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.

A potent and selective activator of large-conductance Ca2+-activated K+ channels induces preservation of mitochondrial function after hypoxia and reoxygenation by handling of calcium and transmembrane potential.

AIMS: Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. METHODS: Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. RESULTS: Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. CONCLUSION: The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.

BK ZERO isoform HEK293 stably transfected cell lines differing 3'UTRs to assess miR-9 regulation.

Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3'untranslated region (3'UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies.

Physiological and Pathophysiological Role of Large-Conductance Calcium-Activated Potassium Channels (BKCa) in HUVECs and Placenta.

BKCa channels (large-conductance Ca2+-activated K+ channels) play a critical role in regulating vascular tone and blood pressure. These channels are present in the smooth muscle cells of blood vessels and are activated by voltage and increased intracellular Ca2+ concentration. More recently, the expression and activity of BKCa have been proposed to be relevant in endothelial cells, too, specifically in human umbilical vein endothelial cells (HUVECs), the more studied cell type in the fetoplacental circulation. The role of BKCa in endothelial cells is not well understood, but in HUVECs or placental endothelium, these channels could be crucial for vascular tone regulation during pregnancy as part of endothelium-derived hyperpolarization (EDH), a key mechanism for an organ that lacks nervous system innervation like the placenta.In this review, we will discuss the evidence about the role of BKCa (and other Ca2+-activated K+ channels) in HUVECs and the placenta to propose a physiological mechanism for fetoplacental vascular regulation and a pathophysiological role of BKCa, mainly associated with pregnancy pathologies that present maternal hypertension and/or placental hypoxia, like preeclampsia.

Ca2+- and Voltage-Activated K+ (BK) Channels in the Nervous System: One Gene, a Myriad of Physiological Functions.

BK channels are large conductance potassium channels characterized by four pore-forming α subunits, often co-assembled with auxiliary ß and γ subunits to regulate Ca2+ sensitivity, voltage dependence and gating properties. BK channels are abundantly expressed throughout the brain and in different compartments within a single neuron, including axons, synaptic terminals, dendritic arbors, and spines. Their activation produces a massive efflux of K+ ions that hyperpolarizes the cellular membrane. Together with their ability to detect changes in intracellular Ca2+ concentration, BK channels control neuronal excitability and synaptic communication through diverse mechanisms. Moreover, increasing evidence indicates that dysfunction of BK channel-mediated effects on neuronal excitability and synaptic function has been implicated in several neurological disorders, including epilepsy, fragile X syndrome, mental retardation, and autism, as well as in motor and cognitive behavior. Here, we discuss current evidence highlighting the physiological importance of this ubiquitous channel in regulating brain function and its role in the pathophysiology of different neurological disorders.

Mechanism of voltage sensing in Ca2+- and voltage-activated K+ (BK) channels.

In neurosecretion, allosteric communication between voltage sensors and Ca2+ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.

State-dependent inhibition of BK channels by the opioid agonist loperamide.

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

BKCa Channel Activation Attenuates the Pathophysiological Progression of Monocrotaline-Induced Pulmonary Arterial Hypertension in Wistar Rats.

PURPOSE: In the present study, the therapeutic efficacy of a selective BKCa channel opener (compound X) in the treatment of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) was investigated. METHODS: PAH was induced in male Wistar rats by a single injection of MCT. After two weeks, the MCT-treated group was divided into two groups that were either treated with compound X or vehicle. Compound X was administered daily at 28 mg/kg. Electrocardiographic, echocardiographic, and haemodynamic analyses were performed; ex vivo evaluations of pulmonary artery reactivity, right ventricle (RV) and lung histology as well as expression levels of α and ß myosin heavy chain, brain natriuretic peptide, and cytokines (TNFα and IL10) in heart tissue were performed. RESULTS: Pulmonary artery rings of the PAH group showed a lower vasodilatation response to acetylcholine, suggesting endothelial dysfunction. Compound X promoted strong vasodilation in pulmonary artery rings of both control and MCT-induced PAH rats. The untreated hypertensive rats presented remodelling of pulmonary arterioles associated with increased resistance to pulmonary flow; increased systolic pressure, hypertrophy and fibrosis of the RV; prolongation of the QT and Tpeak-Tend intervals (evaluated during electrocardiogram); increased lung and liver weights; and autonomic imbalance with predominance of sympathetic activity. On the other hand, treatment with compound X reduced pulmonary vascular remodelling, pulmonary flow resistance and RV hypertrophy and afterload. CONCLUSION: The use of a selective and potent opener to activate the BKCa channels promoted improvement of haemodynamic parameters and consequent prevention of RV maladaptive remodelling in rats with MCT-induced PAH.

Nitric oxide and Ca2+-activated high-conductance K+ channels mediate nothofagin-induced endothelium-dependent vasodilation in the perfused rat kidney.

Nothofagin is a natural 3'-C-ß-D-glucoside of the polyphenol phloretin that is mainly found in Aspalathus linearis, Nothofagus fusca, and Leandra dasytricha. In recent years, nothofagin has been described as a potential therapeutic agent for renal disorders, but the mechanisms that are involved in its renoprotective effects remain unclear. In the present study, perfused rat kidneys were used to test the hypothesis that nothofagin causes the direct relaxation of renal arteries. The molecular mechanisms that underlie these vascular effects were also investigated. The left kidney from Wistar rats was coupled in a perfusion system and continuously perfused with physiological saline solution (PSS). Initially, preparations with and without the endothelium were contracted with phenylephrine and received injections of 1-300 nmol nothofagin. The preparations were then perfused with PSS that contained phenylephrine plus KCl, indomethacin, l-NAME, tetraethylammonium, glibenclamide, 4-aminopyridine, iberiotoxin, charybdotoxin, and apamin. After 15 min under perfusion, nothofagin was injected again. In preparations with an intact endothelium, nothofagin dose-dependently reduced perfusion pressure. Endothelium removal or the inhibition of nitric oxide synthase by l-NAME prevented the vasodilatory effect of nothofagin at all doses tested. Perfusion with PSS that contained KCl or tetraethylammonium chloride also abolished the vasodilatory effect of nothofagin. Treatment with glibenclamide, 4-aminopyridine, and apamin did not affect the vasodilatory effect of nothofagin. Iberiotoxin (selective Ca2+-activated high-conductance K+ channel [KCa1.1] blocker) and charybdotoxin (selective KCa1.1 and Ca2+-activated intermediate-conductance K+ channel [KCa3.1] blocker) application blocked the vasodilatory effect of nothofagin at all doses tested, pointing to a predominant role for KCa1.1 in the action of nothofagin. However, these data cannot exclude a potential contribution of endothelial KCa3.1 channel in the nothofagin-induced vasodilation. Overall, our findings indicate that nothofagin induces vasodilation in renal arteries, an effect that is mediated by Ca2+ -activated high-conductance K+ channels opening and endothelial nitric oxide production.

Functional Postnatal Maturation of the Medial Olivocochlear Efferent-Outer Hair Cell Synapse.

The organ of Corti, the auditory mammalian sensory epithelium, contains two types of mechanotransducer cells, inner hair cells (IHCs) and outer hair cells (OHCs). IHCs are involved in conveying acoustic stimuli to the CNS, while OHCs are implicated in the fine tuning and amplification of sounds. OHCs are innervated by medial olivocochlear (MOC) cholinergic efferent fibers. The functional characteristics of the MOC-OHC synapse during maturation were assessed by electrophysiological and pharmacological methods in mouse organs of Corti at postnatal day 11 (P11)-P13, hearing onset in altricial rodents, and at P20-P22 when the OHCs are morphologically and functionally mature. Synaptic currents were recorded in whole-cell voltage-clamped OHCs while electrically stimulating the MOC fibers. A progressive increase in the number of functional MOC-OHC synapses, as well as in their strength and efficacy, was observed between P11-13 and P20-22. At hearing onset, the MOC-OHC synapse presented facilitation during MOC fibers high-frequency stimulation that disappeared at mature stages. In addition, important changes were found in the VGCC that are coupled to transmitter release. Ca2+ flowing in through L-type VGCCs contribute to trigger ACh release together with P/Q- and R-type VGCCs at P11-P13, but not at P20-P22. Interestingly, N-type VGCCs were found to be involved in this process at P20-P22, but not at hearing onset. Moreover, the degree of compartmentalization of calcium channels with respect to BK channels and presynaptic release components significantly increased from P11-P13 to P20-P22. These results suggest that the MOC-OHC synapse is immature at the onset of hearing.SIGNIFICANCE STATEMENT The functional expression of both VGCCs and BK channels, as well as their localization with respect to the presynaptic components involved in transmitter release, are key elements in determining synaptic efficacy. In this work, we show dynamic changes in the expression of VGCCs and Ca2+-dependent BK K+ channels coupled to ACh release at the MOC-OHC synapse and their shift in compartmentalization during postnatal maturation. These processes most likely set the short-term plasticity pattern and reliability of the MOC-OHC synapse on high-frequency activity.

Molecular mechanism of BK channel activation by the smooth muscle relaxant NS11021.

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at -200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than -60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

The activity of IKCa and BKCa channels contributes to insulin-mediated NO synthesis and vascular tone regulation in human umbilical vein.

Insulin regulates the l-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or NG-nitro-l-arginine methyl ester. l-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l-arginine transport in HUVECs.

A cytoplasmic Slo3 isoform is expressed in somatic tissues.

Slo3 is a pH-sensitive and weakly voltage-sensitive potassium channel that is essential for male fertility in mouse and whose expression is regarded as sperm-specific. These properties have proposed Slo3 as a candidate target for male contraceptive drugs. Nonetheless, the tissue distribution of Slo3 expression has not been rigorously studied yet. Applying computational and RT-PCR approaches, we identified expression of two short Slo3 isoforms in somatic mouse tissues such as brain, kidney and eye. These isoforms, which seem to result of transcription starting sites between exons 20 and 21, have an identical open reading frame, both encoding the terminal 381 amino acids of the cytosolic Slo3 domain. We corroborated the expression of these isoforms in mouse brain and testis by Western-blot. The complete isoform encoding the Slo3 ion channel was uniquely detected in testis, both at transcript and protein level. Although the functional role of the cytosolic Slo3 isoforms remains to be established, we propose that they may have a functional effect by modulating Slo channels trafficking and/or activity. This study confirms that expression of full-length Slo3 is sperm-specific but warns against developing contraceptive drugs targeting the C-terminal tail of Slo3 channels.

Determination of the Stoichiometry between α- and γ1 Subunits of the BK Channel Using LRET.

Two families of accessory proteins, ß and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca2+, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb+3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.

Role of Ion Channels in the Sperm Acrosome Reaction.

The acrosome reaction (AR) is a unique exocytotic process where the acrosome, a single membrane-delimited specialized organelle, overlying the nucleus in the sperm head of many species, fuses with the overlying plasma membrane. This reaction, triggered by physiological inducers from the female gamete, its vicinity, or other stimuli, discharges the acrosomal content modifying the plasma membrane, incorporating the inner acrosomal membrane, and exposing it to the extracellular medium. The AR is essential for sperm-egg coat penetration, fusion with the eggs' plasma membrane, and fertilization. As in most exocytotic processes Ca(2+) is crucial for the AR, as well as intracellular pH and membrane potential changes. Thus, among the required processes needed for this reaction, ion permeability changes involving channels are pivotal. In spite of the key role ion channels play in the AR, their identity and regulation is not fully understood. Though molecular and pharmacological evidence indicates that various ionic channels participate during the AR, such as store-operated Ca(2+) channels and voltage-dependent Ca(2+) channels, whole cell patch clamp recordings have failed to detect some of them until now. Since sperm display a very high resistance and a minute cytoplasmic volume, very few channels are needed to achieve large membrane potential and concentration changes. Functional detection of few channels in the morphologically complex and tiny sperm poses technical problems, especially when their conductance is very small, as in the case of SOCs. Single channel recordings and novel fluorescence microscopy strategies will help to define the participation of ionic channels in the intertwined signaling network that orchestrates the AR.

Sperm Capacitation and Acrosome Reaction in Mammalian Sperm.

Physiological changes that endow mammalian sperm with fertilizing capacity are known as sperm capacitation. As part of capacitation, sperm develop an asymmetrical flagellar beating known as hyperactivation and acquire the ability to undergo the acrosome reaction. Together, these processes promote fertilizing competence in sperm. At the molecular level, capacitation involves a series of signal transduction events which include activation of cAMP-dependent phosphorylation pathways, removal of cholesterol, hyperpolarization of the sperm plasma membrane, and changes in ion permeability. In recent years, new technologies have aided in the study of sperm signaling molecules with better resolution, at both spatial and temporal levels, unraveling how different cascades integrate and cooperate to render a fertilizing sperm. Despite this new information, the molecular mechanisms connecting capacitation with acrosomal exocytosis and hyperactivation are not well understood. This review brings together results obtained in mammalian species in the field of sperm capacitation with special focus on those pathways involved in the preparation to undergo the acrosomal reaction.

Pharmacology of hSlo3 channels and their contribution in the capacitation-associated hyperpolarization of human sperm.

Slo3 channels (mSlo3) primarily mediate mouse sperm K(+) currents and are essential for the capacitation-associated hyperpolarization (CAH). Whether Slo3 and/or Slo1, two Slo family K(+) channels are functionally expressed in human sperm is controversial. Our recent pharmacological studies of the human sperm CAH suggested the participation of both. Lack of a detailed pharmacology of heterologously expressed human Slo3 (hSlo3) prevented precisely identifying the K(+) channel(s) involved. In the present report, we compare the pharmacological profile of expressed hSlo3 in CHO cells with that of the CAH to advance this matter. Whole-cell patch-clamp recordings showed that hSlo3 currents are inhibited: significantly by progesterone, Ba(2+) and quinidine; partially by Penitrem A and Charybdotoxin; and poorly by Iberiotoxin and Slotoxin. Surprisingly, hSlo3 currents were resistant to Clofilium and 60 mM TEA(+) which inhibit mSlo3. Pharmacological comparison of the CAH and hSlo3 profiles indicates in addition to hSlo3, other K(+) channels, possibly Slo1, may participate in CAH. The pharmacological profile of heterologously expressed hSlo3 channels differs from that of mSlo3 K(+) channels, consistent with species-specific differences observed among other sperm ion channels. While the pharmacological correlation analysis of the hSlo3 currents and the CAH confirmed the participation of hSlo3 channels, it suggests that additional K(+) channels may be involved, in particular Slo1 channels.

Ethanol Effect on BK Channels is Modulated by Magnesium.

BACKGROUND: Alcoholics have been reported to have reduced levels of magnesium in both their extracellular and intracellular compartments. Calcium-dependent potassium channels (BK) are known to be one of ethanol (EtOH)'s better known molecular targets. METHODS: Using outside-out patches from hippocampal neuronal cultures, we examined the consequences of altered intracellular Mg(2+) on the effects that EtOH has on BK channels. RESULTS: We find that the effect of EtOH is bimodally influenced by the Mg(2+) concentration on the cytoplasmic side. More specifically, when internal Mg(2+) concentrations are ≤200 µM, EtOH decreases BK activity, whereas it increases activity when Mg(2+) is at 1 mM. Similar results are obtained when using patches from HEK cells expressing only the α-subunit of BK. When patches are made with the actin destabilizer cytochalasin D present on the cytoplasmic side, the potentiation caused by EtOH becomes independent of the Mg(2+) concentration. Furthermore, in the presence of the actin stabilizer phalloidin, EtOH causes inhibition even at Mg(2+) concentrations of 1 mM. CONCLUSIONS: Internal Mg(2+) can modulate the EtOH effects on BK channels only when there is an intact, internal actin interaction with the channel, as is found at synapses. We propose that the EtOH-induced decrease in cytoplasmic Mg(2+) observed in frequent/chronic drinkers would decrease EtOH's actions on synaptic (e.g., actin-bound) BK channels, producing a form of molecular tolerance.

Time-Dependent Effects of Ethanol on BK Channel Expression and Trafficking in Hippocampal Neurons.

BACKGROUND: The large conductance Ca(2+) - and voltage-activated K(+) channel (BK) is an important player in molecular and behavioral alcohol tolerance. Trafficking and surface expression of ion channels contribute to the development of addictive behaviors. We have previously reported that internalization of the BK channel is a component of molecular tolerance to ethanol (EtOH). METHODS: Using primary cultures of hippocampal neurons, we combine total internal reflection fluorescence microscopy, electrophysiology, and biochemical techniques to explore how exposure to EtOH affects the expression and subcellular localization of endogenous BK channels over time. RESULTS: Exposure to EtOH changed the expression of endogenous BK channels in a time-dependent manner at the perimembrane area (plasma membrane and/or the area adjacent to it), while total protein levels of BK remain unchanged. These results suggest a redistribution of the channel within the neurons rather than changes in synthesis or degradation rates. Our results showed a temporally nonlinear effect of EtOH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10 minutes of EtOH exposure that remained evident after 3 hours, although not correlated to increases in functional channel expression. In contrast, after 6 hours of EtOH exposure, we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24 hours of EtOH exposure, perimembrane levels of BK had returned to baseline. CONCLUSIONS: We report a complex time-dependent pattern in the effect of EtOH on BK channel trafficking, including successive increases and decreases in perimembrane expression and a reduction in active BK channels after 3 and 6 hours of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. As molecular tolerance necessarily underlies behavioral tolerance, the time-dependent alterations we see at the level of the channel may be relevant to the influence of drinking patterns on the development of behavioral tolerance.