Voltage-gated Potassium Currents Research Articles

A novel isoquinoline alkaloid HJ-69 isolated from Zanthoxylum bungeanum attenuates inflammatory pain by inhibiting voltage-gated sodium and potassium channels

Ethnopharmacology relevanceZanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. Aim of the studyThe study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. Materials and methodsIsoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. ResultsA novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 μM and 30.19 ± 2.07 μM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 μM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 μM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. ConclusionThe study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.

Early deficits in an in vitro striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation

Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson’s disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.

Inhibition of Kv2.1 potassium channels by the antidepressant drug sertraline

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In this study, we have used the patch-clamp technique to assess the effects of sertraline on Kv2.1 channels heterologously expressed in HEK-293 cells and on the voltage-gated potassium currents (IKv) of Neuro 2a cells, which are predominantly mediated by Kv2.1 channels. Our results reveal that sertraline inhibits Kv2.1 channels in a concentration-dependent manner. The sertraline-induced inhibition was not voltage-dependent and did not require the channels to be open. The kinetics of activation and deactivation were accelerated and decelerated, respectively, by sertraline. Moreover, the inhibition by this drug was use-dependent. Notably, sertraline significantly modified the inactivation mechanism of Kv2.1 channels; the steady-state inactivation was shifted to hyperpolarized potentials, the closed-state inactivation was enhanced and accelerated, and the recovery from inactivation was slowed, suggesting that this is the main mechanism by which sertraline inhibits Kv2.1 channels. Overall, this study provides novel insights into the pharmacological actions of sertraline on Kv2.1 channels, shedding light on the intricate interaction between SSRIs and ion channel function.

Human voltage-gated Na+ and K+ channel properties underlie sustained fast AP signaling.

Human cortical pyramidal neurons are large, have extensive dendritic trees, and yet have unexpectedly fast input-output properties: Rapid subthreshold synaptic membrane potential changes are reliably encoded in timing of action potentials (APs). Here, we tested whether biophysical properties of voltage-gated sodium (Na+) and potassium (K+) currents in human pyramidal neurons can explain their fast input-output properties. Human Na+ and K+ currents exhibited more depolarized voltage dependence, slower inactivation, and faster recovery from inactivation compared with their mouse counterparts. Computational modeling showed that despite lower Na+ channel densities in human neurons, the biophysical properties of Na+ channels resulted in higher channel availability and contributed to fast AP kinetics stability. Last, human Na+ channel properties also resulted in a larger dynamic range for encoding of subthreshold membrane potential changes. Thus, biophysical adaptations of voltage-gated Na+ and K+ channels enable fast input-output properties of large human pyramidal neurons.

Activation and polarization of striatal microglia and astrocytes are involved in bradykinesia and allodynia in early-stage parkinsonian mice

In addition to the cardinal motor symptoms, pain is a major non-motor symptom of Parkinson's disease (PD). Neuroinflammation in the substantia nigra pars compacta and dorsal striatum is involved in neurodegeneration in PD. But the polarization of microglia and astrocytes in the dorsal striatum and their contribution to motor deficits and hyperalgesia in PD have not been characterized. In the present study, we observed that hemiparkinsonian mice established by unilateral 6-OHDA injection in the medial forebrain bundle exhibited motor deficits and mechanical allodynia. In these mice, both microglia and astrocytes in the dorsal striatum were activated and polarized to M1 / M2 microglia and A1 / A2 astrocytes as genes specific to these cells were upregulated. These effects peaked 7 days after 6-OHDA injection. Meanwhile, striatal astrocytes in parkinsonian mice also displayed hyperpolarized membrane potentials, enhanced voltage-gated potassium currents, and dysfunction in inwardly rectifying potassium channels and glutamate transporters. Systemic administration of minocycline, a microglia inhibitor, attenuated the expression of genes specific to M1 microglia and A1 astrocytes in the dorsal striatum (but not those specific to M2 microglia and A2 astrocytes), attenuated the damage in the nigrostriatal dopaminergic system, and alleviated the motor deficits and mechanical allodynia in parkinsonian mice. By contrast, local administration of minocycline into the dorsal striatum of parkinsonian mice mitigated only hyperalgesia. This study suggests that M1 microglia and A1 astrocytes in the dorsal striatum may play important roles in the development of pathophysiology underlying hyperalgesia in the early stages of PD.

Slowly activating voltage-gated potassium current potentiation by ML277 is a novel cardioprotective intervention.

Cardiovascular disease is thought to account for nearly a third of deaths worldwide, with ischemic heart disease, including acute coronary syndromes such as myocardial infarction, accounting for 1.7 million deaths per year. There is a clear need for interventions to impart cardioprotection against ischemia. Here, we show that the slowly activating voltage-gated potassium current (IKs) potentiator ML277 imparts cardioprotection against ischemia in cellular and whole-heart models by modulating the action potential duration. In three different metabolic inhibition and reperfusion models, an increased contractile recovery and cell survival was observed with ML277, indicative of protection. Finally, ML277 reduced infarct size in an ex vivo Langendorff coronary ligation model, including if only applied on reperfusion. In conclusion, potentiation of the IKs with ML277 imparted a cardioprotection that was equivalent to the protection reported previously by ischemic preconditioning. These data suggest that IKs potentiation may be therapeutically useful in acute coronary syndromes.

Estradiol regulates voltage-gated potassium currents in corticotropin-releasing hormone neurons.

Corticotropin-releasing hormone (CRH) neurons are the primary neural population controlling the hypothalamic-pituitary-adrenal (HPA) axis and the secretion of adrenal stress hormones. Previous work has demonstrated that stress hormone secretion can be regulated by circulating levels of estradiol. However, the effect of estradiol on CRH neuron excitability is less clear. Here, we show that chronic estradiol replacement following ovariectomy increases two types of potassium channel currents in CRH neurons: fast inactivating voltage-gated A-type K+ channel currents (IA) and non-inactivating M-type K+ channel currents (IM). Despite the increase in K+ currents following estradiol replacement, there was no overall change in CRH neuron spiking excitability assessed with either frequency-current curves or current ramps. Together, these data reveal a complex picture whereby ovariectomy and estradiol replacement differentially modulate distinct aspects of CRH neuron and HPA axis function.

Ouabain-Induced Changes in the Expression of Voltage-Gated Potassium Channels in Epithelial Cells Depend on Cell-Cell Contacts.

Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical-basolateral polarity and cell-cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell-cell contact-lacking cells, ouabain did not produce a significant change; (4) Na+/K+ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and β-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.

Extracellular S100B inhibits A-type voltage-gated potassium currents and increases L-type voltage-gated calcium channel activity in dopaminergic neurons.

Parkinson's disease (PD) is associated with an increase in secreted S100B within the midbrain and cerebrospinal fluid. In addition, S100B overexpression in mice accelerates the loss of substantia nigra pars compacta dopaminergic (DA) neurons, suggesting a role for this protein in PD pathogenesis. We found that in the mouse SNc, S100B labeled astrocytic processes completely envelop the somata of tyrosine hydroxylase (TH) expressing DA neurons only in male mice. These data suggest that an increase in S100B secretion by astrocytes within the midbrain could play a role in DA dysfunction during early PD. We therefore asked if acute exposure to extracellular S100B alters the activity of identified TH expressing DA neurons in primary mouse midbrain cultures. Acute exposure to 50 pM S100B specifically inhibited A-type voltage-gated potassium currents in TH+ , but not TH- neurons. This was accompanied by ~2-fold increases in the frequency of both intrinsic firing, as well as L-type voltage-gated calcium channel (VGCC)-mediated calcium fluxes only in TH+ neurons. Further, exposure to 100 μM 4-aminopyridine (4-AP), an A-type voltage-gated potassium channel inhibitor, mimicked the S100B mediated increase in intrinsic firing and L-type VGCC-mediated calcium fluxes in TH+ neurons. Taken together, our finding that extracellular S100B alters the activity of native DA neurons via an inhibition of A-type voltage-gated potassium channels has important implications for understanding the pathophysiology of early PD.

Flavonoid-Like Components of Peanut Stem and Leaf Extract Promote Sleep by Decreasing Neuronal Excitability.

Peanut stem and leaf (PSL), a traditional Chinese medicine, is widely used as a dietary supplement to improve sleep quality; however, the underlying mechanism is unclear. Here, the study aims to determine whether active compounds in PSL extract exert their effects by mediating neuronal excitability. Aqueous PSL extract (500mg kg-1 BW) increases the duration of total sleep (TS), slow wave sleep (SWS) and rapid eye movement sleep (REMS) in BALB/c mice after 7 and 14 continuous days of intragastric administration. Two PSL extract components with flavonoid-like structures: 4',7-di-O-methylnaringenin (DMN, 61µg kg-1 BW) and 2'-O-methylisoliquiritigenin (MIL, 12µg kg-1 BW), show similar effects on sleep in BALB/c mice. Moreover, incubation with DMN (50µM) and MIL (50µM) acutely reduces voltage-gated sodium and potassium currents and suppresses the firing of evoked action potential in mouse cortical neurons, indicating the inhibition on neuronal excitability. Meanwhile, RNA-seq analysis predicts the potential regulation of voltage-gated channels, which is according with the molecular docking simulation that both MIL and DMN can bind to voltage gated sodium channels 1.2 (Nav 1.2). DMN and MIL are the active ingredients of PSL that improve sleep quality, suggesting that PSL promotes sleep by regulating the excitability of neurons.

The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Reciprocal Changes in Voltage-Gated Potassium and Subthreshold Inward Currents Help Maintain Firing Dynamics of AVPV Kisspeptin Neurons during the Estrous Cycle.

Kisspeptin-expressing neurons in the anteroventral-periventricular nucleus (AVPV) are part of a neural circuit generating the gonadotropin-releasing hormone (GnRH) surge. This process is estradiol-dependent and occurs on the afternoon of proestrus in female mice. On proestrus, AVPV kisspeptin neurons express more kisspeptin and exhibit higher frequency action potentials and burst firing compared with diestrus, which is characterized by a pulsatile rather than a prolonged surge of GnRH secretion. We hypothesized changes in voltage-gated potassium conductances shape activity profiles of these cells in a cycle-dependent manner. Whole-cell voltage-clamp recordings of GFP-identified AVPV kisspeptin neurons in brain slices from diestrous and proestrous mice revealed three subcomponents of the voltage-sensitive K+ current: fast-transient slow-transient, and residual. During proestrus, the V50 of inactivation of the fast-transient current was depolarized and the amplitude of the slow-transient component was reduced compared with diestrus; the residual component was consistent across both stages. Computational models were fit to experimental data, including published estrous-cycle effects on other voltage-gated currents. Computer simulations suggest proestrus-typical K+ currents are suppressive compared with diestrus. Interestingly, larger T-type, persistent-sodium, and hyperpolarization-activated currents during proestrus compensate for this suppressive effect while also enabling postinhibitory rebound bursting. These findings suggest modulation of voltage-gated K+ and multiple subthreshold depolarizing currents across the negative to positive feedback transition maintain AVPV kisspeptin neuron excitability in response to depolarizing stimuli. These changes also enable firing in response to hyperpolarization, providing a net increase in neuronal excitability, which may contribute to activation of this population leading up to the preovulatory GnRH surge.

Towards maturation of human otic hair cell-like cells in pluripotent stem cell-derived organoid transplants.

Human otic organoids generated from pluripotent stem cells (PSCs) provide a promising platform for modeling, drug testing, and cell-based therapies of inner ear diseases. However, providing the appropriate niche that resembles inner ear development and its vasculature to generate otic organoids is less conspicuous. Here, we devised a strategy to enhance maturation of otic progenitor cells toward human hair cell-like cells (HCLCs) by assembling three-dimensional (3D) otic organoids that contain human PSC-derived otic cells, endothelial cells, and mesenchymal stem cells (MSCs). Heterotopic implantation of otic organoids, designated as grafted otic organoids (GOs), in ex ovo chick embryo chorioallantoic membrane (CAM) stimulated maturation of the HCLCs. Functional analysis revealed the presence of voltage-gated potassium currents without detectable sodium currents in these cells in the GOs. Our results demonstrated that implantation of 3D heterotypic cell mixtures of otic organoids improved maturation of human HCLCs. This GO-derived HCLCs could be an attractive source for drug discovery and other biomedical applications.

The Differentiation Status of Hair Cells That Regenerate Naturally in the Vestibular Inner Ear of the Adult Mouse.

Aging, disease, and trauma can lead to loss of vestibular hair cells and permanent vestibular dysfunction. Previous work showed that, following acute destruction of ∼95% of vestibular hair cells in adult mice, ∼20% regenerate naturally (without exogenous factors) through supporting cell transdifferentiation. There is, however, no evidence for the recovery of vestibular function. To gain insight into the lack of functional recovery, we assessed functional differentiation in regenerated hair cells for up to 15 months, focusing on key stages in stimulus transduction and transmission: hair bundles, voltage-gated conductances, and synaptic contacts. Regenerated hair cells had many features of mature type II vestibular hair cells, including polarized mechanosensitive hair bundles with zone-appropriate stereocilia heights, large voltage-gated potassium currents, basolateral processes, and afferent and efferent synapses. Regeneration failed, however, to recapture the full range of properties of normal populations, and many regenerated hair cells had some properties of immature hair cells, including small transduction currents, voltage-gated sodium currents, and small or absent HCN (hyperpolarization-activated cyclic nucleotide-gated) currents. Furthermore, although mouse vestibular epithelia normally have slightly more type I hair cells than type II hair cells, regenerated hair cells acquired neither the low-voltage-activated potassium channels nor the afferent synaptic calyces that distinguish mature type I hair cells from type II hair cells and confer distinctive physiology. Thus, natural regeneration of vestibular hair cells in adult mice is limited in total cell number, cell type diversity, and extent of cellular differentiation, suggesting that manipulations are needed to promote full regeneration with the potential for recovery of vestibular function.SIGNIFICANCE STATEMENT Death of inner ear hair cells in adult mammals causes permanent loss of hearing and balance. In adult mice, the sudden death of most vestibular hair cells stimulates the production of new hair cells but does not restore balance. We investigated whether the lack of systems-level function reflects functional deficiencies in the regenerated hair cells. The regenerated population acquired mechanosensitivity, voltage-gated channels, and afferent synapses, but did not reproduce the full range of hair cell types. Notably, no regenerated cells acquired the distinctive properties of type I hair cells, a major functional class in amniote vestibular organs. To recover vestibular system function in adults, we may need to solve how to regenerate the normal variety of mature hair cells.

Gonadotropin-Releasing Hormone (GnRH) Neuron Potassium Currents and Excitability in Both Sexes Exhibit Minimal Changes upon Removal of Negative Feedback.

Gonadotropin-releasing hormone (GnRH) drives pituitary secretion of luteinizing hormone and follicle-stimulating hormone, which in turn regulate gonadal functions including steroidogenesis. The pattern of GnRH release and thus fertility depend on gonadal steroid feedback. Under homeostatic (negative) feedback conditions, removal of the gonads from either females or males increases the amplitude and frequency of GnRH release and alters the long-term firing pattern of these neurons in brain slices. The neurobiological mechanisms intrinsic to GnRH neurons that are altered by homeostatic feedback are not well studied and have not been compared between sexes. During estradiol-positive feedback, which is unique to females, there are correlated changes in voltage-gated potassium currents and neuronal excitability. We thus hypothesized that these same mechanisms would be engaged in homeostatic negative feedback. Voltage-gated potassium channels play a direct role in setting excitability and action potential properties. Whole-cell patch-clamp recordings of GFP-identified GnRH neurons in brain slices from sham-operated and castrated adult female and male mice were made to assess fast and slow inactivating potassium currents as well as action potential properties. Surprisingly, no changes were observed among groups in most potassium current properties, input resistance, or capacitance, and this was reflected in a lack of differences in excitability and specific action potential properties. These results support the concept that, in contrast to positive feedback, steroid-negative feedback regulation of GnRH neurons in both sexes is likely conveyed to GnRH neurons via mechanisms that do not induce major changes in the biophysical properties of these cells.

Topographical Organization of M-Current on Dorsal and Median Raphe Serotonergic Neurons.

Dorsal and median raphe nuclei (DR and MR, respectively) are members of the reticular activating system and play important role in the regulation of the sleep-wakefulness cycle, movement, and affective states. M-current is a voltage-gated potassium current under the control of neuromodulatory mechanisms setting neuronal excitability. Our goal was to determine the proportion of DR and MR serotonergic neurons possessing M-current and whether they are organized topographically. Electrophysiological parameters of raphe serotonergic neurons influenced by this current were also investigated. We performed slice electrophysiology on genetically identified serotonergic neurons. Neurons with M-current are located rostrally in the DR and dorsally in the MR. M-current determines firing rate, afterhyperpolarization amplitude, and adaptation index (AI) of these neurons, but does not affect input resistance, action potential width, and high threshold oscillations.These findings indicate that M-current has a strong impact on firing properties of certain serotonergic neuronal subpopulations and it might serve as an effective contributor to cholinergic and local serotonergic neuromodulatory actions.

Aldosterone up-regulates voltage-gated potassium currents and NKCC1 protein membrane fractions

Na+–K+–2Cl− Cotransporter (NKCC1) is a protein that aids in the active transport of sodium, potassium, and chloride ions across cell membranes. It has been shown that long-term systemic treatment with aldosterone (ALD) can enhance NKCC1 protein expression and activity in the aging cochlea resulting in improved hearing. In the present work, we used a cell line with confirmed NKCC1 expression to demonstrate that in vitro application of ALD increased outward voltage-gated potassium currents significantly, and simultaneously upregulated whole lysate and membrane portion NKCC1 protein expression. These ALD-induced changes were blocked by applying the mineralocorticoid receptor antagonist eplerenone. However, application of the NKCC1 inhibitor bumetanide or the potassium channel antagonist Tetraethyl ammonium had no effect. In addition, NKKC1 mRNA levels remained stable, indicating that ALD modulates NKCC1 protein expression via the activation of mineralocorticoid receptors and post-transcriptional modifications. Further, in vitro electrophysiology experiments, with ALD in the presence of NKCC1, K+ channel and mineralocorticoid receptor inhibitors, revealed interactions between NKCC1 and outward K+ channels, mediated by a mineralocorticoid receptor-ALD complex. These results provide evidence of the therapeutic potential of ALD for the prevention/treatment of inner ear disorders such as age-related hearing loss.

A CRH Receptor Type 1 Agonist Increases GABA Transmission to GnRH Neurons in a Circulating-Estradiol-Dependent Manner.

GnRH neurons are central regulators of reproduction and respond to factors affecting fertility, such as stress. Corticotropin-releasing hormone (CRH) is released during stress response. In brain slices from unstressed controls, CRH has opposite, estradiol-dependent effects on GnRH neuron firing depending on the CRH receptor activated; activating CRHR-1 stimulates whereas activating CRHR-2 suppresses activity. We investigated possible direct and indirect mechanisms. Mice were ovariectomized and either not treated further (OVX) or given a capsule producing high positive feedback (OVX + E) or low negative feedback (OVX + low E) physiologic circulating estradiol levels. We tested possible direct effects on GnRH neurons by altering voltage-gated potassium currents. Two types of voltage-gated potassium currents (transient IA and sustained IK) were measured; neither CRHR-1 nor CRHR-2 agonists altered potassium current density in GnRH neurons from OVX + E mice. Further, neither CRH nor receptor-specific agonists altered action potential generation in response to current injection in GnRH neurons from OVX + E mice. To test the possible indirect actions, GABAergic postsynaptic currents were monitored. A CRHR-1 agonist increased GABAergic transmission frequency to GnRH neurons from OVX + E, but not OVX, mice, whereas a CRHR-2 agonist had no effect. Finally, we tested if CRH alters the firing rate of arcuate kisspeptin neurons, which provide an important excitatory neuromodulatory input to GnRH neurons. CRH did not acutely alter firing activity of these neurons from OVX, OVX + E or OVX + low E mice. These results suggest CRH increases GnRH neuron activity in an estradiol-dependent manner in part by activating GABAergic afferents. Mechanisms underlying inhibitory effects of CRH remain unknown.

Characteristics of voltage-gated potassium currents in monosodium urate induced gouty arthritis in mice.

To evaluate the role of K+ channels in pain following gouty arthritis. The model of acute gouty arthritis was induced by monosodium urate (MSU) in mice. The swelling degree was determined by measuring the circumference of the ankle joint. Mechanical hyperalgesia was detected by von Frey filaments. Two types of K+ currents, A-type currents (IA) and delayed rectifier currents (IK), were recorded in dorsal root ganglion (DRG) neurons using patch-clamp techniques. The swelling degree reached its maximum at 10h and the minimum pain threshold was maintained between 8 and 48h after MSU treatment in mice. The amplitudes of IA and IK in DRG neurons were moderately increased on day 1 after MSU treatment, and then, they were gradually decreased with times and reached their minimums on day 4 (for IA) or 5 (for IK). Compared with control group, the activation curve of IA was significantly shifted to more positive potential and the recovery time of IA from inactivation was markedly prolonged, but inactivation and frequency dependence of IA appeared unaffected in MSU-treated group. Additionally, no change was observed in the activation curve of IK after MSU treatment. The excitability was significantly higher in the MSU group than in the control group. MSU-induced gout pain may be related to the hyperexcitability of DRG neurons elicited by decreasing K+ currents.