaluated; in addition, changes in myocardial infarct size, cTnI from coronary effluent and myocardial ultrastructure, as well as ATP, MDA and SOD in myocardial tissues, were detected. In the hypoxia/reoxygenation injury model of neonatal rat cardiomyocyte, cTnI release in the culture medium and levels of ATP, MDA and SOD in cardiomyocytes and mitochondrial membrane potential, were analyzed. Overall, anisodamine could significantly improve the hemodynamic indexes of isolated rat heart injured by ischemia/reperfusion, reduce the occurrence of ventricular reperfusion arrhythmia and myocardial infarction area, and improve the ultrastructural damage of myocardium and mitochondria. The in vitro results demonstrated that anisodamine could improve mitochondrial energy metabolism, reduce oxidative stress and stabilize mitochondrial membrane potential. The cardioprotective effects were significantly inhibited by 5-hydroxydecanoic acid. In conclusion, this study suggests that the opening of mitoK ATP could play an important role in the protective effect of anisodamine against myocardial ischemia/reperfusion injury.

Analysis of Resibufogenin on Cardiac conduction reveals a species difference in the cardiac electrophysiology: Rats versus guinea pigs.

Resibufogenin (RBG) is a chemical ingredient of Chan Su. In our research, we found RBG affected cardiac rhythm in a negative chronotropic way in vivo. The cardiac Mapping system ex vivo and the patch clamp in vitro were used to explore how RBG influenced the cardiac electrophysiological properties. The negative chronotropic action of RBG at 100 µM might be attribute to prolongation in the atrioventricular conduction time and reduction in the ventricular conduction velocity. Using whole-cell patch clamp in ventricular myocytes of adult rats, we found that RBG prolonged the action potential duration (APD) in APD20, APD50, and APD90 at 100 µM and inhibited calcium currents (ICa), total outward potassium currents (IK), and transient outward potassium current (Ito) in a concentration-dependent manner, but not on the inward rectifying potassium current (IK1). Notably, RBG had a potent proarrhythmic action ex vivo in the isolated perfused guinea pig hearts at 10 µM, but not in rats. To avoid the potential cardiotoxicity derived from the distributional differences of ion channels among species, the effect of RGB on IKr in hERG-HEK293 cells was detected. The IC50 of RGB on IKr was more than 100 µM. In summary, all these results indicated that the negative chronotropic action of RBG relied on the blocking activities on multiple ion channels, and the species-difference of proarrhythmic effects might result from lack of the Ito on the myocardial membrane of guinea pigs. Anyhow, the cardiotoxicity observed in guinea pigs required further detailed studies to mitigate the potential risks in the clinical application of Chan Su.

Ivabradine augments high-frequency dynamic gain of the heart rate response to low- and moderate-intensity vagal nerve stimulation under ß-blockade.

Our previous study indicated that intravenously administered ivabradine (IVA) augmented the dynamic heart rate (HR) response to moderate-intensity vagal nerve stimulation (VNS). Considering an accentuated antagonism, the results were somewhat paradoxical; i.e., the accentuated antagonism indicates that an activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels via the accumulation of intracellular cyclic adenosine monophosphate (cAMP) augments the HR response to VNS, whereas the inhibition of HCN channels by IVA also augmented the HR response to VNS. To remove the possible influence from the accentuated antagonism, we examined the effects of IVA on the dynamic vagal control of HR under ß-blockade. In anesthetized rats (n = 7), the right vagal nerve was stimulated for 10 min according to binary white noise signals between 0 and 10 Hz (V0-10), between 0 and 20 Hz (V0-20), and between 0 and 40 Hz (V0-40). The transfer function from VNS to HR was estimated. Under ß-blockade (propranolol, 2 mg/kg iv), IVA (2 mg/kg iv) did not augment the asymptotic low-frequency gain but increased the asymptotic high-frequency gain in V0-10 (0.53 ± 0.10 vs. 1.74 ± 0.40 beats/min/Hz, P < 0.01) and V0-20 (0.79 ± 0.14 vs. 2.06 ± 0.47 beats/min/Hz, P < 0.001). These changes, which were observed under a minimal influence from sympathetic background tone, may reflect an increased contribution of the acetylcholine-sensitive potassium channel (IK,ACh) pathway after IVA, because the HR control via the IK,ACh pathway is faster and acts in the frequency range higher than the cAMP-mediated pathway.NEW & NOTEWORTHY Since ivabradine (IVA) inhibits hyperpolarization-activated cyclic nucleotide-gated channels, interactions among the sympathetic effect, vagal effect, and IVA can occur in the control of heart rate (HR). To remove the sympathetic effect, we estimated the transfer function from vagal nerve stimulation to HR under ß-blockade in anesthetized rats. IVA augmented the high-frequency dynamic gain during low- and moderate-intensity vagal nerve stimulation. Untethering the hyperpolarizing effect of acetylcholine-sensitive potassium channels after IVA may be a possible underlying mechanism.

Analgesic effects of cuminic alcohol (4-isopropylbenzyl alcohol), a monocyclic terpenoid, in animal models of nociceptive and neuropathic pain: Role of opioid receptors, L-arginine/NO/cGMP pathway, and inflammatory cytokines.

Cuminic alcohol (4-isopropylbenzyl alcohol; 4-IPBA) is a monocyclic terpenoid found in the analgesic medicinal plants Cuminum cyminum and Bunium persicum. The current study assessed the analgesic effects of 4-IPBA in different animal models of pain. Hot plate, formalin, and acetic acid tests were used to evaluate nociceptive pain in mice. The involvement of opioid receptors and the L-arginine/NO/cGMP/K+ channel pathway in 4-IPBA effects were investigated. Allodynia and hyperalgesia were assessed following peripheral neuropathy induced by chronic constriction of the sciatic nerve in rats. The spinal levels of inflammatory cytokines were measured using the ELISA method. The drugs and compounds were administered intraperitoneally. The results showed that 4-IPBA (200 and 400 mg/kg) significantly prolonged the hot plate latency. This effect was antagonized by naloxone (2 mg/kg). 4-IPBA (25-100 mg/kg) also significantly attenuated formalin- and acetic acid-induced nociceptive pain. L-arginine (200 mg/kg), sodium nitroprusside (0.25 mg/kg), and sildenafil (0.5 mg/kg) reversed while L-NAME (30 mg/kg) and methylene blue (20 mg/kg) potentiated the antinociceptive effects of 4-IPBA in the writhing test. Glibenclamide (10 mg/kg) and tetraethylammonium chloride (4 mg/kg) did not have any influence on the 4-IPBA effect. Furthermore, 4-IPBA (6.25-25 mg/kg) significantly relieved mechanical allodynia, cold allodynia, and hyperalgesia in rats. The concentrations of TNF-α and IL-1ß in the spinal cord of rats were decreased by 4-IPBA. No evidence of 4-IPBA-induced toxicity was found in behavioral or histopathological examinations. These results demonstrate that 4-IPBA attenuates nociceptive and neuropathic pain through the involvement of opioid receptors, the L-arginine/NO/cGMP pathway, and anti-inflammatory functions.

Potassium (K+) channels in the pulmonary vasculature: Implications in pulmonary hypertension Physiological, pathophysiological and pharmacological regulation.

The large K+ channel functional diversity in the pulmonary vasculature results from the multitude of genes expressed encoding K+ channels, alternative RNA splicing, the post-transcriptional modifications, the presence of homomeric or heteromeric assemblies of the pore-forming α-subunits and the existence of accessory ß-subunits modulating the functional properties of the channel. K+ channels can also be regulated at multiple levels by different factors controlling channel activity, trafficking, recycling and degradation. The activity of these channels is the primary determinant of membrane potential (Em) in pulmonary artery smooth muscle cells (PASMC), providing an essential regulatory mechanism to dilate or contract pulmonary arteries (PA). K+ channels are also expressed in pulmonary artery endothelial cells (PAEC) where they control resting Em, Ca2+ entry and the production of different vasoactive factors. The activity of K+ channels is also important in regulating the population and phenotype of PASMC in the pulmonary vasculature, since they are involved in cell apoptosis, survival and proliferation. Notably, K+ channels play a major role in the development of pulmonary hypertension (PH). Impaired K+ channel activity in PH results from: 1) loss of function mutations, 2) downregulation of its expression, which involves transcription factors and microRNAs, or 3) decreased channel current as a result of increased vasoactive factors (e.g., hypoxia, 5-HT, endothelin-1 or thromboxane), exposure to drugs with channel-blocking properties, or by a reduction in factors that positively regulate K+ channel activity (e.g., NO and prostacyclin). Restoring K+ channel expression, its intracellular trafficking and the channel activity is an attractive therapeutic strategy in PH.

Activation of Oxytocin Receptors Excites Subicular Neurons by Multiple Signaling and Ionic Mechanisms.

Oxytocin (OXT) is a nonapeptide that serves as a neuromodulator in the brain and a hormone participating in parturition and lactation in the periphery. The subiculum is the major output region of the hippocampus and an integral component in the networks that process sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information. Whilst the subiculum expresses the highest OXT-binding sites and is the first brain region to be activated by peripheral application of OXT, the precise actions of OXT in the subiculum have not been determined. Our results demonstrate that application of the selective OXT receptor (OXTR) agonist, [Thr4,Gly7]-oxytocin (TGOT), excited subicular neurons via activation of TRPV1 channels, and depression of K+ channels. The OXTR-mediated excitation of subicular neurons required the functions of phospholipase Cß, protein kinase C, and degradation of phosphatidylinositol 4,5-bisphosphate (PIP2). OXTR-elicited excitation of subicular neurons enhanced long-term potentiation via activation of TRPV1 channels. Our results provide a cellular and molecular mechanism to explain the physiological functions of OXT in the brain.

Potassium channels in intestinal epithelial cells and their pharmacological modulation: a systematic review.

Several potassium channels (KCs) have been described throughout the gastrointestinal tract. Notwithstanding, their contribution to both physiologic and pathophysiologic conditions, as inflammatory bowel disease (IBD), remains underexplored. Therefore, we aim to systematically review, for the first time, the evidence on the characteristics and modulation of KCs in intestinal epithelial cells (IECs). PubMed, Scopus, and Web of Science were searched to identify studies focusing on KCs and their modulation in IECs. The included studies were assessed using a reporting inclusiveness checklist. From the 745 identified records, 73 met the inclusion criteria; their reporting inclusiveness was moderate-high. Some studies described the physiological role of KCs, while others explored their importance in pathological settings. Globally, in IBD animal models, apical KCa1.1 channels, responsible for luminal secretion, were upregulated. In human colonocytes, basolateral KCa3.1 channels were downregulated. The pharmacological inhibition of K2P and Kv influenced intestinal barrier function, promoting inflammation. Evidence suggests a strong association between KCs expression and secretory mechanisms in human and animal IECs. Further research is warranted to explore the usefulness of KC pharmacological modulation as a therapeutic target.

Delta-opioid receptor-mediated modulation of excitability of individual hippocampal neurons: mechanisms involved.

BACKGROUND: Delta-opioid receptor (DOR)-mediated modulation of hippocampal neural networks is involved in emotions, cognition, and in pathophysiology and treatment of mood disorders. In this study, we examined the effects of DOR agonist (SNC80) and antagonist (naltrindole) on the excitability of individual hippocampal neurons. METHODS: Primary neuronal cultures were prepared from hippocampi of newborn rats and cultivated in vitro for 8-14 days (DIV8-14). The effects of SNC80 naltrindole on evoked and spontaneous action potentials (APs) were measured at DIV8-9 and DIV13-14, respectively. RESULTS: SNC80 (100 µM) potentiated spontaneous AP firing and stimulated sodium current; naltrindole had opposite effects. The stimulatory effect of 100 µM of SNC80 was revoked by pre-administration of 1 µM of naltrindole. SNC80 and naltrindole induced similar inhibitory effects on the evoked AP firing and on the calcium current. Further, SNC80 inhibited both peak and sustained potassium currents. Naltrindole had no effect on potassium currents. CONCLUSION: We suggest that the effects of naltrindole and high concentration of SNC80 on the sodium currents are mediated via DORs and underlying the changes in spontaneous activity. The inhibitory effects of SNC80 on calcium and potassium currents might also be DOR-dependent; these currents might mediate SNC80 effect on the evoked AP firing. The inhibitory effects of naltrindole on calcium and of low doses of SNC80 on sodium currents might be however DOR independent. The behavioral effects of SNC80 and naltrindole, observed in previous studies, might be mediated, at least in part, via the modulatory effect of these ligands on the excitability of hippocampal neurons.

Mechanistic insights into volatile anesthetic modulation of K2P channels.

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA-binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamics simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3, and TM4 that contribute to anesthetic binding. The identified VA-binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.

Inhibition of cardiac potassium currents by oxidation-activated protein kinase A contributes to early afterdepolarizations in the heart.

Reactive oxygen species (ROS) have been shown to prolong cardiac action potential duration resulting in afterdepolarizations, the cellular basis of triggered arrhythmias. As previously shown, protein kinase A type I (PKA I) is readily activated by oxidation of its regulatory subunits. However, the relevance of this mechanism of activation for cardiac pathophysiology is still elusive. In this study, we investigated the effects of oxidation-activated PKA I on cardiac electrophysiology. Ventricular cardiomyocytes were isolated from redox-dead PKA-RI Cys17Ser knock-in (KI) and wild-type (WT) mice and exposed to H2O2 (200 µmol/L) or vehicle (Veh) solution. In WT myocytes, exposure to H2O2 significantly increased oxidation of the regulatory subunit I (RI) and thus its dimerization (threefold increase in PKA RI dimer). Whole cell current clamp and voltage clamp were used to measure cardiac action potentials (APs), transient outward potassium current (Ito) and inward rectifying potassium current (IK1), respectively. In WT myocytes, H2O2 exposure significantly prolonged AP duration due to significantly decreased Ito and IK1 resulting in frequent early afterdepolarizations (EADs). Preincubation with the PKA-specific inhibitor Rp-8-Br-cAMPS (10 µmol/L) completely abolished the H2O2-dependent decrease in Ito and IK1 in WT myocytes. Intriguingly, H2O2 exposure did not prolong AP duration, nor did it decrease Ito, and only slightly enhanced EAD frequency in KI myocytes. Treatment of WT and KI cardiomyocytes with the late INa inhibitor TTX (1 µmol/L) completely abolished EAD formation. Our results suggest that redox-activated PKA may be important for H2O2-dependent arrhythmias and could be important for the development of specific antiarrhythmic drugs.NEW & NOTEWORTHY Oxidation-activated PKA type I inhibits transient outward potassium current (Ito) and inward rectifying potassium current (IK1) and contributes to ROS-induced APD prolongation as well as generation of early afterdepolarizations in murine ventricular cardiomyocytes.

Testing broad-spectrum and isoform-preferring HCN channel blockers for anticonvulsant properties in mice.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have been implicated in the pathogenesis of epilepsy and consequently as targets for anticonvulsant drugs. Consistent with this, broad-spectrum block of HCN-mediated current (Ih) reduces seizure susceptibility in a variety of epilepsy models. However, HCN channel isoforms have distinct biophysical characteristics and anatomical expression suggesting that they may play different roles in setting neuronal excitability. Here we confirm that the broad-spectrum blocker ivabradine is effective at reducing seizure susceptibility in the s.c.PTZ seizure assay and extend this, showing efficacy of this drug in a thermogenic assay that models febrile seizures. Ivabradine is also effective at reducing thermogenic seizures in the Scn1a mouse model of Dravet syndrome in which febrile seizures are a feature. HCN isoform-preferring drugs were tested in the s.c.PTZ seizure assay. We confirm that the HCN4-preferring drug, EC18, is efficacious in reducing seizure susceptibility. Conversely, the HCN2/1-preferring drug, MEL55A, increased seizure susceptibility in the s.c.PTZ seizure assay. MEL57A, an HCN1-preferring drug, had no effect on seizure susceptibility. Mouse pharmacokinetic studies (for MEL55A and MEL57A) and screening against additional ion channels have not been thoroughly investigated on the HCN isoform-preferring compounds. Our results need to be considered in this light. Nevertheless, these data suggest that HCN isoform-selective block can have a differential impact on seizure susceptibility. This motivates the need to develop more HCN isoform-selective compounds to better explore this idea.

Histamine depolarizes rat intracardiac ganglion neurons through the activation of TRPC non-selective cation channels.

The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 µM. This depolarization was markedly inhibited by the H1 receptor antagonist triprolidine and mimicked by the H1 receptor agonist 2-pyridylethylamine, thus implicating histamine H1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca2+ removal and attenuated by extracellular Na+ replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H1 receptors and Gq/11-protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either Gq/11 signalling or intracellular Ca2+ release and by the application of the TRPC blockers Pyr3, Gd3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine.

Sevoflurane depresses neurons in the medial parabrachial nucleus by potentiating postsynaptic GABAA receptors and background potassium channels.

Despite persistent clinical use for over 170 years, the neuronal mechanisms by which general anesthetics produce hypnosis remain unclear. Previous studies suggest that anesthetics exert hypnotic effects by acting on endogenous arousal circuits. Recently, it has been shown that the medial parabrachial nucleus (MPB) is a novel wake-promoting component in the dorsolateral pons. However, it is not known whether and how the MPB contributes to anesthetic-induced hypnosis. Here, we investigated the action of sevoflurane, a widely used volatile anesthetic agent that best represents the drug class of halogenated ethers, on MPB neurons in mice. Using in vivo fiber photometry, we found that the population activities of MPB neurons were inhibited during sevoflurane-induced loss of consciousness. Using in vitro whole-cell patch-clamp recordings, we revealed that sevoflurane suppressed the firing rate of MPB neurons in concentration-dependent and reversible manners. At a concentration equal to MAC of hypnosis, sevoflurane potentiated synaptic GABAA receptors (GABAA-Rs), and the inhibitory effect of sevoflurane on the firing rate of MPB neurons was completely abolished by picrotoxin, which is a selective GABAA-R antagonist. At a concentration equivalent to MAC of immobility, sevoflurane directly hyperpolarized MPB neurons and induced a significant decrease in membrane input resistance by increasing a basal potassium conductance. Moreover, pharmacological blockade of GABAA-Rs in the MPB prolongs induction and shortens emergence under sevoflurane inhalation at MAC of hypnosis. These results indicate that sevoflurane inhibits MPB neurons through postsynaptic GABAA-Rs and background potassium channels, which contributes to sevoflurane-induced hypnosis.