Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa.

Cannabidiol (CBD), one of the main Cannabis sativa bioactive compounds, is utilized in the treatment of major epileptic syndromes. Its efficacy can be attributed to a multimodal mechanism of action that includes, as potential targets, several types of ion channels. In the brain, CBD reduces the firing frequency in rat hippocampal neurons, partly prolonging the duration of action potentials, suggesting a potential blockade of voltage-operated K+ channels. We postulate that this effect might involve the inhibition of the large-conductance voltage- and Ca2+-operated K+ channel (BK channel), which plays a role in the neuronal action potential's repolarization. Thus, we assessed the impact of CBD on the BK channel activity, heterologously expressed in HEK293 cells. Our findings, using the patch-clamp technique, revealed that CBD inhibits BK channel currents in a concentration-dependent manner with an IC50 of 280 nM. The inhibition is through a direct interaction, reducing both the unitary conductance and voltage-dependent activation of the channel. Additionally, the cannabinoid significantly delays channel activation kinetics, indicating stabilization of the closed state. These effects could explain the changes induced by CBD in action potential shape and duration, and they may contribute to the observed anticonvulsant activity of this cannabinoid.

Neuronal mechanism of a BK channelopathy in absence epilepsy and dyskinesia.

A growing number of gain-of-function (GOF) BK channelopathies have been identified in patients with epilepsy and movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here, we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and dyskinesia. The BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits and dyskinesia-like behaviors. The cortical pyramidal neurons and cerebellar Purkinje cells from the BK-D434G mice show hyperexcitability, which likely contributes to the pathogenesis of absence seizures and paroxysmal dyskinesia. A BK channel blocker, paxilline, potently suppresses BK-D434G­induced hyperexcitability and effectively mitigates absence seizures and locomotor deficits in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and dyskinesia. Our findings also suggest that BK inhibition is a promising therapeutic strategy for mitigating BK GOF-induced neurological disorders.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

Cytoprotective effects of the flavonoid quercetin by activating mitochondrial BKCa channels in endothelial cells.

Mitochondrial potassium channels have been implicated in cytoprotective mechanisms. Activation of the mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channel is important for protecting brain tissue against stroke damage as well as heart tissue against ischemia damage. In this paper, we examine the effect of the natural flavonoid quercetin as an activator of the mitoBKCa channel. Quercetin has a beneficial effect on many processes in the human body and interacts with many receptors and signaling pathways. We found that quercetin acts on mitochondria as a mitoBKCa channel opener. The activation observed with the patch-clamp technique was potent and increased the channel open probability from approximately 0.35 to 0.95 at + 40 mV in the micromolar concentration range. Moreover, quercetin at a concentration of 10 µM protected cells by reducing damage from treatment factors (tumor necrosis factor α and cycloheximide) by 40%, enhancing cellular migration and depolarizing the mitochondrial membrane. Moreover, the presence of quercetin increased the gene expression and protein level of the mitoBKCa ß3 regulatory subunit. The observed cytoprotective effects suggested the involvement of BKCa channel activation. Additionally, the newly discovered mitoBKCa activator quercetin elucidates a new mitochondrial pathway that is beneficial for vascular endothelial cells.

Emodepside targets SLO-1 channels of Onchocerca ochengi and induces broad anthelmintic effects in a bovine model of onchocerciasis.

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness.

Molecular mechanism of apelin-13 regulation of colonic motility in rats.

Apelin is a novel neuropeptide identified as the endogenous ligand for the apelin receptor. Apelin and its receptor are widely distributed in the gastrointestinal tract. Studies have reported that apelin-13 is involved in modulating gastrointestinal motility; however, the evidence is insufficient and the relevant mechanism is still not fully clear. Consequently, our study designed to explore the effect induced by exogenous apelin-13, to analyze the mechanism of action in isolated rat colons and colonic smooth muscle cells. The spontaneous contractions of colonic smooth muscle strips from rat were measured in an organ bath system. L-type calcium currents and large conductance Ca2+-activated K+ (BKCa) currents in rat colonic smooth muscle cells were investigated using the electrophysiological patch-clamp technique. Apelin-13 decreased the spontaneous contractile activity of colonic smooth muscle strips in a dose-dependent manner, and the inhibitory effect was not abolished by tetrodotoxin. The electrophysiological recordings revealed that apelin-13 reduced the crest currents of L-type calcium in a concentration-dependent manner in colonic smooth muscle cells at the test potential of 0 mV. Moreover, apelin-13 moved the current-voltage (I-V) curves of L-type calcium channels upward, but did not change their contour. Furthermore, the characteristics of L-type calcium channels with steady-state activation and steady-state inactivation were not significantly changed. Similarly, application of apelin-13 also significantly decreased BKCa currents in a concentration-dependent manner. In conclusion, apelin-13 inhibited the spontaneous contractile activity of isolated rat colons via the suppression of L-type calcium channels and BKCa channels in colonic smooth muscle cells.

Vasodilator effects of sulforaphane in cerebral circulation: A critical role of endogenously produced hydrogen sulfide and arteriolar smooth muscle KATP and BK channels in the brain.

We investigated the effects of sulforaphane (SFN), an isothiocyanate from cruciferous vegetables, in the regulation of cerebral blood flow using cranial windows in newborn pigs. SFN administered topically (10 µM-1 mM) or systemically (0.4 mg/kg ip) caused immediate and sustained dilation of pial arterioles concomitantly with elevated H2S in periarachnoid cortical cerebrospinal fluid. H2S is a potent vasodilator of cerebral arterioles. SFN is not a H2S donor but it acts via stimulating H2S generation in the brain catalyzed by cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS). CSE/CBS inhibitors propargylglycine, ß-cyano-L-alanine, and aminooxyacetic acid blocked brain H2S generation and cerebral vasodilation caused by SFN. The SFN-elicited vasodilation requires activation of potassium channels in cerebral arterioles. The inhibitors of KATP and BK channels glibenclamide, paxilline, and iberiotoxin blocked the vasodilator effects of topical and systemic SFN, supporting the concept that H2S is the mediator of the vasodilator properties of SFN in cerebral circulation. Overall, we provide first evidence that SFN is a brain permeable compound that increases cerebral blood flow via a non-genomic mechanism that is mediated via activation of CSE/CBS-catalyzed H2S formation in neurovascular cells followed by H2S-induced activation of KATP and BK channels in arteriolar smooth muscle.

Comparative gain-of-function effects of the KCNMA1-N999S mutation on human BK channel properties.

KCNMA1, encoding the voltage- and calcium-activated potassium channel, has a pivotal role in brain physiology. Mutations in KCNMA1 are associated with epilepsy and/or dyskinesia (PNKD3). Two KCNMA1 mutations correlated with these phenotypes, D434G and N999S, were previously identified as producing gain-of-function (GOF) effects on BK channel activity. Three new patients have been reported harboring N999S, one carrying a second mutation, R1128W, but the effects of these mutations have not yet been reported under physiological K+ conditions or compared to D434G. In this study, we characterize N999S, the novel N999S/R1128W double mutation, and D434G in a brain BK channel splice variant, comparing the effects on BK current properties under a physiological K+ gradient with action potential voltage commands. N999S, N999S/R1128W, and D434G cDNAs were expressed in HEK293T cells and characterized by patch-clamp electrophysiology. N999S BK currents were shifted to negative potentials, with faster activation and slower deactivation compared with wild type (WT) and D434G. The double mutation N999S/R1128W did not show any additional changes in current properties compared with N999S alone. The antiepileptic drug acetazolamide was assessed for its ability to directly modulate WT and N999S channels. Neither the WT nor N999S channels were sensitive to the antiepileptic drug acetazolamide, but both were sensitive to the inhibitor paxilline. We conclude that N999S is a strong GOF mutation that surpasses the D434G phenotype, without mitigation by R1128W. Acetazolamide has no direct modulatory action on either WT or N999S channels, indicating that its use may not be contraindicated in patients harboring GOF KCNMA1 mutations.NEW & NOTEWORTHYKCNMA1-linked channelopathy is a new neurological disorder characterized by mutations in the BK voltage- and calcium-activated potassium channel. The epilepsy- and dyskinesia-associated gain-of-function mutations N999S and D434G comprise the largest number of patients in the cohort. This study provides the first direct comparison between D434G and N999S BK channel properties as well as a novel double mutation, N999S/R1128W, from another patient, defining the functional effects during an action potential stimulus.

The functionally relevant site for paxilline inhibition of BK channels.

The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca2+-dependent BK-type K+ channel. PAX inhibits BK channels by selective interaction with closed states. BK inhibition by PAX is best characterized by the idea that PAX gains access to the channel through the central cavity of the BK channel, and that only a single PAX molecule can interact with the BK channel at a time. The notion that PAX reaches its binding site via the central cavity and involves only a single PAX molecule would be consistent with binding on the axis of the permeation pathway, similar to classical open channel block and inconsistent with the observation that PAX selectively inhibits closed channels. To explore the potential sites of interaction of PAX with the BK channel, we undertook a computational analysis of the interaction of PAX with the BK channel pore gate domain guided by recently available liganded (open) and metal-free (closed) Aplysia BK channel structures. The analysis unambiguously identified a preferred position of PAX occupancy that accounts for all previously described features of PAX inhibition, including state dependence, G311 sensitivity, stoichiometry, and central cavity accessibility. This PAX-binding pose in closed BK channels is supported by additional functional results.

LRRC52 regulates BK channel function and localization in mouse cochlear inner hair cells.

The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca2+- and voltage-activated large conductance Ca2+-activated K+ channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (-60 mV) even in the absence of intracellular Ca2+ elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca2+ channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat-containing (LRRC) regulatory γ subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs from Lrrc26 knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs from Lrrc52 KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK α subunits shifts BK current gating about -90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning.

The absence of NIPA2 enhances neural excitability through BK (big potassium) channels.

AIM: To reveal the pathogenesis and find the precision treatment for the childhood absence epilepsy (CAE) patients with NIPA2 mutations. METHODS: We performed whole-cell patch-clamp recordings to measure the electrophysiological properties of layer V neocortical somatosensory pyramidal neurons in wild-type (WT) and NIPA2-knockout mice. RESULTS: We identified that layer V neocortical somatosensory pyramidal neurons isolated from the NIPA2-knockout mice displayed higher frequency of spontaneous and evoked action potential, broader half-width of evoked action potential, and smaller currents of BK channels than those from the WT mice. NS11021, a specific BK channel opener, reduced neuronal excitability in the NIPA2-knockout mice. Paxilline, a selective BK channel blocker, treated WT neurons and could simulate the situation of NIPA2-knockout group, thereby suggesting that the absence of NIPA2 enhanced the excitability of neocortical somatosensory pyramidal neurons by decreasing the currents of BK channels. Zonisamide, an anti-epilepsy drug, reduced action potential firing in NIPA2-knockout mice through increasing BK channel currents. CONCLUSION: The results indicate that the absence of NIPA2 enhances neural excitability through BK channels. Zonisamide is probably a potential treatment for NIPA2 mutation-induced epilepsy, which may provide a basis for the development of new treatment strategies for epilepsy.

Cannabinoids-induced peripheral analgesia depends on activation of BK channels.

The endogenous cannabinoid system is involved in the physiological inhibitory control of pain and is of particular interest for the development of therapeutic approaches for pain management. Selective activation of the peripheral CB1 cannabinoid receptor has been shown to suppress the heightened firing of primary afferents, which is the peripheral mechanism underlying neuropathic pain after nerve injury. However, the mechanism underlying this effect of CB1 receptor remains unclear. The large-conductance calcium-activated potassium (BK) channels have been reported to participate in anticonvulsant and vasorelaxant effects of cannabinoids. We asked whether BK channels participate in cannabinoids-induced analgesia and firing-suppressing effects in primary afferents after nerve injury. Here, using mice with chronic constriction injury (CCI)-induced neuropathic pain, antinociception action and firing-suppressing effect of HU210 were measured before and after BK channel blocker application. We found that local peripheral application of HU210 alleviated CCI-induced pain behavior and suppressed the heightened firing of injured fibers. Co-administration of IBTX with HU210 significantly reversed the analgesia and the firing-suppressing effect of HU210. This result indicated that the peripheral analgesic effects of cannabinoids depends on activation of BK channels.

Liposomal quercetin potentiates maxi-K channel openings in smooth muscles and restores its activity after oxidative stress.

The effects of quercetin-loaded liposomes (PCL-Q) and their constituents, that is, free quercetin (Q) and 'empty' phosphatidylcholine vesicles (PCL), on maxi-K channel activity were studied in single mouse ileal myocytes before and after H2O2-induced oxidative stress. Macroscopic Maxi-K channel currents were recorded using whole-cell patch clamp techniques, while single BKCa channel currents were recorded in the cell-attached configuration. Bath application of PCL-Q (100 µg/ml of lipid and 3 µg/ml of quercetin) increased single Maxi-K channel activity more than threefold, from 0.010 ± 0.003 to 0.034 ± 0.004 (n = 5; p < 0.05), whereas single-channel conductance increased non-significantly from 138 to 146 pS. In the presence of PCL-Q multiple simultaneous channel openings were observed, with up to eight active channels in the membrane patch. Surprisingly, 'empty' PCL (100 µg/ml) also produced some channel activation, although it was less potent compared to PCL-Q, that is, these increased NPo from 0.010 ± 0.003 to 0.019 ± 0.003 (n = 5; p < 0.05) and did not affect single-channel conductance (139 pS). Application of PCL-Q restored macroscopic Maxi-K currents suppressed by H2O2-induced oxidative stress in ileal smooth muscle cells. We conclude that PCL-Q can activate Maxi-K channels in ileal myocytes mainly by increasing channel open probability, as well as maintain Maxi-K-mediated whole-cell current under the conditions of oxidative stress. While fusion of the 'pure' liposomes with the plasma membrane may indirectly activate Maxi-K channels by altering channel's phospholipids environment, the additional potentiating action of quercetin may be due to its better bioavailability.

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.

Milrinone-Induced Postconditioning Requires Activation of Mitochondrial Ca2+-sensitive Potassium (mBKCa) Channels.

OBJECTIVES: Cardioprotection by postconditioning requires activation of mitochondrial large-conductance Ca2+-sensitive potassium (mBKCa) channels. The involvement of these channels in milrinone-induced postconditioning is unknown. The authors determined whether cardioprotection by milrinone-induced postconditioning involves activation of mBKCa channels in the rat heart in vitro. DESIGN: Randomized, prospective, blinded laboratory investigation. SETTING: Experimental laboratory. PARTICIPANTS: Male Wistar rats. INTERVENTIONS: Hearts of male Wistar rats were randomized, placed on a Langendorff system, and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 minutes of global ischemia and 60 minutes of reperfusion. At the onset of reperfusion, hearts were perfused with different concentrations of milrinone (0.3-100 µM) for determination of a dose-effect curve. In a second set of experiments, 3 µM milrinone was administered in combination with the mBKCa channel inhibitor paxilline (1 µM). Infarct size was determined by triphenyltetrazoliumchloride staining. MEASUREMENTS AND MAIN RESULTS: In control animals, infarct size was 37 ± 7%. Milrinone at a concentration of 3 µM reduced infarct size to 22 ± 7% (p < 0.05 v control). Higher milrinone concentrations did not confer stronger protection. Paxilline completely blocked milrinone-induced cardioprotection whereas paxilline alone had no effect on infarct size. CONCLUSIONS: This study shows that activation of mBKCa channels plays a pivotal role in milrinone-induced postconditioning.

Hydrogen sulfide-induced vasodilation mediated by endothelial TRPV4 channels.

Hydrogen sulfide (H2S) is a recently described gaseous vasodilator produced within the vasculature by the enzymes cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase. Previous data demonstrate that endothelial cells (EC) are the source of endogenous H2S production and are required for H2S-induced dilation. However, the signal transduction pathway activated by H2S within EC has not been elucidated. TRPV4 and large-conductance Ca2+-activated K channels (BK channels) are expressed in EC. H2S-induced dilation is inhibited by luminal administration of iberiotoxin and disruption of the endothelium. Calcium influx through TRPV4 may activate these endothelial BK channels (eBK). We hypothesized that H2S-mediated vasodilation involves activation of TRPV4 within the endothelium. In pressurized, phenylephrine-constricted mesenteric arteries, H2S elicited a dose-dependent vasodilation blocked by inhibition of TRPV4 channels (GSK2193874A, 300 nM). H2S (1 µM) increased TRPV4-dependent (1.8-fold) localized calcium events in EC of pressurized arteries loaded with fluo-4 and Oregon Green. In pressurized EC tubes, H2S (1 µM) and the TRPV4 activator, GSK101679A (30 nM), increased calcium events 1.8- and 1.5-fold, respectively. H2S-induced an iberiotoxin-sensitive outward current measured using whole cell patch-clamp techniques in freshly dispersed EC. H2S increased K+ currents from 10 to 30 pA/pF at +150 mV. Treatment with Na2S increased the level of sulfhydration of TRPV4 channels in aortic ECs. These results demonstrate that H2S-mediated vasodilation involves activation of TRPV4-dependent Ca2+ influx and BK channel activation within EC. Activation of TRPV4 channels appears to cause calcium events that result in the opening of eBK channels, endothelial hyperpolarization, and subsequent vasodilation.

Alcohol Regulates BK Surface Expression via Wnt/ß-Catenin Signaling.

It has been suggested that drug tolerance represents a form of learning and memory, but this has not been experimentally established at the molecular level. We show that a component of alcohol molecular tolerance (channel internalization) from rat hippocampal neurons requires protein synthesis, in common with other forms of learning and memory. We identify ß-catenin as a primary necessary protein. Alcohol increases ß-catenin, and blocking accumulation of ß-catenin blocks alcohol-induced internalization in these neurons. In transfected HEK293 cells, suppression of Wnt/ß-catenin signaling blocks ethanol-induced internalization. Conversely, activation of Wnt/ß-catenin reduces BK current density. A point mutation in a putative glycogen synthase kinase phosophorylation site within the S10 region of BK blocks internalization, suggesting that Wnt/ß-catenin directly regulates alcohol-induced BK internalization via glycogen synthase kinase phosphorylation. These findings establish de novo protein synthesis and Wnt/ß-catenin signaling as critical in mediating a persistent form of BK molecular alcohol tolerance establishing a commonality with other forms of long-term plasticity. SIGNIFICANCE STATEMENT: Alcohol tolerance is a key step toward escalating alcohol consumption and subsequent dependence. Our research aims to make significant contributions toward novel, therapeutic approaches to prevent and treat alcohol misuse by understanding the molecular mechanisms of alcohol tolerance. In our current study, we identify the role of a key regulatory pathway in alcohol-induced persistent molecular changes within the hippocampus. The canonical Wnt/ß-catenin pathway regulates BK channel surface expression in a protein synthesis-dependent manner reminiscent of other forms of long-term hippocampal neuronal adaptations. This unique insight opens the possibility of using clinically tested drugs, targeting the Wnt/ß-catenin pathway, for the novel use of preventing and treating alcohol dependency.

The earliest neuronal responses to hypoxia in the neocortical circuit are glutamate-dependent.

Soon after exposure to hypoxia or ischemia, neurons in cortical tissues undergo massive anoxic depolarization (AD). This precipitous event is preceded by more subtle neuronal changes, including enhanced excitatory and inhibitory synaptic transmitter release. Here, we have used patch-in-slice techniques to identify the earliest effects of acute hypoxia on the synaptic and intrinsic properties of Layer 5 neurons, to determine their time course and to evaluate the role of glutamate receptors in their generation. Coronal slices of mouse somatosensory cortex were maintained at 36°C in an interface chamber and challenged with episodes of hypoxia. In recordings with cell-attached electrodes, the open probability of Ca(2+)-dependent BK channels began to increase within seconds of hypoxia onset, indicating a sharp rise in [Ca(2+)]i just beneath the membrane. By using a high concentration of K(+) in the pipette, we simultaneously monitored the membrane potential and showed that the [Ca(2+)]i rise was not associated with membrane depolarization. The earliest hypoxia-induced synaptic disturbance was a marked increase in the frequency of sPSCs, which also began soon after the removal of oxygen and long before AD. This synaptic effect was accompanied by depletion of the readily releasable transmitter pools, as demonstrated by a decreased response to hyperosmotic solutions. The early [Ca(2+)]i rise, the early increase in transmitter release and the subsequent AD itself were all prevented by bathing in a cocktail containing blockers of ionotropic glutamate receptors. We found no evidence for involvement of pannexin hemichannels or TRPM7 channels in the early responses to hypoxia in this experimental preparation. Our data indicate that the earliest cellular consequences of cortical hypoxia are triggered by activation of glutamate-gated channels.

Modulation of BK Channels by Small Endogenous Molecules and Pharmaceutical Channel Openers.

Voltage- and Ca(2+)-activated K(+) channels of big conductance (BK channels) are abundantly found in various organs and their relevance for smooth muscle tone and neuronal signaling is well documented. Dysfunction of BK channels is implicated in an array of human diseases involving many organs including the nervous, pulmonary, cardiovascular, renal, and urinary systems. In humans a single gene (KCNMA1) encodes the pore-forming α subunit (Slo1) of BK channels, but the channel properties are variable because of alternative splicing, tissue- and subcellular-specific auxiliary subunits (ß, γ), posttranslational modifications, and a multitude of endogenous signaling molecules directly affecting the channel function. Initiatives to develop drugs capable of activating BK channels (channel openers) therefore need to consider the tissue-specific variability of BK channel structure and the potential interference with endogenously produced regulatory factors. The atomic structural basis of BK channel function is only beginning to be revealed. However, building on detailed knowledge of BK channel function, including its single-channel characteristics, voltage- and Ca(2+) dependence of channel gating, and modulation by diffusible messengers, a multi-tier allosteric model of BK channel gating (Horrigan and Aldrich (HA) model) has become a valuable tool in studying modulation of the channel. Using the conceptual framework of the HA model, we here review the functional impact of endogenous modulatory factors and select small synthetic compounds that regulate BK channel activity. Furthermore, we devise experimental approaches for studying BK channel-drug interactions with the aim to classify BK-modulating substances according to their molecular mode of action.

Modulation of BK Channels by Ethanol.

In alcohol-naïve systems, ethanol (<100mM) exposure of calcium-gated BK channels perturbs physiology and behavior. Brief (several minutes) ethanol exposure usually leads to increased BK current, which results from ethanol interaction with a pocket mapped to the BK channel-forming slo1 protein cytosolic tail domain. The importance of this region in ethanol-induced intoxication has been independently supported by an unbiased screen of Caenorhabditis elegans slo1 mutants. However, ethanol-induced BK activation is not universal as refractoriness and inhibition have been reported. The final effect depends on many factors, including intracellular calcium levels, slo1 isoform, BK beta subunit composition, posttranslational modification of BK proteins, channel lipid microenvironment, and type of ethanol administration. Studies in Drosophila melanogaster, C. elegans, and rodents show that protracted/repeated ethanol administration leads to tolerance to ethanol-induced modification of BK-driven physiology and behavior. Unveiling the mechanisms underlying tolerance is of major importance, as tolerance to ethanol has been proposed as predictor of risk for alcoholism.