Sorbs2 Deficiency and Vascular BK Channelopathy in Diabetes.

BACKGROUND: Vascular large conductance Ca2+-activated K+ (BK) channel, composed of the α-subunit (BK-α) and the ß1-subunit (BK-ß1), is a key determinant of coronary vasorelaxation and its function is impaired in diabetic vessels. However, our knowledge of diabetic BK channel dysregulation is incomplete. The Sorbs2 (Sorbin homology [SoHo] and Src homology 3 [SH3] domains-containing protein 2), is ubiquitously expressed in arteries, but its role in vascular pathophysiology is unknown. METHODS: The role of Sorbs2 in regulating vascular BK channel activity was determined using patch-clamp recordings, molecular biological techniques, and in silico analysis. RESULTS: Sorbs2 is not only a cytoskeletal protein but also an RNA-binding protein that binds to BK channel proteins and BK-α mRNA, regulating BK channel expression and function in coronary smooth muscle cells. Molecular biological studies reveal that the SH3 domain of Sorbs2 is necessary for Sorbs2 interaction with BK-α subunits, while both the SH3 and SoHo domains of Sorbs2 interact with BK-ß1 subunits. Deletion of the SH3 or SoHo domains abolishes the Sorbs2 effect on the BK-α/BK-ß1 channel current density. Additionally, Sorbs2 is a target gene of the Nrf2 (nuclear factor erythroid-2-related factor 2), which binds to the promoter of Sorbs2 and regulates Sorbs2 expression in coronary smooth muscle cells. In vivo studies demonstrate that Sorbs2 knockout mice at 4 months of age display a significant decrease in BK channel expression and function, accompanied by impaired BK channel Ca2+-sensitivity and BK channel-mediated vasodilation in coronary arteries, without altering their body weights and blood glucose levels. Importantly, Sorbs2 expression is significantly downregulated in the coronary arteries of db/db type 2 diabetic mice. CONCLUSIONS: Sorbs2, a downstream target of Nrf2, plays an important role in regulating BK channel expression and function in vascular smooth muscle cells. Vascular Sorbs2 is downregulated in diabetes. Genetic knockout of Sorbs2 manifests coronary BK channelopathy and vasculopathy observed in diabetic mice, independent of obesity and glucotoxicity.

Dbx1 controls the development of astrocytes of the intermediate spinal cord by modulating Notch signaling.

Significant progress has been made in elucidating the basic principles that govern neuronal specification in the developing central nervous system. In contrast, much less is known about the origin of astrocytic diversity. Here, we demonstrate that a restricted pool of progenitors in the mouse spinal cord, expressing the transcription factor Dbx1, produces a subset of astrocytes, in addition to interneurons. Ventral p0-derived astrocytes (vA0 cells) exclusively populate intermediate regions of spinal cord with extraordinary precision. The postnatal vA0 population comprises gray matter protoplasmic and white matter fibrous astrocytes and a group of cells with strict radial morphology contacting the pia. We identified that vA0 cells in the lateral funiculus are distinguished by the expression of reelin and Kcnmb4. We show that Dbx1 mutants have an increased number of vA0 cells at the expense of p0-derived interneurons. Manipulation of the Notch pathway, together with the alteration in their ligands seen in Dbx1 knockouts, suggest that Dbx1 controls neuron-glial balance by modulating Notch-dependent cell interactions. In summary, this study highlights that restricted progenitors in the dorsal-ventral neural tube produce region-specific astrocytic subgroups and that progenitor transcriptional programs highly influence glial fate and are instrumental in creating astrocyte diversity.

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.

The potassium channel subunit Kvß1 serves as a major control point for synaptic facilitation.

Analysis of the presynaptic action potential's (APsyn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for Kv1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic Kv1 channel inactivation was mediated by the Kvß1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of Kvß1 blocked all broadening of the APsyn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic Kv channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery.

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.

Antenatal Dexamethasone Exposure Impairs the High-Conductance Ca2+-Activated K+ Channels via Epigenetic Alteration at Gene Promoter in Male Offspring.

OBJECTIVE: Antenatal exposure to glucocorticoids increases cardiovascular risks related to vascular dysfunctions in offspring, although underlying mechanisms are still unknown. As an important vascular mediator, high-conductance Ca2+-activated K+ channels (BK) plays an essential role in determining vascular tone. Long-term effects of antenatal glucocorticoids on BK in offspring are largely unknown. This study examined the effects and mechanisms of antenatal exposure to clinically relevant doses of glucocorticoids on vascular BK in offspring. Approach and Results: Pregnant Sprague-Dawley rats received synthetic glucocorticoids dexamethasone or vehicle during the last week of pregnancy. Vascular functions, cellular electrophysiology, target gene expression, and promoter methylation were examined in mesenteric arteries of male offspring (gestational day 21 [fetus] and postnatal day 120 [adult offspring]). Antenatal dexamethasone exposure impaired BK activators-mediated relaxation and reduced whole-cell BK currents in mesenteric arteries. Antenatal dexamethasone exposure did not alter Ca2+/voltage-sensitivity of BK but downregulated the expressions of BK α and ß1 subunits in both fetal and adult mesenteric arteries. In addition, increased promoter methylations within BKα and BKß1 were compatible with reduced expressions of the 2 genes. CONCLUSIONS: Our findings showed a profound and long-term impact of antenatal dexamethasone exposure on vascular BK via an altered epigenetic pattern from fetal stage to adulthood, advancing understanding of prolonged adverse effects and mechanisms of antenatal glucocorticoids exposure on vascular health in offspring.

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.

The Role of KCNMB1 and BK Channels in Myofibroblast Differentiation and Pulmonary Fibrosis.

The differentiation of fibroblasts into myofibroblasts is critical for the development of fibrotic disorders, including idiopathic pulmonary fibrosis (IPF). Previously, we demonstrated that fibroblasts from patients with IPF exhibit changes in DNA methylation across the genome that contribute to a profibrotic phenotype. One of the top differentially methylated genes identified in our previous study was KCNMB1, which codes for the ß subunit of the large-conductance potassium (BK, also known as MaxiK or KCa1.1) channel. Here, we examined how the expression of KCNMB1 differed between IPF fibroblasts and normal cells, and how BK channels affected myofibroblast differentiation. Fibroblasts from patients with IPF exhibited increased expression of KCNMB1, which corresponded to increased DNA methylation within the gene body. Patch-clamp experiments demonstrated that IPF fibroblasts had increased BK channel activity. Knockdown of KCNMB1 attenuated the ability of fibroblasts to contract collagen gels, and this was associated with a loss of α-smooth muscle actin (SMA) expression. Pharmacologic activation of BK channels stimulated α-SMA expression, whereas BK channel inhibitors blocked the upregulation of α-SMA. The ability of BK channels to enhance α-SMA expression was dependent on intracellular calcium, as activation of BK channels resulted in increased levels of intracellular calcium and the effects of BK agonists were abolished when calcium was removed. Together, our findings demonstrate that epigenetic upregulation of KCNMB1 contributes to increased BK channel activity in IPF fibroblasts, and identify a newfound role for BK channels in myofibroblast differentiation.

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.

MitoBKCa channel is functionally associated with its regulatory ß1 subunit in cardiac mitochondria.

KEY POINTS: Association of plasma membrane BKCa channels with BK-ß subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa ) by BK-ß subunits is not established. MitoBKCa -α and the regulatory BK-ß1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK-ß1 (left-shifted voltage dependence of activation, V1/2  = -55 mV, 12 µm matrix Ca2+ ). In BK-ß1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+ ) Co-expression of BKCa with the BK-ß1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK-ß1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. ABSTRACT: Association of the plasma membrane BKCa channel with auxiliary BK-ß1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa ) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK-ß1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po ) and voltage half-activation (V1/2  = -55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK-ß1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa -α and its BK-ß1 subunit. Mitochondria from the BK-ß1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+ ). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK-ß1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK-ß1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-ß1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK-ß1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.

Furosemide reduces BK-αß4-mediated K+ secretion in mice on an alkaline high-K+ diet.

Special high-K diets have cardioprotective effects and are often warranted in conjunction with diuretics such as furosemide for treating hypertension. However, it is not understood how a high-K diet (HK) influences the actions of diuretics on renal K+ handling. Furosemide acidifies the urine by increasing acid secretion via the Na+-H+ exchanger 3 (NHE3) in TAL and vacuolar H+-ATPase (V-ATPase) in the distal nephron. We previously found that an alkaline urine is required for large conductance Ca2+-activated K+ (BK)-αß4-mediated K+ secretion in mice on HK. We therefore hypothesized that furosemide could reduce BK-αß4-mediated K+ secretion by acidifying the urine. Treating with furosemide (drinking water) for 11 days led to decreased urine pH in both wild-type (WT) and BK-ß4-knockout mice (BK-ß4-KO) with increased V-ATPase expression and elevated plasma aldosterone levels. However, furosemide decreased renal K+ clearance and elevated plasma [K+] in WT but not BK-ß4-KO. Western blotting and immunofluorescence staining showed that furosemide treatment decreased cortical expression of BK-ß4 and reduced apical localization of BK-α in connecting tubules. Addition of the carbonic anhydrase inhibitor, acetazolamide, to furosemide water restored urine pH along with renal K+ clearance and plasma [K+] to control levels. Acetazolamide plus furosemide also restored the cortical expression of BK-ß4 and BK-α in connecting tubules. These results indicate that in mice adapted to HK, furosemide reduces BK-αß4-mediated K+ secretion by acidifying the urine.

Doxorubicin induces detrusor smooth muscle impairments through myosin dysregulation, leading to a risk of lower urinary tract dysfunction.

Cytotoxic chemotherapy is the foundation for the treatment of the wide variety of childhood malignancies; however, these therapies are known to have a variety of deleterious side effects. One common chemotherapy used in children, doxorubicin (DOX), is well known to cause cardiotoxicity and cardiomyopathy. Recent studies have revealed that DOX impairs skeletal and smooth muscle function and contributes to fatigue and abnormal intestinal motility in patients. In this study, we tested the hypothesis that systemic DOX administration also affects detrusor smooth muscle (DSM) function in the urinary bladder, especially when administered at a young age. The effects on the DSM and bladder function were assessed in BALB/cJ mice that received six weekly intravenous injections of DOX (3 mg·kg-1·wk-1) or saline for the control group. Systemic DOX administration resulted in DSM hypertrophy, increased voiding frequency, and a significant attenuation of DSM contractility, followed by a slower relaxation compared with the control group. Gene expression analyses revealed that unlike DOX-induced cardiotoxicity, the bladders from DOX-administered animals showed no changes in oxidative stress markers; instead, downregulation of large-conductance Ca2+-activated K+ channels and altered expression of myosin light-chain kinase coincided with reduced myosin light-chain phosphorylation. These results indicate that in vivo DOX exposure caused DSM dysfunction by dysregulation of molecules involved in the detrusor contractile-relaxation mechanisms. Collectively, our findings suggest that survivors of childhood cancer treated with DOX may be at increased risk of bladder dysfunction and benefit from followup surveillance of bladder function.

ß1-Subunit of the calcium-sensitive potassium channel modulates the pulmonary vascular smooth muscle cell response to hypoxia.

Accessory subunits associated with the calcium-sensitive potassium channel (BKCa), a major determinant of vascular tone, confer functional and anatomical diversity. The ß1 subunit increases Ca2+ and voltagesensitivity of the BKCa channel and is expressed exclusively in smooth muscle cells. Evidence supporting the physiological significance of the ß1 subunit includes the observations that murine models with deletion of the ß1 subunit are hypertensive and that humans with a gain-of-function ß1 mutation are at a decreased risk of diastolic hypertension. However, whether the ß1 subunit of the BKCa channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BKCa channel ß1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the ß1 subunit ( Kcnmb1-/-) were placed in chronic hypoxia (10% O2) for 21-24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1+/+ (controls) and Kcnmb1-/- mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1-/- mice compared with Kcnmb1+/+ mice, without increased vascular remodeling. ß1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMCs) from vessels ≤ 150 µm. Peripheral PASMCs contracted collagen gels irrespective of ß1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1-/- compared with Kcnmb1+/+ PASMCs. Compromised PASMC ß1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension.

Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle.

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.

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.

Regulation of Coronary Arterial Large Conductance Ca2+-Activated K+ Channel Protein Expression and Function by n-3 Polyunsaturated Fatty Acids in Diabetic Rats.

AIM: The objective of this study was to examine the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on coronary arterial large conductance Ca2+-activated K+ (BK) channel function in coronary smooth muscle cells (SMCs) of streptozotocin-induced diabetic rats. METHODS: The effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on coronary BK channel open probabilities were determined using the patch clamp technique. The mRNA and protein expressions of BK channel subunits were measured using qRT-PCR and Western blots. The coronary artery tension and coronary SMC Ca2+ concentrations were measured using a myograph system and fluorescence Ca2+ indicator. RESULTS: Compared to nondiabetic control rats, the BK channel function was impaired with a reduced response to EPA and DHA in freshly isolated SMCs of diabetic rats. Oral administration of n-3 PUFAs had no effects on protein expressions of BK channel subunits in nondiabetic rats, but significantly enhanced those of BK-ß1 in diabetic rats without altering BK-α protein levels. Moreover, coronary ring tension induced by iberiotoxin (a specific BK channel blocker) was increased and cytosolic Ca2+ concentrations in coronary SMCs were decreased in diabetic rats, but no changes were found in nondiabetic rats. CONCLUSIONS: n-3 PUFAs protect the coronary BK channel function and coronary vasoreactivity in diabetic rats as a result of not only increasing BK-ß1 protein expressions, but also decreasing coronary artery tension and coronary smooth muscle cytosolic Ca2+ concentrations.

Exercise Prevents Upregulation of RyRs-BKCa Coupling in Cerebral Arterial Smooth Muscle Cells From Spontaneously Hypertensive Rats.

OBJECTIVE: Regular exercise is an effective nonpharmacological means of preventing and controlling hypertension. However, the molecular mechanisms underlying its effects remain undetermined. The hypothesis that hypertension increases the functional coupling of large-conductance Ca(2+)-activated K(+) (BKCa) channels with ryanodine receptors in spontaneously hypertensive rats (SHR) as a compensatory response to an increase in intracellular Ca(2+) concentration in cerebral artery smooth muscle cells was assessed here. It was further hypothesized that exercise training would prevent this increase in functional coupling. APPROACH AND RESULTS: SHR and Wistar-Kyoto (WKY) rats were randomly assigned to sedentary groups (SHR-SED and WKY-SED) and exercise training groups (SHR-EX and WKY-EX). Cerebral artery smooth muscle cells displayed spontaneous transient outward currents at membrane potentials more positive than -40 mV. The amplitude of spontaneous transient outward currents together with the spontaneous Ca(2+) sparks in isolated cerebral artery smooth muscle cells was significantly higher in SHR-SED than in WKY-SED. Moreover, hypertension displayed increased whole-cell BKCa, voltage-gated Ca(2+) channel, but decreased KV currents in cerebral artery smooth muscle cells. In SHRs, the activity of the single BKCa channel increased markedly, and the protein expression of BKCa (ß1, but not α-subunit) also increased, but KV1.2 decreased significantly. Exercise training ameliorated all of these functional and molecular alterations in hypertensive rats. CONCLUSIONS: These data indicate that hypertension leads to enhanced functional coupling of ryanodine receptors-BKCa to buffer pressure-induced constriction of cerebral arteries, which attributes not only to an upregulation of BKCa ß1-subunit function but also to an increase of Ca(2+) release from ryanodine receptors. However, regular aerobic exercise efficiently prevents augmented coupling and so alleviates the pathological compensation and restores cerebral arterial function.

Genetic upregulation of BK channel activity normalizes multiple synaptic and circuit defects in a mouse model of fragile X syndrome.

KEY POINTS: Single-channel recordings in CA3 pyramidal neurons revealed that large-conductance calcium-activated K(+) (BK) channel open probability was reduced by loss of fragile X mental retardation protein (FMRP) and that FMRP acts on BK channels by modulating the channel's gating kinetics. Fmr1/BKß4 double knockout mice were generated to genetically upregulate BK channel activity in the absence of FMRP. Deletion of the BKß4 subunit alleviated reduced BK channel open probability via increasing BK channel open frequency, but not through prolonging its open duration. Genetic upregulation of BK channel activity via deletion of BKß4 normalized action potential duration, excessive glutamate release and short-term synaptic plasticity during naturalistic stimulus trains in excitatory hippocampal neurons in the absence of FMRP. Genetic upregulation of BK channel activity via deletion of BKß4 was sufficient to normalize excessive epileptiform activity in an in vitro model of seizure activity in the hippocampal circuit in the absence of FMRP. Loss of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), yet the mechanisms underlying the pathophysiology of FXS are incompletely understood. Recent studies identified important new functions of FMRP in regulating neural excitability and synaptic transmission via both translation-dependent mechanisms and direct interactions of FMRP with a number of ion channels in the axons and presynaptic terminals. Among these presynaptic FMRP functions, FMRP interaction with large-conductance calcium-activated K(+) (BK) channels, specifically their auxiliary ß4 subunit, regulates action potential waveform and glutamate release in hippocampal and cortical pyramidal neurons. Given the multitude of ion channels and mechanisms that mediate presynaptic FMRP actions, it remains unclear, however, to what extent FMRP-BK channel interactions contribute to synaptic and circuit defects in FXS. To examine this question, we generated Fmr1/ß4 double knockout (dKO) mice to genetically upregulate BK channel activity in the absence of FMRP and determine its ability to normalize multilevel defects caused by FMRP loss. Single-channel analyses revealed that FMRP loss reduced BK channel open probability, and this defect was compensated in dKO mice. Furthermore, dKO mice exhibited normalized action potential duration, glutamate release and short-term dynamics during naturalistic stimulus trains in hippocampal pyramidal neurons. BK channel upregulation was also sufficient to correct excessive seizure susceptibility in an in vitro model of seizure activity in hippocampal slices. Our studies thus suggest that upregulation of BK channel activity normalizes multi-level deficits caused by FMRP loss.

AKAP150 contributes to enhanced vascular tone by facilitating large-conductance Ca2+-activated K+ channel remodeling in hyperglycemia and diabetes mellitus.

RATIONALE: Increased contractility of arterial myocytes and enhanced vascular tone during hyperglycemia and diabetes mellitus may arise from impaired large-conductance Ca(2+)-activated K(+) (BKCa) channel function. The scaffolding protein A-kinase anchoring protein 150 (AKAP150) is a key regulator of calcineurin (CaN), a phosphatase known to modulate the expression of the regulatory BKCa ß1 subunit. Whether AKAP150 mediates BKCa channel suppression during hyperglycemia and diabetes mellitus is unknown. OBJECTIVE: To test the hypothesis that AKAP150-dependent CaN signaling mediates BKCa ß1 downregulation and impaired vascular BKCa channel function during hyperglycemia and diabetes mellitus. METHODS AND RESULTS: We found that AKAP150 is an important determinant of BKCa channel remodeling, CaN/nuclear factor of activated T-cells c3 (NFATc3) activation, and resistance artery constriction in hyperglycemic animals on high-fat diet. Genetic ablation of AKAP150 protected against these alterations, including augmented vasoconstriction. d-glucose-dependent suppression of BKCa channel ß1 subunits required Ca(2+) influx via voltage-gated L-type Ca(2+) channels and mobilization of a CaN/NFATc3 signaling pathway. Remarkably, high-fat diet mice expressing a mutant AKAP150 unable to anchor CaN resisted activation of NFATc3 and downregulation of BKCa ß1 subunits and attenuated high-fat diet-induced elevation in arterial blood pressure. CONCLUSIONS: Our results support a model whereby subcellular anchoring of CaN by AKAP150 is a key molecular determinant of vascular BKCa channel remodeling, which contributes to vasoconstriction during diabetes mellitus.

N-terminal isoforms of the large-conductance Ca²âº-activated K⁺ channel are differentially modulated by the auxiliary ß1-subunit.

The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK(Ca) α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary ß1-subunits, we examined whether ß1 differentially modulates the N-terminal BK(Ca) α-subunit isoforms. In the absence of ß1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas ß1 did not modulate the voltage-activation curve of MSSN, ß1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 µM and 100 µM Ca(2+). The ß1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the ß1-subunit may be due to the differential effect of ß1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related ß2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to ß1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK(Ca) channel activity in cells, highlighting a novel mechanism of BK(Ca) channel regulation.