A molecular switch controls the impact of cholesterol on a Kir channel.

SignificanceCholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.

Dual-color miniscope imaging of microvessels and neuronal activity in the hippocampus CA1 region of freely moving mice following alcohol administration.

Moderate-to-heavy episodic ("binge") drinking is the most common form of alcohol consumption in the United States. Alcohol at binge drinking concentrations reduces brain artery diameter in vivo and in vitro in many species including rats, mice, and humans. Despite the critical role played by brain vessels in maintaining neuronal function, there is a shortage of methodologies to simultaneously assess neuron and blood vessel function in deep brain regions. Here, we investigate cerebrovascular responses to ethanol by choosing a deep brain region that is implicated in alcohol disruption of brain function, the hippocampal CA1, and describe the process for obtaining simultaneous imaging of pyramidal neuron activity and diameter of nearby microvessels in freely moving mice via a dual-color miniscope. Recordings of neurovascular events were performed upon intraperitoneal injection of saline versus 3 g/kg ethanol in the same mouse. In male mice, ethanol mildly increased the amplitude of calcium signals while robustly decreasing their frequency. Simultaneously, ethanol decreased microvessel diameter. In females, ethanol did not change the amplitude or frequency of calcium signals from CA1 neurons but decreased microvessel diameter. A linear regression of ethanol-induced reduction in number of active neurons and microvessel constriction revealed a positive correlation (R = 0.981) in females. Together, these data demonstrate the feasibility of simultaneously evaluating neuronal and vascular components of alcohol actions in a deep brain area in freely moving mice, as well as the sexual dimorphism of hippocampal neurovascular responses to alcohol.

Interspecies and regional variability of alcohol action on large cerebral arteries: regulation by KCNMB1 proteins.

Alcohol intake leading to blood ethanol concentrations (BEC) ≥ legal intoxication modifies brain blood flow with increases in some regions and decreases in others. Brain regions receive blood from the Willis' circle branches: anterior, middle (MCA) and posterior cerebral (PCA), and basilar (BA) arteries. Rats and mice have been used to identify the targets mediating ethanol-induced effects on cerebral arteries, with conclusions being freely interchanged, albeit data were obtained in different species/arterial branches. We tested whether ethanol action on cerebral arteries differed between male rat and mouse and/or across different brain regions and identified the targets of alcohol action. In both species and all Willis' circle branches, ethanol evoked reversible and concentration-dependent constriction (EC50s ≈ 37-86 mM; below lethal BEC in alcohol-naïve humans). Although showing similar constriction to depolarization, both species displayed differential responses to ethanol: in mice, MCA constriction was highly sensitive to the presence/absence of the endothelium, whereas in rat PCA was significantly more sensitive to ethanol than its mouse counterpart. In the rat, but not the mouse, BA was more ethanol sensitive than other branches. Both interspecies and regional variability were ameliorated by endothelium. Selective large conductance (BK) channel block in de-endothelialized vessels demonstrated that these channels were the effectors of alcohol-induced cerebral artery constriction across regions and species. Variabilities in alcohol actions did not fully matched KCNMB1 expression across vessels. However, immunofluorescence data from KCNMB1-/- mouse arteries electroporated with KCNMB1-coding cDNA demonstrate that KCNMB1 proteins, which regulate smooth muscle (SM) BK channel function and vasodilation, regulate interspecies and regional variability of brain artery responses to alcohol.

Commonly used anesthetics modify alcohol and (-)-trans-delta9-tetrahydrocannabinol in vivo effects on rat cerebral arterioles.

BACKGROUND: Ethyl alcohol and cannabis are widely used recreational substances with distinct effects on the brain. These drugs increase accidental injuries requiring treatment under anesthesia. Moreover, alcohol and cannabis are often used in anesthetized rodents for biomedical research. Here, we compared the influence of commonly used forms of anesthesia, injectable ketamine/xylazine (KX) versus inhalant isoflurane, on alcohol- and (-)-trans-delta9-tetrahydrocannabinol (THC) effects on cerebral arteriole diameter evaluated in vivo. METHODS: Studies were performed on male and female Sprague-Dawley rats subjected to intracarotid catheter placement for drug infusion, and cranial window surgery for monitoring pial arteriole diameter. Depth of anesthesia was monitored every 10-15 min by toe-pinch. Under KX, the number of toe-pinch responders was maximal after the first dose of anesthesia and diminished over time in both males and females. In contrast, the number of toe-pinch responders under isoflurane slowly raised over time, leading to increase in isoflurane percentage until deep anesthesia was re-established. Rectal temperature under KX remained stable in males while dropping in females. As expected for gaseous anesthesia, both males and females exhibited rectal temperature drops under isoflurane. RESULTS: Infusion of 50 mM alcohol (ethanol, EtOH) into the cerebral circulation rendered robust constriction in males under KX anesthesia, this alcohol action being significantly smaller, but still present under isoflurane anesthesia. In females, EtOH did not cause measurable changes in pial arteriole diameter regardless of the anesthetic. These findings indicate a strong sex bias with regards to EtOH induced vasoconstriction. Infusion of 42 nM THC in males and females under isoflurane tended to constrict cerebral arterioles in both males and females when compared to isovolumic infusion of THC vehicle (dimethyl sulfoxide in saline). Moreover, THC-driven changes in arteriole diameter significantly differed in magnitude depending on the anesthetic used. Simultaneous administration of 50 mM alcohol and 42 nM THC to males constricted cerebral arterioles regardless of the anesthetic used. In females, constriction by the combined drugs was also observed, with limited influence by anesthetic presence. CONCLUSIONS: We demonstrate that two commonly used anesthetic formulations differentially influence the level of vasoconstriction caused by alcohol and THC actions in cerebral arterioles.

From Crosstalk to Synergism: The Combined Effect of Cholesterol and PI(4,5)P2 on Inwardly Rectifying Potassium Channels.

Inwardly rectifying potassium (Kir) channels are integral membrane proteins that control the flux of potassium ions across cell membranes and regulate membrane permeability. All eukaryotic Kir channels require the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for activation. In recent years, it has become evident that the function of many members of this family of channels is also mediated by another essential lipid-cholesterol. Here, we focus on members of the Kir2 and Kir3 subfamilies and their modulation by these two key lipids. We discuss how PI(4,5)P2 and cholesterol bind to Kir2 and Kir3 channels and how they affect channel activity. We also discuss the accumulating evidence indicating that there is interplay between PI(4,5)P2 and cholesterol in the modulation of Kir2 and Kir3 channels. In particular, we review the crosstalk between PI(4,5)P2 and cholesterol in the modulation of the ubiquitously expressed Kir2.1 channel and the synergy between these two lipids in the modulation of the Kir3.4 channel, which is primarily expressed in the heart. Additionally, we demonstrate that there is also synergy in the modulation of Kir3.2 channels, which are expressed in the brain. These observations suggest that alterations in the relative levels PI(4,5)P2 and cholesterol may fine-tune Kir channel activity.

Cholesterol and PIP2 Modulation of BKCa Channels.

Ca2+/voltage-gated, large conductance K+ channels (BKCa) are formed by homotetrameric association of α (slo1) subunits. Their activity, however, is suited to tissue-specific physiology largely due to their association with regulatory subunits (ß and γ types), chaperone proteins, localized signaling, and the channel's lipid microenvironment. PIP2 and cholesterol can modulate BKCa activity independently of downstream signaling, yet activating Ca2+i levels and regulatory subunits control ligand action. At physiological Ca2+i and voltages, cholesterol and PIP2 reduce and increase slo1 channel activity, respectively. Moreover, slo1 proteins provide sites that seem to recognize cholesterol and PIP2: seven CRAC motifs in the slo1 cytosolic tail and a string of positively charged residues (Arg329, Lys330, Lys331) immediately after S6, respectively. A model that could explain the modulation of BKCa activity by cholesterol and/or PIP2 is hypothesized. The roles of additional sites, whether in slo1 or BKCa regulatory subunits, for PIP2 and/or cholesterol to modulate BKCa function are also discussed.

Differential Functional Contribution of BK Channel Subunits to Aldosterone-Induced Channel Activation in Vascular Smooth Muscle and Eventual Cerebral Artery Dilation.

Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory ß1 subunits, the latter being highly expressed in SM. Both subunits participate in steroid-induced modification of BK activity: ß1 provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone's cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3-1 µM and ≥100 µM for ß1. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from ß1-/- vs. wt mice. Data showed that ß1 leftward-shifted aldosterone-induced BK activation, rendering EC50~3 µM and ECMAX ≥ 10 µM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in ß1-/- mice. Therefore, ß1 enables BK activation and MCA dilation by low µM aldosterone.

Cholesterol Inhibition of Slo1 Channels Is Calcium-Dependent and Can Be Mediated by Either High-Affinity Calcium-Sensing Site in the Slo1 Cytosolic Tail.

Calcium- and voltage-gated K+ channels of large conductance (BKs) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in calcium (Ca2+) nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity site locate to the regulator of conductance for K+ (RCK) 1 domain, whereas another high-affinity site locates within the RCK2 domain. Here, we first evaluated the crosstalking between Ca2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca2+ CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of magnesium (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ sites were mutated (RCK1:D367A,D372A and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. SIGNIFICANCE STATEMENT: Results reveal that inhibition of calcium/voltage-gated K+ channel of large conductance (BK) (slo1) channels by membrane cholesterol requires a physiologically range of internal calcium (Ca2+) and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.

Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma.

Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (ß1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 µM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.

Discovery of agonist-antagonist pairs for the modulation of Ca [2]+ and voltage-gated K+ channels of large conductance that contain beta1 subunits.

Large conductance, calcium/voltage-gated potassium channels (BK) regulate critical body processes, including neuronal, secretory and smooth muscle (SM) function. While BK-forming alpha subunits are ubiquitous, accessory beta1 subunits are highly expressed in SM. This makes beta1 an attractive target for pharmaceutical development to treat SM disorders, such as hypertension or cerebrovascular spasm. Compounds activating BK via beta1 have been identified, yet they exhibit low potency and off-target effects while antagonists that limit agonist activity via beta 1 remain unexplored. Beta1-dependent BK ligand-based pharmacophore modeling and ZINC database searches identified 15 commercially available hits. Concentration-response curves on BK alpha + beta1 subunit-mediated currents were obtained in CHO cells. One potent (EC50 = 20 nM) and highly efficacious activator (maximal activation = ×10.3 of control) was identified along with a potent antagonist (KB = 3.02 nM), both of which were dependent on beta1. Our study provides the first proof-of-principle that an agonist/antagonist pair can be used to control beta1-containing BK activity.

Proteomic Analysis of Baboon Cerebral Artery Reveals Potential Pathways of Damage by Prenatal Alcohol Exposure.

Alcohol is one of the most widely misused substances in the world. Alcohol consumption by pregnant women often results in an array of fetal developmental abnormalities, but the damage to the fetus by alcohol remains poorly understood. The limited knowledge regarding the molecular targets of alcohol in the developing fetus constitutes one of the major obstacles in developing effective pharmacological interventions that could prevent fetal damage after alcohol consumption by pregnant women. The fetal cerebral artery is emerging as an important mediator of fetal cerebral damage by maternal alcohol drinking. In the present work, we conduct proteomics analysis of cerebral (basilar) artery lysates of near-term fetal baboons to search for protein targets of fetal alcohol exposure. Our study demonstrates that 3 episodes of binge alcohol exposure during the second trimester-equivalent of human pregnancy are sufficient to render profound changes in fetal cerebral artery proteome. These changes persisted, as they were detected in near-term fetuses. In particular, the relative abundance of 238 proteins differed significantly between control and alcohol-exposed fetuses. Enrichment analysis pointed at the group of metabolic activity proteins as a major class targeted by alcohol. Western blotting confirmed upregulation of the aldehyde dehydrogenase 6 family member A1 (ALDH6A1) in cerebral artery lysates from alcohol-exposed fetuses. This upregulation translated to greater ALDH activity of cerebral artery lysate of near-term fetuses following prenatal alcohol exposure when compared with controls.

Celastrol Dilates and Counteracts Ethanol-Induced Constriction of Cerebral Arteries.

The increasing recognition of the role played by cerebral artery dysfunction in brain disorders has fueled the search for new cerebrovascular dilators. Celastrol, a natural triterpene undergoing clinical trials for treating obesity, exerts neuroprotection, which was linked to its antioxidant/anti-inflammatory activities. We previously showed that celastrol fit pharmacophore criteria for activating calcium- and voltage-gated potassium channels of large conductance (BK channels) made of subunits cloned from cerebrovascular smooth muscle (SM). These recombinant BK channels expressed in a heterologous system were activated by celastrol. Activation of native SM BK channels is well known to evoke cerebral artery dilation. Current data demonstrate that celastrol (1-100 µM) dilates de-endothelialized, ex vivo pressurized middle cerebral arteries (MCAs) from rats, with EC50 = 45 µM and maximal effective concentration (Emax)= 100 µM and with MCA diameter reaching a 10% increase over vehicle-containing, time-matched values (P < 0.05). A similar vasodilatory efficacy is achieved when celastrol is probed on MCA segments with intact endothelium. Selective BK blocking with 1 µM paxilline blunts celastrol vasodilation. Similar blunting is achieved with 0.8 mM 4-aminopirydine, which blocks voltage-gated K+ channels other than BK. Using an in vivo rat cranial window, we further demonstrate that intracarotid injections of 45 µM celastrol into pial arteries branching from MCA mimics celastrol ex vivo action. MCA constriction by ethanol concentrations reached in blood during moderate-heavy alcohol drinking (50 mM), which involves SM BK inhibition, is both prevented and reverted by celastrol. We conclude that celastrol could be an effective cerebrovascular dilator and antagonist of alcohol-induced cerebrovascular constriction, with its efficacy being uncompromised by conditions that disrupt endothelial and/or BK function. SIGNIFICANCE STATEMENT: Our study demonstrates for the first time that celastrol significantly dilates rat cerebral arteries both ex vivo and in vivo and both prevents and reverses ethanol-induced cerebral artery constriction. Celastrol actions are endothelium-independent but mediated through voltage-gated (KV) and calcium- and voltage-gated potassium channel of large conductance (BK) K+ channels. This makes celastrol an appealing new agent to evoke cerebrovascular dilation under conditions in which endothelial and/or BK channel function are impaired.

Cholesterol intake and statin use regulate neuronal G protein-gated inwardly rectifying potassium channels.

Cholesterol, a critical component of the cellular plasma membrane, is essential for normal neuronal function. Cholesterol content is highest in the brain, where most cholesterol is synthesized de novo; HMG-CoA reductase controls the synthesis rate. Despite strict control, elevated blood cholesterol levels are common and are associated with various neurological disorders. G protein-gated inwardly rectifying potassium (GIRK) channels mediate the actions of inhibitory brain neurotransmitters. Loss of GIRK function enhances neuron excitability; gain of function reduces neuronal activity. However, the effect of dietary cholesterol or HMG-CoA reductase inhibition (i.e., statin therapy) on GIRK function remains unknown. Using a rat model, we compared the effects of a high-cholesterol versus normal diet both with and without atorvastatin, a widely prescribed HMG-CoA reductase inhibitor, on neuronal GIRK currents. The high-cholesterol diet increased hippocampal CA1 region cholesterol levels and correspondingly increased neuronal GIRK currents. Both phenomena were reversed by cholesterol depletion in vitro. Atorvastatin countered the high-cholesterol diet effects on neuronal cholesterol content and GIRK currents; these effects were reversed by cholesterol enrichment in vitro. Our findings suggest that high-cholesterol diet and atorvastatin therapy affect ion channel function in the brain by modulating neuronal cholesterol levels.

Physiology of the Endocannabinoid System During Development.

The endocannabinoid (eCB) system comprises endogenously produced cannabinoids (CBs), enzymes of their production and degradation, and CB-sensing receptors and transporters. The eCB system plays a critical role in virtually all stages of animal development. Studies on eCB system components and their physiological role have gained increasing attention with the rising legalization and medical use of marijuana products. The latter represent exogenous interventions that target the eCB system. This chapter summarizes knowledge in the field of CB contribution to gametogenesis, fertilization, embryo implantation, fetal development, birth, and adolescence-equivalent periods of ontogenesis. The material is complemented by the overview of data from our laboratory documenting the functional presence of the eCB system within cerebral arteries of baboons at different stages of development.

Cannabinoid Interactions with Proteins: Insights from Structural Studies.

Cannabinoids have been widely used for recreational and medicinal purposes. The increasing legalization of cannabinoid use and the growing success in Medicinal Chemistry of cannabinoids have fueled recent interest in cannabinoid-sensing sites in receptor proteins. Here, we review structural data from high-resolution cryo-EM and crystallography studies that depict phytocannabinoid, endocannabinoid, and synthetic cannabinoid molecules bound to various proteins. The latter include antigen-binding fragment (Fab), cellular retinol binding protein 2 (CRBP2), fatty acid-binding protein 5 (FABP5), peroxisome proliferator-activated receptor γ (PPAR γ), and cannabinoid receptor types 1 and 2 (CB1 and CB2). Cannabinoid-protein complexes reveal the complex design of cannabinoid binding sites that are usually presented by conventional ligand-binding pockets on respective proteins. However, subtle differences in cannabinoid interaction with amino acids within the binding pocket often result in diverse consequences for protein function. The rapid increase in available structural data on cannabinoid-protein interactions will ultimately direct drug design efforts toward rendering highly potent cannabinoid-related pharmacotherapies that are devoid of side effects.

Regulation of BK Channel Activity by Cholesterol and Its Derivatives.

Cholesterol (CLR) is an essential structural lipid in the plasma membrane of animal cells. In addition, CLR has been widely recognized as a critical modulator of protein function, including ion channels. Voltage- and Ca2+-gated K+ (BK) channels control a wide variety of physiological processes, including cell excitability, smooth muscle contractility, sensory perception, neurotransmitter release, and hormone secretion. Thus, disruption of BK currents has been implicated in the pathophysiology of prevalent human diseases. The current chapter reviews the literature documenting CLR modulation of BK channel function at a variety of levels ranging from organ systems to artificial lipid bilayers. We discuss the use of CLR isomers and structural analogs as a tool to help in discerning the mechanisms underlying CLR-driven modification of BK current. The chapter is finalized with an overview of the phenomenology and potential mechanisms that govern CLR control over the alcohol (ethyl alcohol, ethanol) sensitivity of BK channels. Studies on CLR regulation of BK currents may ultimately pave the way for novel therapeutic approaches to combat prevalent pathophysiological and morbid conditions.

Molecular Determinants of Cholesterol Binding to Soluble and Transmembrane Protein Domains.

Cholesterol-protein interactions play a critical role in lipid metabolism and maintenance of cell integrity. To elucidate the molecular mechanisms underlying these interactions, a growing number of studies have focused on determining the crystal structures of a variety of proteins complexed with cholesterol. These include structures in which cholesterol binds to transmembrane domains, and structures in which cholesterol interacts with soluble ones. However, it remains unknown whether there are differences in the prerequisites for cholesterol binding to these two types of domains. Thus, to define the molecular determinants that characterize the binding of cholesterol to these two distinct protein domains, we employed the database of crystal structures of proteins complexed with cholesterol. Our analysis suggests that cholesterol may bind more strongly to soluble domains than to transmembrane domains. The interactions between cholesterol and the protein in both cases critically depends on hydrophobic and aromatic residues. In addition, cholesterol binding sites in both types of domains involve polar and/or charged residues. However, the percentage of appearance of the different types of polar/charged residues in cholesterol binding sites differs between soluble and transmembrane domains. No differences were observed in the conformational characteristics of the cholesterol molecules bound to soluble versus transmembrane protein domains suggesting that cholesterol is insensitive to the environment provided by the different protein domains.

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus.

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease.

Calcium- and voltage-gated BK channels in vascular smooth muscle.

Ion channels in vascular smooth muscle regulate myogenic tone and vessel contractility. In particular, activation of calcium- and voltage-gated potassium channels of large conductance (BK channels) results in outward current that shifts the membrane potential toward more negative values, triggering a negative feed-back loop on depolarization-induced calcium influx and SM contraction. In this short review, we first present the molecular basis of vascular smooth muscle BK channels and the role of subunit composition and trafficking in the regulation of myogenic tone and vascular contractility. BK channel modulation by endogenous signaling molecules, and paracrine and endocrine mediators follows. Lastly, we describe the functional changes in smooth muscle BK channels that contribute to, or are triggered by, common physiological conditions and pathologies, including obesity, diabetes, and systemic hypertension.

Tyrosine 450 in the Voltage- and Calcium-Gated Potassium Channel of Large Conductance Channel Pore-Forming (slo1) Subunit Mediates Cholesterol Protection against Alcohol-Induced Constriction of Cerebral Arteries.

Alcohol (ethanol) at physiologically relevant concentrations (<100 mM) constricts cerebral arteries via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) located in vascular smooth muscle (VSM). These channels consist of channel-forming slo1 (cbv1, KCNMA1) and accessory beta1 (KCNMB1) subunits. An increase in VSM cholesterol (CLR) via either dietary CLR intake or in vitro CLR enrichment was shown to protect against endothelium-independent, alcohol-induced constriction of cerebral arteries. The molecular mechanism(s) of this protection remains unknown. Here, we demonstrate that CLR enrichment of de-endothelialized middle cerebral arteries (MCAs) of rat increased CLR content in the VSM in a concentration-dependent manner. CLR enrichment blunted MCA constriction evoked by 18-75 mM but not by 100 mM alcohol. MCA enrichment with coprostanol (COPR) also blunted vasoconstriction by 50 mM alcohol, despite the fact that COPR and CLR differ in their ability to modify several major physical properties of the bilayer. CLR protection against 50 but not 100 mM alcohol was also observed in C57BL/6 and KCNMB1 knockout (KO) mice. Permeabilization of KCNMA1 KO MCAs with Y450Fcbv1 totally ablated CLR, but not COPR protection against vasoconstriction by 50 mM alcohol. Thus, CLR and alcohol interact at the level of the BK channel slo1 subunit, with Y450 being critical for CLR protection against alcohol-induced vasoconstriction. We document for the first time a functional competition between CLR and alcohol in regulating cerebral artery diameter and a critical role of a single amino acid within the BK channel pore-forming subunit in controlling CLR-alcohol interaction at the organ level.