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.

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.

Site-specific deacylation by ABHD17a controls BK channel splice variant activity.

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/ß-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0-S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

Regulation of KCNMA1 transcription by Nrf2 in coronary arterial smooth muscle cells.

The large conductance Ca2+-activated K+ (BK) channels, composed of the pore-forming α subunits (BK-α, encoded by KCNMA1 gene) and the regulatory ß1 subunits (BK-ß1, encoded by KCNMB1 gene), play a unique role in the regulation of coronary vascular tone and myocardial perfusion by linking intracellular Ca2+ homeostasis with excitation-contraction coupling in coronary arterial smooth muscle cells (SMCs). The nuclear factor erythroid 2-related factor 2 (Nrf2) belongs to a member of basic leucine zipper transcription factor family that regulates the expression of antioxidant and detoxification enzymes by binding to the antioxidant response elements (AREs) of these target genes. We have previously reported that vascular BK-ß1 protein expression was tightly regulated by Nrf2. However, the molecular mechanism underlying the regulation of BK channel expression by Nrf2, particularly at transcription level, is unknown. In this study, we hypothesized that KCNMA1 and KCNMB1 are the target genes of Nrf2 transcriptional regulation. We found that BK channel protein expression and current density were diminished in freshly isolated coronary arterial SMCs of Nrf2 knockout (KO) mice. However, BK-α mRNA expression was reduced, but not that of BK-ß1 mRNA expression, in the arteries of Nrf2 KO mice. Promoter-Nrf2 luciferase reporter assay confirmed that Nrf2 binds to the ARE of KCNMA1 promoter, but not that of KCNMB1. Adenoviral expression and pharmacological activation of Nrf2 increased BK-α and BK-ß1 protein levels and enhanced BK channel activity in coronary arterial SMCs. Hence, our results indicate that Nrf2 is a key determinant of BK channel expression and function in vascular SMCs. Nrf2 facilitates BK-α expression through a direct increase in gene transcription, whereas that on BK-ß1 is through a different mechanism.

S-Acylation controls functional coupling of BK channel pore-forming α-subunits and ß1-subunits.

The properties and physiological function of pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are potently modified by their functional coupling with regulatory subunits in many tissues. However, mechanisms that might control functional coupling are very poorly understood. Here we show that S-acylation, a dynamic post-translational lipid modification of proteins, of the intracellular S0-S1 loop of the BK channel pore-forming α-subunit controls functional coupling to regulatory ß1-subunits. In HEK293 cells, α-subunits that cannot be S-acylated show attenuated cell surface expression, but expression was restored by co-expression with the ß1-subunit. However, we also found that nonacylation of the S0-S1 loop reduces functional coupling between α- and ß1-subunits by attenuating the ß1-subunit-induced left shift in the voltage for half-maximal activation. In mouse vascular smooth muscle cells expressing both α- and ß1-subunits, BK channel α-subunits were endogenously S-acylated. We further noted that S-acylation is significantly reduced in mice with a genetic deletion of the palmitoyl acyltransferase (Zdhhc23) that controls S-acylation of the S0-S1 loop. Genetic deletion of Zdhhc23 or broad-spectrum pharmacological inhibition of S-acylation attenuated endogenous BK channel currents independently of changes in cell surface expression of the α-subunit. We conclude that functional effects of S-acylation on BK channels depend on the presence of ß1-subunits. In the absence of ß1-subunits, S-acylation promotes cell surface expression, whereas in its presence, S-acylation controls functional coupling. S-Acylation thus provides a mechanism that dynamically regulates the functional coupling with ß1-subunits, enabling an additional level of conditional, cell-specific control of ion-channel physiology.

MicroRNA-mediated downregulation of K+ channels in pulmonary arterial hypertension.

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel ß1 subunit (BKCaß1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaß1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

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.

Distinct mechanisms underlying cholesterol protection against alcohol-induced BK channel inhibition and resulting vasoconstriction.

Alcohol (ethanol) at concentrations reached in blood following moderate to heavy drinking (30-80mM) reduces cerebral artery diameter via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) in cerebral artery smooth muscle. These channels consist of channel-forming α and regulatory ß1 subunits. A high-cholesterol diet protects against ethanol-induced constriction via accumulation of cholesterol within the vasculature. The molecular mechanisms of this protection remain unknown. In the present work, we demonstrate that in vitro cholesterol enrichment of rat middle cerebral arteries significantly increased cholesterol within arterial tissues and blunted constriction by 50mM of ethanol. Ethanol-induced BK channel inhibition in inside-out patches excised from freshly isolated cerebral artery myocytes was also abolished by cholesterol enrichment. Enrichment of arteries with enantiomeric cholesterol (ent-cholesterol) also blunted BK channel inhibition and cerebral artery constriction in response to ethanol. The similar protection of cholesterol and ent-cholesterol against ethanol action indicates that this protection does not require protein site(s) that specifically sense natural cholesterol. Cholesterol-driven protection against ethanol-induced BK channel inhibition and vasoconstriction was replicated in myocytes and middle cerebral arteries of C57BL/6 mice. BK ß1 subunits are known to regulate vascular diameter and its modification by ethanol. However, blunting of an ethanol effect by in vitro cholesterol enrichment was observed in arteries and myocyte membrane patches from BK ß1 (KCNMB1) knockout mice. Thus, BK ß1 subunits are not needed for cholesterol protection against ethanol effect on BK channel function and cerebral artery diameter.

Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat.

Large conductance, Ca2+i- and voltage-gated K+ (BK) channels regulate myogenic tone and, thus, arterial diameter. In smooth muscle (SM), BK channels include channel-forming α and auxiliary ß1 subunits. BK ß1 increases the channel's Ca2+ sensitivity, allowing BK channels to negatively feedback on depolarization-induced Ca2+ entry, oppose SM contraction and favor vasodilation. Thus, endothelial-independent vasodilation can be evoked though targeting of SM BK ß1 by endogenous ligands, including lithocholate (LCA). Here, we investigated the expression of BK ß1 across arteries of the cerebral and peripheral circulations, and the contribution of such expression to channel function and BK ß1-mediated vasodilation. Data demonstrate that endothelium-independent, BK ß1-mediated vasodilation by LCA is larger in coronary (CA) and basilar (BA) arteries than in anterior cerebral (ACA), middle cerebral (MCA), posterior cerebral (PCA), and mesenteric (MA) arteries, all arterial segments having a similar diameter. Thus, differential dilation occurs in extracranial arteries which are subjected to similar vascular pressure (CA vs. MA) and in arteries that irrigate different brain regions (BA vs. ACA, MCA, and PCA). SM BK channels from BA and CA displayed increased basal activity and LCA responses, indicating increased BK ß1 functional presence. Indeed, in the absence of detectable changes in BK α, BA and CA myocytes showed an increased location of BK ß1 in the plasmalemma/subplasmalemma. Moreover, these myocytes distinctly showed increased BK ß1 messenger RNA (mRNA) levels. Supporting a major role of enhanced BK ß1 transcripts in artery dilation, LCA-induced dilation of MCA transfected with BK ß1 complementary DNA (cDNA) was as high as LCA-induced dilation of untransfected BA or CA.

7-Ketocholesterol induces the reduction of KCNMB1 in atherosclerotic blood vessels.

Hypertension is a high-risk symptom in atherosclerotic patients, and vascular rigidity is one of the main factors leading to hypertension. ß1-Subunit of BKCa channel (KCNMB1; MaxiKß1) has been reported as a modulator of vascular flexibility. To determine the relationship between atherosclerosis and KCNMB1, we studied some atherogenic factors affecting vascular tone. Blood of atherosclerotic patients shows increased concentration of 7-ketocholesterol (7K), which has been studied as a harmful lipid to blood vessels. Our data showed that KCNMB1 was significantly down-regulated in the presence of 7K, in a dose-/time-dependent manner in vascular smooth muscle cells (VSMCs). And, the reduction of KCNMB1 was confirmed in cell images of 7K-stimulated VSMCs and in vessel tissue images of ApoE knock-out mice. To determine whether aryl hydrocarbon receptor (AhR) was involved in the reduction of KCNMB1 by 7K-stimulation, protein level of AhR was analyzed by Western blot. Our data showed that the reduction of KCNMB1 was modulated through the AhR pathway. In conclusion, results of our study suggest that 7K induces the reduction of KCNMB1 through the AhR pathway.

Hypoxia and ischemia-reperfusion: a BiK contribution?

Over the last decades, cardiovascular disease has become the primary cause of death in the Western world, and this trend is expanding throughout the world. In particular, atherosclerosis and the subsequent vessel obliterations are the primary cause of ischemic disease (stroke and coronary heart disease). Excess calcium influx into the cells is one of the major pathophysiological mechanisms important for ischemic injury in the brain and heart in humans. The large-conductance calcium-activated K(+) channels (BK) are thus interesting candidates to protect against excess calcium influx and the events leading to ischemic injury. Indeed, the mitochondrial BK channels (mitoBK) have recently been shown to play a protective function against ischemia-reperfusion injury both in vitro and in animal models, although the exact mechanism of this protection is still under scrutiny. In addition, in both the plasma membrane and mitochondrial BK channel, the α-subunit itself is sensitive to hypoxia. This sensitivity is tissue specific and conferred by a highly conserved motif within an alternatively spliced cysteine-rich insert (STREX) in the intracellular C terminus of the channel. This review describes recent developments of the increasing relevance of BK channels in hypoxia and ischemia-reperfusion injury.

DiBAC4(3) hits a "sweet spot" for the activation of arterial large-conductance Ca²âº-activated potassium channels independently of the ß1-subunit.

The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] has been reported as a novel large-conductance Ca²âº-activated K⁺ (BK) channel activator with selectivity for its ß1- or ß4-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory ß1-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the ß1-subunit, we explored the effects of DiBAC4(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC4(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic ß1-knockout mice in excised patches. BK channels from brain arteries, with or without the ß1-subunit, were similarly activated by DiBAC4(3). In addition, we found that saturating concentrations of DiBAC4(3) (~30 µM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as -300 mV. This "sweet spot" for persistent activation is independent of Ca²âº and/or the ß1₋4-subunits and is fully achieved when DiBAC4(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC4(3) varies across species and/or vascular beds. DiBAC4(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators.

Revisiting the Large-Conductance Calcium-Activated Potassium (BKCa) Channels in the Pulmonary Circulation.

Potassium ion concentrations, controlled by ion pumps and potassium channels, predominantly govern a cell's membrane potential and the tone in the vessels. Calcium-activated potassium channels respond to two different stimuli-changes in voltage and/or changes in intracellular free calcium. Large conductance calcium-activated potassium (BKCa) channels assemble from pore forming and various modulatory and auxiliary subunits. They are of vital significance due to their very high unitary conductance and hence their ability to rapidly cause extreme changes in the membrane potential. The pathophysiology of lung diseases in general and pulmonary hypertension, in particular, show the implication of either decreased expression and partial inactivation of BKCa channel and its subunits or mutations in the genes encoding different subunits of the channel. Signaling molecules, circulating humoral molecules, vasorelaxant agents, etc., have an influence on the open probability of the channel in pulmonary arterial vascular cells. BKCa channel is a possible therapeutic target, aimed to cause vasodilation in constricted or chronically stiffened vessels, as shown in various animal models. This review is a comprehensive collation of studies on BKCa channels in the pulmonary circulation under hypoxia (hypoxic pulmonary vasoconstriction; HPV), lung pathology, and fetal to neonatal transition, emphasising pharmacological interventions as viable therapeutic options.

Calcium-activated chloride channel regulator 1 (CLCA1): More than a regulator of chloride transport and mucus production.

CLCA1 is a member of the CLCA (calcium-activated chloride channel regulator) family and plays an essential role in goblet cell mucus production from the respiratory tract epithelium. CLCA1 also regulates Ca2+-dependent Cl- transport that involves the channel protein transmembrane protein 16A (TMEM16A) and its accessary molecules. CLCA1 modulates epithelial cell chloride current and participates in the pathogenesis of mucus hypersecretory-associated respiratory and gastrointestinal diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, colon colitis, cystic fibrosis intestinal mucous disease, ulcerative colitis, and gastrointestinal parasitic infection. Most studies have been focused on the expression regulation of CLCA1 in human specimens. Limited studies used the CLCA1-deficient mice and CLCA1 blocking agents and yielded inconsistent conclusions regarding its role in these diseases. CLCA1 not only regulates mucin expression, but also participates in innate immune responses by binding to yet unidentified molecules on inflammatory cells for cytokine and chemokine production. CLCA1 also targets lymphatic endothelial cells and cancer cells by regulating lymphatic cell proliferation and lymphatic sinus growth in the lymphatic organs and controlling cancer cell differentiation, proliferation, and apoptosis, all which depend on the location of the lymphatic vessels, the type of cancers, the presence of Th2 cytokines, and possibly the availability and type of CLCA1-binding proteins. Here we summarize available studies related to these different activities of CLCA1 to assist our understanding of how this secreted modifier of calcium-activated chloride channels (CaCCs) affects mucus production and innate immunity during the pathogenesis of respiratory, gastrointestinal, and malignant diseases.

BK Channels in the Vertebrate Inner Ear.

The perception of complex acoustic stimuli begins with the deconstruction of sound into its frequency components. This spectral processing occurs first and foremost in the inner ear. In vertebrates, two very different strategies of frequency analysis have evolved. In nonmammalian vertebrates, the sensory hair cells of the inner ear are intrinsically electrically tuned to a narrow band of acoustic frequencies. This electrical tuning relies on the interplay between BK channels and voltage-gated calcium channels. Systematic variations in BK channel density and kinetics establish a gradient in electrical resonance that enables the coding of a broad range of acoustic frequencies. In contrast, mammalian hair cells are extrinsically tuned by mechanical properties of the cochlear duct. Even so, mammalian hair cells also express BK channels. These BK channels play critical roles in various aspects of mammalian auditory signaling, from developmental maturation to protection against acoustic trauma. This review summarizes the anatomical localization, biophysical properties, and functional contributions of BK channels in vertebrate inner ears. Areas of future research, based on an updated understanding of the biology of both BK channels and the inner ear, are also highlighted. Investigation of BK channels in the inner ear continues to provide fertile research grounds for examining both BK channel biophysics and the molecular mechanisms underlying signal processing in the auditory periphery.

The smooth muscle-type ß1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels.

Large-conductance Ca(2+)-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC4(3) does not depend, as initially proposed, on the ß 1 or ß 4 subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the ß 1 subunit is not essential to this activation but exerts a large potentiating effect. DiBAC4(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca(2+). Kd values for BK channel activation by DiBAC4(3) in 0 mM Ca(2+) are approximately 20 µM (α) and 5 µM (α+ß 1), and G-V curves shift up to -40 mV and -110 mV, respectively. ß1 to ß2 mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the ß 1 subunit in cardiovascular pharmacology.