[Effect of Erxian Decoction-containing serum on H_2O_2-induced proliferation and osteogenic differentiation of MC3T3-E1 cells via BK channels].

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 µmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.

Er-xian decoction drug-containing serum promotes Mc3t3-e1 cell proliferation and osteogenic differentiation via regulating BK channel.

ETHNOPHARMACOLOGICAL RELEVANCE: Er-xian Decoction (EXD) is a well-known prescription widely used to prevent and treat climacteric syndrome and osteoporosis in China. BK channel positively affects osteoblast bone formation in vitro. However, it is still unclear whether the effect of EXD on promoting osteoblasts osteogenic differentiation is related to BK channel. AIM OF THE STUDY: The study is aimed at determining whether the EXD-containing serum promotes the proliferation of osteoblasts and their differentiation through BK channel. MATERIALS AND METHODS: The chemical compounds of EXD were analyzed by UPLC-Q-TOF/MS. An osteogenic induction medium (OM) was used to induce MC3T3-E1 cells' osteogenic differentiation. The effects of EXD-containing serum and tetraethylammonium (TEA) on the proliferation activity of Mc3t3-e1 cells were detected by CCK-8 assay. ALP activity was determined by an alkaline phosphatase kit. The protein expression (BMP2, OPG, and COL1) was analyzed by Western blot, and the mRNA expression (Runx2, OPG, and BMP2) was assessed by real-time PCR. Alizarin red was used to stain the mineralized region of osteoblasts. In addition, we analyzed the relationship between BK channel and its downstream PTEN/Akt/Foxo1 signaling pathway. RESULTS: 72 compounds were identified by UPLC-Q-TOF/MS analysis in EXD. Mangiferin, ferulic acid, berberine, and icariin were main active components of EXD. EXD-containing serum could enhance the cell viability of MC3T3-E1 cells by decreasing the expression of BKα protein. EXD-containing serum increased ALP activity and calcium nodule formation of Mc3t3-e1 cells, promoted the protein expression of BKα, COL1, BMP2, OPG, and the mRNA expression of RUNX2, OPG, and BMP2, however, these effects can be reversed after adding TEA. In addition, EXD-containing serum could upregulate phosphorylation of Akt and Foxo1 in osteogenic-induced Mc3t3-e1 cells, and lower the expression of PTEN. And these effects of EXD-containing serum could be reduced by TEA. CONCLUSIONS: The effect of EXD-containing serum on promoting cell proliferation and osteogenic differentiation of Mc3t3-e1 cells might be linked to the regulation of BK channel.

The Effect of 40-Hz White LED Therapy on Structure-Function of Brain Mitochondrial ATP-Sensitive Ca-Activated Large-Conductance Potassium Channel in Amyloid Beta Toxicity.

Photobiomodulation therapy has become the focus of medical research in many areas such as Alzheimer's disease (AD), because of its modulatory effect on cellular processes through light energy absorption via photoreceptors/chromophores located in the mitochondria. However, there are still many questions around the underlying mechanisms. This study was carried out to unravel whether the function-structure of ATP-sensitive mitoBKCa channels, as crucial components for maintenance of mitochondrial homeostasis, can be altered subsequent to light therapy in AD. Induction of Aß neurotoxicity in male Wistar rats was done by intracerebroventricular injection of Aß1-42. After a week, light-treated rats were exposed to 40-Hz white light LEDs, 15 min for 7 days. Electrophysiological properties of mitoBKCa channel were investigated using a channel incorporated into the bilayer lipid membrane, and mitoBKCa-ß2 subunit expression was determined using western blot analysis in Aß-induced toxicity and light-treated rats. Our results describe that conductance and open probability (Po) of mitoBKCa channel decreased significantly and was accompanied by a Po curve rightward shift in mitochondrial preparation in Aß-induced toxicity rats. We also showed a significant reduction in expression of mitoBKCa-ß2 subunit, which is partly responsible for a leftward shift in BKCa Po curve in low calcium status. Interestingly, we provided evidence of a significant improvement in channel conductance and Po after light therapy. We also found that light therapy improved mitoBKCa-ß2 subunit expression, increasing it close to saline group. The current study explains a light therapy improvement in brain mitoBKCa channel function in the Aß-induced neurotoxicity rat model, an effect that can be linked to increased expression of ß2 subunit.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

Glabridin Alleviates Inflammation and Nociception in Rodents by Activating BKCa Channels and Reducing NO Levels.

Inflammation, and the pain that accompanies it, is a natural response of the body. The licorice plant (Glycyrrhiza glabra) have demonstrated anti-inflammatory, anti-edematous, and anti-nociceptive effects of its extracts. The effective ingredient remains unidentified; however, one possibility is the unique isoflavone glabridin. The anti-nociceptive, and anti-inflammatory effects of glabridin and its possible mechanism with focus on the large conductance Ca2+-activated K+ (BKCa) channels and L-arginine-nitric oxide (NO) pathway were examined by using different tests. In order to determine the anti-edematous, anti-nociceptive, and anti-oxidative effects of glabradin, some tests such as the tail flick, hotplate, carrageenan-induced paw edema, air pouch, acetic-acid-induced writhing, formalin, and capsaicin tests, as well as toxicity and open field tests were made. Glabridin was administered to rats (n = 8) or mice (n = 8) for 3 d at 3 doses (10, 20, and 40 mg/kg). Glabridin inhibited cytokine production and showed an anti-nociceptive response via the activating of BKCa channels and downregulating NO level and partially transient receptor potential vanilloid-1 pathways. It also demonstrated anti-inflammatory effects by inhibiting cyclooxygenase (COX) activity, while showing no cytotoxicity. Glabridin, however, showed no anti-nociceptive effect in the neurogenic phase. Glabridin is a promising substance in terms of its anti-nociceptive and anti-inflammatory effects by disrupting peripheral NO production, inhibiting cyclic guanosine monophosphate (cGMP) activation and activating BKCa channels and its lack of acute and subacute toxic effects.

Herbal components of Japanese Kampo medicines exert laxative actions in colonic epithelium cells via activation of BK and CFTR channels.

Japanese Kampo medicines Junchoto and Mashiningan are mixtures of numerous herbal plant extracts and empirically known to exert laxative actions by stimulating fluid secretion in the colonic epithelium. However, it is unknown which and how the herbal components of these crude Kampo drugs are effective to stimulate ion effluxes causing fluid secretion. Here, we selected four herbal components of Junchoto and Mashiningan, Mashinin (MSN), Kyonin (KYN), Tonin (TON), and Daio (DIO), which are putatively laxatives, and examined their effects on the ion channel activity of human colonic epithelial Caco-2 cells. Patch clamp analyses revealed that MSN activated whole-cell current characteristics of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, whereas KYN, TON, and DIO activated the large-conductance and voltage-activated K+ (BK) channel. Furthermore, electronic cell sizing showed that MSN induced secretory volume decrease (SVD) sensitivity to a CFTR blocker, whereas TON, KYN, and DIO induced SVD sensitivity to a K+ channel blocker. In conclusion, MSN and TON, KYN, and DIO promote fluid secretion from colonic epithelial cells by activating CFTR and BK channels. Thus, Japanese Kampo medicines, Junchoto and Mashiningan, exert anti-constipation actions by inducing KCl efflux through the combined actions of CFTR- and BK-stimulating herbal components.

Ginkgo biloba Pretreatment Attenuates Myocardial Ischemia-Reperfusion Injury via mitoBKCa.

Ginkgo biloba extracts (EGb) alleviate myocardial ischemia/reperfusion (MI/R) injury. However, the underlying mechanisms have not yet been characterized. This study aimed to investigate whether activation of large-conductance Ca2+-activated K+ channels at the inner mitochondrial membrane (mitoBKCa) of cardiomyocytes is involved in Ginkgo biloba extract-mediated cardioprotection. Shuxuening injection (SXNI, 12.5ml/kg/d), a widely prescribed herbal medicine containing Ginkgo biloba extracts in China, or vehicle, was administered to C57BL/6 mice via tail vein injection for one week prior to surgical procedures. The mitoBKCa blocker paxilline (PAX) (1ml/kg, 115 nM) was administered via tail vein injection 30min prior to the onset of ischemia. The mice were randomly divided into the following groups: Sham, MI/R, MI/R+SXNI, and MI/R+SXNI+PAX. MI/R was induced by ligating the left anterior descending coronary artery for 30min with subsequent reperfusion for 24h. SXNI pretreatment conferred cardioprotective effects against MI/R injury as evidenced by reduced infarct size, improved cardiac function, and improved mitochondrial function. However, these effects were abrogated by co-administration with PAX. In addition, activation of mitoBKCa by Ginkgo biloba extract EGb761 reduced hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury in vitro through the inhibition of mitochondrial fragmentation, restoration of the mitochondrial membrane potential, decreased generation of superoxide, and inhibition of apoptosis which is associated with alleviating mitochondrial Ca2+ overload. These results indicated that Ginkgo biloba extracts pretreatment protected against MI/R injury via activation of mitoBKCa.

Serotonergic Signaling Controls Input-Specific Synaptic Plasticity at Striatal Circuits.

Monoaminergic modulation of cortical and thalamic inputs to the dorsal striatum (DS) is crucial for reward-based learning and action control. While dopamine has been extensively investigated in this context, the synaptic effects of serotonin (5-HT) have been largely unexplored. Here, we investigated how serotonergic signaling affects associative plasticity at glutamatergic synapses on the striatal projection neurons of the direct pathway (dSPNs). Combining chemogenetic and optogenetic approaches reveals that impeding serotonergic signaling preferentially gates spike-timing-dependent long-term depression (t-LTD) at thalamostriatal synapses. This t-LTD requires dampened activity of the 5-HT4 receptor subtype, which we demonstrate controls dendritic Ca2+ signals by regulating BK channel activity, and which preferentially localizes at the dendritic shaft. The synaptic effects of 5-HT signaling at thalamostriatal inputs provide insights into how changes in serotonergic levels associated with behavioral states or pathology affect striatal-dependent processes.

[Downregulation of large conductance calcium-activated potassium channels in paraventricular nucleus contributes to sympathoexcitation in rats with chronic heart failure].

Objective: To elucidate the association between large conductance calcium-activated potassium channels (BKCa) in the paraventricular hypothalamic nucleus (PVN) and sympathetic outflow in rats with chronic heart failure (CHF) . Methods: Male Wistar rats (6-7 weeks old) were randomized to sham operated group and CHF group (coronary artery ligation) . Two weeks after operation, BKCa inhibitor Iberiotoxin (IBTX) was infused into PVN by osmotic minipumps, rats were divided into following groups: sham+aCSF, CHF+aCSF, sham+low dose IBTX (0.125 nmol/nl) , CHF+low dose IBTX, sham+moderate dose IBTX (1.25 nmol/nl) , CHF+moderate dose IBTX, sham+ high dose IBTX (12.5 nmol/nl) , and CHF+high dose IBTX (n=6 each) . Additional rats were grouped as follows: sham+vehicle, sham+KCNMB4 knockdown (by rAAV2-KCNMB4 shRNA virus injection in PVN) , CHF+vehicle, CHF+ KCNMB4 knockdown group (n=6 each) . The cardiac function was determined by echocardiography, left ventricular hemodynamics were measured invasively, renal sympathetic nerve activity (RSNA) was recorded at 6 weeks after coronary artery ligation or sham operation. The contents of norepinephrine (NE) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in plasma were determined by enzyme-linked immunosorbent assay. The protein and mRNA expression of KCNMB4 in PVN were measured by immunofluorescence staining, Western blot, and real-time PCR, mRNA expression of BKCa in PVN was detected by real-time PCR. Results: Compared with the sham operation group, the cardiac function of the heart failure group was significantly reduced (P<0.05) , and the plasma NE and the serum NT-proBNP were significantly elevated (P<0.05) . The protein and mRNA expression of KCNMB4 in PVN were obviously down-regulated in CHF rats (P<0.05) . After perfusion of IBTX or KCNMB4 knockdown by microinjection of rAAV2-KCNMB4 shRNA virus,right ventricular weight/body weight and lung weight/body weight ratio as well as left ventricular end-diastolic diameter were increased and left ventricular ejection fraction was decreased (all P<0.05) , the sympathetic driving indexes was increased in sham rats, changes of these parameters further aggravated in CHF rats (P<0.05) . KCNMB4 knockdown further downregulated protein expression in PVN of CHF rats. Conclusion: Downregulation and blunted function of BKCa in PVN may contribute to sympathoexcitation and deterioration of cardiac function in rats with chronic heart failure.

The Yin and Yang of BK Channels in Epilepsy.

BACKGROUND & OBJECTIVE: The large conductance calcium-activated potassium (BK) channel, extensively distributed in the central nervous system (CNS), is considered as a vital player in the pathogenesis of epilepsy, with evidence implicating derangement of K+ as well as regulating action potential shape and duration. However, unlike other channels implicated in epilepsy whose function in neurons could clearly be labeled "excitatory" or "inhibitory", the unique physiological behavior of the BK channel allows it to both augment and decrease the excitability of neurons. Thus, the role of BK in epilepsy is controversial so far, and a growing area of intense investigation. CONCLUSION: Here, this review aims to highlight recent discoveries on the dichotomous role of BK channels in epilepsy, focusing on relevant BK-dependent pro- as well as antiepileptic pathways, and discuss the potential of BK specific modulators for the treatment of epilepsy.

BK Channels Mediate Synaptic Plasticity Underlying Habituation in Rats.

Habituation is a basic form of implicit learning and represents a sensory filter that is disrupted in autism, schizophrenia, and several other mental disorders. Despite extensive research in the past decades on habituation of startle and other escape responses, the underlying neural mechanisms are still not fully understood. There is evidence from previous studies indicating that BK channels might play a critical role in habituation. We here used a wide array of approaches to test this hypothesis. We show that BK channel activation and subsequent phosphorylation of these channels are essential for synaptic depression presumably underlying startle habituation in rats, using patch-clamp recordings and voltage-sensitive dye imaging in slices. Furthermore, positive modulation of BK channels in vivo can enhance short-term habituation. Although results using different approaches do not always perfectly align, together they provide convincing evidence for a crucial role of BK channel phosphorylation in synaptic depression underlying short-term habituation of startle. We also show that this mechanism can be targeted to enhance short-term habituation and therefore to potentially ameliorate sensory filtering deficits associated with psychiatric disorders.SIGNIFICANCE STATEMENT Short-term habituation is the most fundamental form of implicit learning. Habituation also represents a filter for inundating sensory information, which is disrupted in autism, schizophrenia, and other psychiatric disorders. Habituation has been studied in different organisms and behavioral models and is thought to be caused by synaptic depression in respective pathways. The underlying molecular mechanisms, however, are poorly understood. We here identify, for the first time, a BK channel-dependent molecular synaptic mechanism leading to synaptic depression that is crucial for habituation, and we discuss the significance of our findings for potential treatments enhancing habituation.

Active integration of glutamatergic input to the inferior olive generates bidirectional postsynaptic potentials.

KEY POINTS: We establish experimental preparations for optogenetic investigation of glutamatergic input to the inferior olive. Neurones in the principal olivary nucleus receive monosynaptic extra-somatic glutamatergic input from the neocortex. Glutamatergic inputs to neurones in the inferior olive generate bidirectional postsynaptic potentials (PSPs), with a fast excitatory component followed by a slower inhibitory component. Small conductance calcium-activated potassium (SK) channels are required for the slow inhibitory component of glutamatergic PSPs and oppose temporal summation of inputs at intervals ≤ 20 ms. Active integration of synaptic input within the inferior olive may play a central role in control of olivo-cerebellar climbing fibre signals. ABSTRACT: The inferior olive plays a critical role in motor coordination and learning by integrating diverse afferent signals to generate climbing fibre inputs to the cerebellar cortex. While it is well established that climbing fibre signals are important for motor coordination, the mechanisms by which neurones in the inferior olive integrate synaptic inputs and the roles of particular ion channels are unclear. Here, we test the hypothesis that neurones in the inferior olive actively integrate glutamatergic synaptic inputs. We demonstrate that optogenetically activated long-range synaptic inputs to the inferior olive, including projections from the motor cortex, generate rapid excitatory potentials followed by slower inhibitory potentials. Synaptic projections from the motor cortex preferentially target the principal olivary nucleus. We show that inhibitory and excitatory components of the bidirectional synaptic potentials are dependent upon AMPA (GluA) receptors, are GABAA independent, and originate from the same presynaptic axons. Consistent with models that predict active integration of synaptic inputs by inferior olive neurones, we find that the inhibitory component is reduced by blocking large conductance calcium-activated potassium channels with iberiotoxin, and is abolished by blocking small conductance calcium-activated potassium channels with apamin. Summation of excitatory components of synaptic responses to inputs at intervals ≤ 20 ms is increased by apamin, suggesting a role for the inhibitory component of glutamatergic responses in temporal integration. Our results indicate that neurones in the inferior olive implement novel rules for synaptic integration and suggest new principles for the contribution of inferior olive neurones to coordinated motor behaviours.

Urinary Bladder-Relaxant Effect of Kurarinone Depending on Potentiation of Large-Conductance Ca2+-Activated K+ Channels.

The large-conductance calcium-activated potassium channel (BKCa channel) plays critical roles in smooth muscle relaxation. In urinary bladder smooth muscle, BKCa channel activity underlies the maintenance of the resting membrane potential and repolarization of the spontaneous action potential triggering the phasic contraction. To identify novel BKCa channel activators, we screened a library of natural compounds using a cell-based fluorescence assay and a hyperactive mutant BKCa channel (Lee et al., 2013). From 794 natural compounds, kurarinone, a flavanone from Sophora flavescens, strongly potentiated BKCa channels. When treated from the extracellular side, this compound progressively shifted the conductance-voltage relationship of BKCa channels to more negative voltages and increased the maximum conductance in a dose-dependent manner. Whereas kurarinone strongly potentiated the homomeric BKCa channel composed of only the α subunit, its effects were much smaller on heteromeric channels coassembled with auxiliary ß subunits. Although the activation kinetics was not altered significantly, the deactivation of BKCa channels was dramatically slowed by kurarinone treatment. At the single-channel level, kurarinone increased the open probability of the BKCa channel without affecting its single-channel conductance. Kurarinone potently relaxed acetylcholine-induced contraction of rat bladder smooth muscle and thus decreased the micturition frequency of rats with overactive bladder symptoms. These results indicate that kurarinone can directly potentiate BKCa channels and demonstrate the therapeutic potentials of kurarinone and its derivatives for developing antioveractive bladder medications and supplements.

BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function.

Large-conductance Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly isolated human DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 µM) increased TBKC activity in human DSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 µM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 µM). 8MM-IBMX (10 µM) hyperpolarized the DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.

Chronic fetal exposure to caffeine altered resistance vessel functions via RyRs-BKCa down-regulation in rat offspring.

Caffeine modifies vascular/cardiac contractility. Embryonic exposure to caffeine altered cardiac functions in offspring. This study determined chronic influence of prenatal caffeine on vessel functions in offspring. Pregnant Sprague-Dawley rats (5-month-old) were exposed to high dose of caffeine, their offspring (5-month-old) were tested for vascular functions in mesenteric arteries (MA) and ion channel activities in smooth muscle cells. Prenatal exposure to caffeine increased pressor responses and vasoconstrictions to phenylephrine, accompanied by enhanced membrane depolarization. Large conductance Ca2(+)-activated K(+) (BKCa) channels in buffering phenylephrine-induced vasoconstrictions was decreased, whole cell BKCa currents and spontaneous transient outward currents (STOCs) were decreased. Single channel recordings revealed reduced voltage/Ca(2+) sensitivity of BKCa channels. BKCa α-subunit expression was unchanged, BKCa ß1-subunit and sensitivity of BKCa to tamoxifen were reduced in the caffeine offspring as altered biophysical properties of BKCa in the MA. Simultaneous [Ca(2+)]i fluorescence and vasoconstriction testing showed reduced Ca(2+), leading to diminished BKCa activation via ryanodine receptor Ca(2+) release channels (RyRs), causing enhanced vascular tone. Reduced RyR1 was greater than that of RyR3. The results suggest that the altered STOCs activity in the caffeine offspring could attribute to down-regulation of RyRs-BKCa, providing new information for further understanding increased risks of hypertension in developmental origins.

Cognitive Improvement by Photic Stimulation in a Mouse Model of Alzheimer's Disease.

We previously reported that activity of the large conductance calcium-activated potassium (big-K, BK) channel is suppressed by intracellular Aß in cortical pyramidal cells, and that this suppression was reversed by expression of the scaffold protein Homer1a in 3xTg Alzheimer's disease model mice. Homer1a is known to be expressed by physiological photic stimulation (PS) as well. The possibility thus arises that PS also reverses Aß-induced suppression of BK channels, and thereby improves cognition in 3xTg mice. This possibility was tested here. Chronic application of 6-hour-long PS (frequency, 2 Hz; duty cycle, about 1/10; luminance, 300 lx) daily for 4 weeks improved contextual and tone-dependent fear memory in 3xTg mice and, to a lesser extent, Morris water maze performance as well. Hippocampal long-term potentiation was also enhanced after PS. BK channel activity in cingulate cortex pyramidal cells and lateral amygdalar principal cells, suppressed in 3xTg mice, were facilitated. In parallel, neuronal excitability, elevated in 3xTg mice, was recovered to the control level. Gene expression of BK channel, as well as that of the scaffold protein Homer1a, was found decreased in 3xTg mice and reversed by PS. It is known that Homer1a is an activity-dependently inducible immediate early gene product. Consistently, our previous findings showed that Homer1a induced by electrical stimulation facilitated BK channels. By using Homer1a knockouts, we showed that the present PS-induced BK channel facilitation is mediated by Homer1a expression. We thus propose that PS might be potentially useful as a non-invasive therapeutic measure against Alzheimer's disease.

Structure-activity relationship of Baifuzi-cerebrosides on BKCa channel activation.

Our previous study reported that a mixture of cerebrosides from traditional Chinese medicine Baifuzi could activate BKCa channel. It is curious to know the effect of each single cerebroside on the channel. Here we isolated 5 pure cerebrosides from the mixture and determined their chemical structures. The most potent one increased the single channel open probability 6 folds with EC50 value of 1.0 µM. The structure-activity relationship revealed that acyl chain length of cerebrosides has potent effect, while configuration of double bond at C8-C9 on their long chain base has weak effect on the channel activity. Thus, this research provides a guide for design and synthesis of a lead cerebroside with more potent effect on the BKCa channel.

Plasticity in ion channel expression underlies variation in hearing during reproductive cycles.

Sensory plasticity related to reproductive state, hormonal profiles, and experience is widespread among vertebrates, including humans. Improvements in audio-vocal coupling that heighten the detection of conspecifics are part of the reproductive strategy of many nonmammalian vertebrates. Although seasonal changes in hearing are known, molecular mechanisms determining this form of adult sensory plasticity remain elusive. Among both nonmammals and mammals, large-conductance, calcium-activated potassium (BK) channels underlie a primary outward current having a predominant influence on frequency tuning in auditory hair cells. We now report an example from fish showing that increased BK channel abundance can improve an individual's ability to hear vocalizations during the breeding season. Pharmacological manipulations targeting BK channels, together with measures of BK transcript abundance, can explain the seasonal enhancement of auditory hair cell sensitivity to the frequency content of calls. Plasticity in ion channel expression is a simple, evolutionarily labile solution for sculpting sensory bandwidth to maximize the detection of conspecific signals during reproductive cycles.

Ca(2+)-dependent large conductance K(+) currents in thalamocortical relay neurons of different rat strains.

Mutations in genes coding for Ca(2+) channels were found in patients with childhood absence epilepsy (CAE) indicating a contribution of Ca(2+)-dependent mechanisms to the generation of spike-wave discharges (SWD) in humans. Since the involvement of Ca(2+) signals remains unclear, the aim of the present study was to elucidate the function of a Ca(2+)-dependent K(+) channel (BKCa) under physiological conditions and in the pathophysiological state of CAE. The activation of BKCa channels is dependent on both voltage and intracellular Ca(2+) concentrations. Moreover, these channels exhibit an outstandingly high level of regulatory heterogeneity that builds the basis for the influence of BKCa channels on different aspects of neuronal activity. Here, we analyse the contribution of BKCa channels to firing of thalamocortical relay neurons, and we test the hypothesis that BKCa channel activity affects the phenotype of a genetic rat model of CAE. We found that the activation of the ß2-adrenergic receptor/protein kinase A pathway resulted in BKCa channel inhibition. Furthermore, BKCa channels affect the number of action potentials fired in a burst and produced spike frequency adaptation during tonic activity. The latter result was confirmed by a computer modelling approach. We demonstrate that the ß2-adrenergic inhibition of BKCa channels prevents spike frequency adaptation and, thus, might significantly support the tonic firing mode of thalamocortical relay neurons. In addition, we show that BKCa channel functioning differs in epileptic WAG/Rij and thereby likely contributes to highly synchronised, epileptic network activity.

Effects of ketamine and its metabolites on ion currents in differentiated hippocampal H19-7 neuronal cells and in HEK293T cells transfected with α-hslo subunit.

Ketamine (KT), a dissociative anesthetic, is known to induce schizophrenia-like psychosis. The percentage of KT abuse has recently grown fast despite KT being a controlled drug. The mechanism of KT actions is related to the inhibition of NMDA receptors. Whether KT produces other effects on ion currents in hippocampal neurons remains unclear. In this study, we attempted to evaluate the possible effects of KT and other related compounds on ion currents in hippocampal neuron-derived H19-7 cells. This drug exerted an inhibitory effect on Ca(2+)-activated K(+) current (IK(Ca)) in these cells with an IC(50) value of 274 µM. Pimaric acid (30 µM) or abietic acid (30 µM), known to stimulate large-conductance Ca(2+)-activated K(+) channels, reversed KT-induced inhibition of I(K)(Ca). In HEK293T cells expressing a-humans low poke, KT-induced inhibition of I(K)(Ca) still existed. Dehydronorketamine (300 µM) had little or no effect on the IK(Ca) amplitude, while norketamine (300 µM) slightly but significantly suppressed it. In inside­out configuration, KT applied to the intracellular face of the membrane did not alter single channel conductance of large-conductance Ca(2+)-activated K(+) (BKCa) channels; however, it did significantly reduce the probability of channel openings. Addition of KT was effective in depressing the peak amplitude of voltage-gated Na(+) current. Moreover, the presence of KT was noted to enhance the amplitude of membrane electroporation-induced inward currents (IMEP) in differentiated H19-7 cells. KT-stimulated IMEP was reversed by further application of LaCl(3) (100 µM), but not by NMDA (30 µM). The modulations by this compound of ion channels may contribute to the underlying mechanisms through which KT and its metabolites influence the electrical behavior of hippocampal neurons if similar findings occur in vivo.