BK Channel Function Research Articles

BK Channels in the Central Nervous System.

Large conductance Ca(2+)- and voltage-activated K(+) (BK) channels are widely distributed in the postnatal central nervous system (CNS). BK channels play a pleiotropic role in regulating the activity of brain and spinal cord neural circuits by providing a negative feedback mechanism for local increases in intracellular Ca(2+) concentrations. In neurons, they regulate the timing and duration of K(+) influx such that they can either increase or decrease firing depending on the cellular context, and they can suppress neurotransmitter release from presynaptic terminals. In addition, BK channels located in astrocytes and arterial myocytes modulate cerebral blood flow. Not surprisingly, both loss and gain of BK channel function have been associated with CNS disorders such as epilepsy, ataxia, mental retardation, and chronic pain. On the other hand, the neuroprotective role played by BK channels in a number of pathological situations could potentially be leveraged to correct neurological dysfunction.

Posttranscriptional and Posttranslational Regulation of BK Channels.

Large conductance calcium- and voltage-activated potassium (BK) channels are ubiquitously expressed and play an important role in the regulation of an eclectic array of physiological processes. Their diverse functional role requires channels with a wide variety of properties even though the pore-forming α-subunit is encoded by a single gene, KCNMA1. To achieve this, BK channels exploit some of the most fundamental posttranscriptional and posttranslational mechanisms that allow proteomic diversity to be generated from a single gene. These include mechanisms that diversify mRNA variants and abundance such as alternative pre-mRNA splicing, editing, and control by miRNA. The BK channel is also subject to a diverse array of posttranslational modifications including protein phosphorylation, lipidation, glycosylation, and ubiquitination to control the number, properties, and regulation of BK channels in specific cell types. Importantly, "cross talk" between these posttranscriptional and posttranslational modifications typically converge on disordered domains of the BK channel α-subunit. This allows both wide physiological diversity to be generated and a diversity of mechanisms to allow conditional regulation of BK channels and is emerging as an important determinant of BK channel function in health and disease.

Transient BK outward current enhances motoneurone firing rates during Drosophila larval locomotion.

We combine in situ electrophysiology with genetic manipulation in Drosophila larvae aiming to investigate the role of fast calcium-activated potassium currents for motoneurone firing patterns during locomotion. We first demonstrate that slowpoke channels underlie fast calcium-activated potassium currents in these motoneurones. By conducting recordings in semi-intact animals that produce crawling-like movements, we show that slowpoke channels are required specifically in motoneurones for maximum firing rates during locomotion. Such enhancement of maximum firing rates occurs because slowpoke channels prevent depolarization block by limiting the amplitude of motoneurone depolarization in response to synaptic drive. In addition, slowpoke channels mediate a fast afterhyperpolarization that ensures the efficient recovery of sodium channels from inactivation during high frequency firing. The results of the present study provide new insights into the mechanisms by which outward conductances facilitate neuronal excitability and also provide direct confirmation of the functional relevance of precisely regulated slowpoke channel properties in motor control. A large number of voltage-gated ion channels, their interactions with accessory subunits, and their post-transcriptional modifications generate an immense functional diversity of neurones. Therefore, a key challenge is to understand the genetic basis and precise function of specific ionic conductances for neuronal firing properties in the context of behaviour. The present study identifies slowpoke (slo) as exclusively mediating fast activating, fast inactivating BK current (ICF ) in larval Drosophila crawling motoneurones. Combining in vivo patch clamp recordings during larval crawling with pharmacology and targeted genetic manipulations reveals that ICF acts specifically in motoneurones to sculpt their firing patterns in response to a given input from the central pattern generating (CPG) networks. First, ICF curtails motoneurone postsynaptic depolarizations during rhythmical CPG drive. Second, ICF is activated during the rising phase of the action potential and mediates a fast afterhyperpolarization. Consequently, ICF is required for maximal intraburst firing rates during locomotion, probably by allowing recovery from inactivation of fast sodium channels and decreased potassium channel activation. This contrasts the common view that outward conductances oppose excitability but is in accordance with reports on transient BK and Kv3 channel function in multiple types of vertebrate neurones. Therefore, our finding that ICF enhances firing rates specifically during bursting patterns relevant to behaviour is probably of relevance to all brains.

BK channel β1-subunit deficiency exacerbates vascular fibrosis and remodelling but does not promote hypertension in high-fat fed obesity in mice.

Reduced expression or increased degradation of BK (large conductance Ca-activated K) channel β1-subunits has been associated with increased vascular tone and hypertension in some metabolic diseases. The contribution of BK channel function to control of blood pressure (BP), heart rate (HR) and vascular function/structure was determined in wild-type and BK channel β1-subunit knockout mice fed a high-fat or control diet. After 24 weeks of high-fat diet, wild-type and BK β1-knockout mice were obese, diabetic, but normotensive. High-fat-BK β1-knockout mice had decreased HR, while high-fat-wild-type mice had increased HR compared with mice on the control diet. Ganglion blockade caused a greater fall in BP and HR in mice on a high-fat diet than in mice on the control diet. β1-adrenergic receptor blockade reduced BP and HR equally in all groups. α1-adrenergic receptor blockade decreased BP in high-fat-BK β1-knockout mice only. Echocardiographic evaluation revealed left ventricular hypertrophy in high-fat-BK β1-knockout mice. Although under anaesthesia, mice on a high-fat diet had higher absolute stroke volume and cardiac output, these measures were similar to control mice when adjusted for body weight. Mesenteric arteries from high-fat-BK β1-knockout mice had higher norepinephrine reactivity, greater wall thickness and collagen accumulation than high-fat-wild-type mesenteric arteries. Compared with control-wild-type mesenteric arteries, high-fat-wild-type mesenteric arteries had blunted contractile responses to a BK channel blocker, although BK α-subunit (protein) and β1-subunit (mRNA) expression were unchanged. BK channel deficiency promotes increased sympathetic control of BP, and vascular dysfunction, remodelling and fibrosis, but does not cause hypertension in high-fat fed mice.

Molecular mechanisms of diabetic coronary dysfunction due to large conductance Ca2⁺-activated K⁺ channel impairment.

Diabetes mellitus is associated with coronary dysfunction, contributing to a 2- to 4-fold increase in the risk of coronary heart diseases. The mechanisms by which diabetes induces vasculopathy involve endothelial-dependent and -independent vascular dysfunction in both type 1 and type 2 diabetes mellitus. The purpose of this study is to determine the role of vascular large conductance Ca(2+)-activated K(+) (BK) channel activities in coronary dysfunction in streptozotocin-induced diabetic rats. Using videomicroscopy, immunoblotting, fluorescent assay and patch clamp techniques, we investigated the coronary BK channel activities and BK channel-mediated coronary vasoreactivity in streptozotocin-induced diabetic rats. BK currents (defined as the iberiotoxin-sensitive K(+) component) contribute (65 ± 4)% of the total K(+) currents in freshly isolated coronary smooth muscle cells and > 50% of the contraction of the inner diameter of coronary arteries from normal rats. However, BK current density is remarkably reduced in coronary smooth muscle cells of streptozotocin-induced diabetic rats, leading to an increase in coronary artery tension. BK channel activity in response to free Ca(2+) is impaired in diabetic rats. Moreover, cytoplasmic application of DHS-1 (a specific BK channel b(1) subunit activator) robustly enhanced the open probability of BK channels in coronary smooth muscle cells of normal rats. In diabetic rats, the DHS-1 effect was diminished in the presence of 200 nmol/L Ca(2+) and was significantly attenuated in the presence of high free calcium concentration, i.e., 1 mmol/L Ca(2+). Immunoblotting experiments confirmed that there was a 2-fold decrease in BK-b(1) protein expression in diabetic vessels, without altering the BK channel α-subunit expression. Although the cytosolic Ca(2+) concentration of coronary arterial smooth muscle cells was increased from (103 ± 23) nmol/L (n = 5) of control rats to (193 ± 22) nmol/L (n = 6, P < 0.05) of STZ-induced diabetic rats, reduced BK-b(1) expression made these channels less sensitive to intracellular Ca(2+), which in turn led to enhanced smooth muscle contraction. Our results indicated that BK channels are the key determinant of coronary arterial tone. Impaired BK channel function in diabetes mellitus is associated with down-regulation of BK-b(1) expression and reduction of the b(1)-mediated BK channel activation in diabetic vessels.

Abstract 616: Diminished K Channel Inhibition Impairs Serotonin-Mediated Vasoconstriction in the Middle Cerebral Arteries From the Fawn Hooded Hypertensive Rat

Fawn Hooded Hypertensive (FHH) rat exhibit impaired pressure-mediated myogenic response (MR) in the middle cerebral arteries (MCAs) that is associated with an increase in large conductance potassium (BK) channel function. Introgression of 2.4 Mbp region of BN (Brown Norway) chromosome 1 into FHH rats (FHH.1BN) restored MR and BK channel function. The present study assessed the hypothesis that FHH rats also exhibit impaired serotonin (5-HT)-mediated vasoconstriction due to diminished BK channel inhibition compared to FHH.1BN rats. We used pressure myography and patch-clamp to measure vessel response and K channel function respectively and Fluo 4 method to measure cytosolic calcium. Basal myogenic tone of MCAs as measured by change in diameter from 22oC to 37oC temperature was ~2.6 fold lower in FHH rats compared to FHH.1BN rats (FHH, 8.1 ± 2% and FHH.1BN, 21.7 ± 3%, n = 4; p&lt;0.05). Vascular smooth muscle cells (VSMCs) of FHH rats have significantly more negative membrane potentials (Em) (–32 ± 1 mV; n=9) compared to FHH.1BN rats (-16 ± 2mV; n=10). 5-HT-mediated vasoconstriction was lower in MCAs isolated from FHH rats compared to FHH.1BN rats (5-HT: 1μM; FHH, 36 ± 5%; FHH.1BN rats, 58 ± 9%; n = 4; p&lt;0.05). 5-HT-mediated BK channel inhibition was less in FHH rats (3μM 5-HT: FHH, 8 ± 3%; FHH.1BN, 39 ± 4%; n = 4; p&lt;0.05). 5-HT did not affect VSMC membrane potential in FHH rats compared to FHH.1BN rats (delta change in Em: FHH, -2 ± 2mV; n = 4; FHH.1BN, -9 ± 1 mV; n = 5; p&lt;0.05). 5-HT-mediated increase in calcium fluorescence (F/Fo) during plateau phase was blunted in the VSMCs isolated from FHH rats compared to FHH.1BN rats (FHH: 1 + 0.01% Vs 1.22 + 0.02% in FHH.1BN (3 rats)). Finally, inhibition of BK channel restored 5-HT-mediated vasoconstriction in MCAs of FHH rats but did not affect FHH.1BN vessels (5-HT + Paxilline; FHH, 2.6 ± 0.6 fold; FHH.1BN, 1.4 ± 0.4 fold; n = 5; p&lt;0.05). In conclusion, mutation in the genes located in 2.4 Mbp region of FHH rats disrupts 5-HT-mediated BK channel inhibition that prevents the raise in [Ca2+]i and this may contribute to impaired 5HT-mediated vasoconstriction.

Maternal high-salt diets affected pressor responses and microvasoconstriction via PKC/BK channel signaling pathways in rat offspring.

High-salt (HS) intake is linked to hypertension, and prenatal exposure to maternal HS diets may have long-term impact on cardiovascular systems. The relationship between HS diets and cardiovascular disease has received extensive attention. This study determined pressor responses and microvessel functions in the adult offspring rats exposed to prenatal HS. The offspring of 5-month old as young adults in rats were used. Blood pressure, vascular tone, intracellular Ca(2+), and BK channels in mesenteric arteries were measured in the offspring. Phenylephrine (Phe)-induced pressor responses were significantly higher in the prenatal HS offspring. Vessel tension and intracellular Ca(2+) concentrations associated with Phe-induced pressor responses were increased in the mesenteric arteries of the HS offspring. PKC α- and δ-isoforms were upregulated in mesenteric arteries of the HS offspring. The enhanced Phe-mediated vascular activity was linked to the altered PKC-modulated BK channel functions. The results suggested that prenatal exposure to HS altered microvascular activity probably via changes in PKC/BK signaling pathways, which may lead to increased risks of hypertension in the offspring.

Enhanced Serotonin (5‐HT)‐Mediated Carotid Vessel Constriction From The Fawn Hooded Hypertensive (FHH) Rat

Impaired myogenic response associated with an increase in BK (large conductance potassium) channel function was suggested in cerebral resistance vessels of FHH rats. Introgression of BN (Brown Norway) 2.4 Mbp region of chromosome 1 into FHH rats restored the function of these vessels by normalizing BK channel function in an FHH.1BN congenic rat. However, conclusions derived from resistance vessels cannot be extrapolated to conduit vessels such as the carotid or aorta since they differ in structure, function, and expression of contractile proteins. Here we tested agonist‐mediated contraction and the regulatory role of PKC in mediating K channel function using isometric measurement of tension in carotid vessels isolated from FHH and FHH.1BN rats. Serotonin (5‐HT)‐mediated tension is enhanced in carotid rings isolated from FHH rats when compared to FHH.1BN rats. 5‐HT‐mediated constriction is enhanced further more by inhibition of Kv channel (4AP; 1mM) but not BK channel (Paxilline or Iberiotoxin; 100nM) in FHH rats. 5‐HT‐mediated constriction is not affected by PKC activation (PMA; 100nM) in FHH rats but significantly enhanced in FHH.1BN rats. While inhibition of PKC (GF 109203X, 3µM) diminished 5‐HT‐mediated contraction in both strains it has significantly less effect in FHH rats compared to FHH.1BN rats. Together, these results suggest that mutation in the genes located in 2.4 Mbp region of FHH rat may enhance PKC function that inhibit K channels contributing to augmented 5‐HT‐mediated carotid vessel constriction. This research has received partial funding support from the American Heart Association, Scientist Development Grant (13SDG14000006).

New insights into the complex physiologic role of the cilium in secretory epithelia

The cortical collecting duct (CCD) of the distal nephron is a major site for the final renal regulation of K homeostasis. Within this segment, principal cells (PCs) possess a single apical cilium whereas the surfaces of adjacent intercalated cells (ICs), devoid of cilia, are decorated with abundant microvilli and microplicae.Increases in tubular (urinary) flow rate, induced by volume expansion, diuretics, or a high K diet, subject CCD cells to hydrodynamic forces (fluid shear stress, circumferential stretch, and drag/torque on apical cilia and presumably microvilli/microplicae) that are transduced into increases in PC and IC cytoplasmic Ca2+ concentration that activate apical voltage-, stretch- and Ca2+-activated BK channels, which mediate flow-induced K secretion (FIKS). Functional and immunoreactive apical BK channels are more abundant in ICs than PCs, but appear to be constitutively closed under low flow conditions in a cell-specific manner by PKA/AKAP and MAPK elements in PCs, and WNK4 in ICs. We have recently identified a high density of immunoreactive and conducting BK channels in PC cilia. Pharmacologic deciliation of CCDs does not affect FIKS, leading us to conclude thatBK channels in PC cilia do not mediate FIKS. We speculate that cilia BK channels are responsible for localized hyperpolarization of membrane potential, facilitating sustained mechanoinduced Ca2+ entry through TRPP Ca2+-selective channels in this organelle and subsequent autocrine/paracrine signaling events.

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

The role of cAMP in synaptic homeostasis in response to environmental temperature challenges and hyperexcitability mutations.

Homeostasis is the ability of physiological systems to regain functional balance following environment or experimental insults and synaptic homeostasis has been demonstrated in various species following genetic or pharmacological disruptions. Among environmental challenges, homeostatic responses to temperature extremes are critical to animal survival under natural conditions. We previously reported that axon terminal arborization in Drosophila larval neuromuscular junctions (NMJs) is enhanced at elevated temperatures; however, the amplitude of excitatory junctional potentials (EJPs) remains unaltered despite the increase in synaptic bouton numbers. Here we determine the cellular basis of this homeostatic adjustment in larvae reared at high temperature (HT, 29°C). We found that synaptic current focally recorded from individual synaptic boutons was unaffected by rearing temperature (<15°C to >30°C). However, HT rearing decreased the quantal size (amplitude of spontaneous miniature EJPs, or mEJPs), which compensates for the increased number of synaptic releasing sites to retain a normal EJP size. The quantal size decrease is accounted for by a decrease in input resistance of the postsynaptic muscle fiber, indicating an increase in membrane area that matches the synaptic growth at HT. Interestingly, a mutation in rutabaga (rut) encoding adenylyl cyclase (AC) exhibited no obvious changes in quantal size or input resistance of postsynaptic muscle cells after HT rearing, suggesting an important role for rut AC in temperature-induced synaptic homeostasis in Drosophila. This extends our previous finding of rut-dependent synaptic homeostasis in hyperexcitable mutants, e.g., slowpoke (slo). In slo larvae, the lack of BK channel function is partially ameliorated by upregulation of presynaptic Shaker (Sh) IA current to limit excessive transmitter release in addition to postsynaptic glutamate receptor recomposition that reduces the quantal size.

Different expressions of large-conductance Ca2+-activated K+ channels in the mouse renal cortex and hippocampus during postnatal development.

Large-conductance Ca(+)-activated K(+) (BKCa) channels are widely distributed in a variety of cells and play a pivotal and specific role in many pathophysiological conditions. However, the function of BK(Ca) channels in the kidney cortex and hippocampus during the postnatal development has not received attention. In this study, to elucidate the role of BK(Ca) channels during the development, it is essential to establish the location and quantitation of expression of BK(Ca). The expressions of BK(Ca) were detected in the kidney and hippocampus on postnatal days (P) 1, 3, 5, 7, 14, 21, 28, and 49 by immunohistochemical and Western blot analysis. Our results showed that expressions of BK(Ca) channels were found in tubules and corpuscles at all time points. The expression was also observed at all developmental stages of the renal corpuscles, such as comma-shaped body, S-shaped body, renal corpuscles of stage III, and renal corpuscles of stage IV. During the development, the expression of BK(Ca) channels was decreased and the most prominent change of BK(Ca) protein level appeared between P14 and P21. In contrast, BK(Ca) channels were expressed in all regions of the hippocampus at every time point with the level increasing during the early development (P1 to P14). The findings of the present study suggest that BKCa channels play an important role during the postnatal development in both the renal cortex and hippocampus.

Modulation of BK channels contributes to activity-dependent increase of excitability through MTORC1 activity in CA1 pyramidal cells of mouse hippocampus.

Memory acquisition and synaptic plasticity are accompanied by changes in the intrinsic excitability of CA1 pyramidal neurons. These activity-dependent changes in excitability are mediated by modulation of intrinsic currents which alters the responsiveness of the cell to synaptic inputs. The afterhyperpolarization (AHP), a major contributor to the regulation of neuronal excitability, is reduced in animals that have acquired several types of hippocampus-dependent memory tasks and also following synaptic potentiation by high frequency stimulation. BK channels underlie the fast AHP and contribute to spike repolarization, and this AHP is reduced in animals that successfully acquired trace-eyeblink conditioning. This suggests that BK channel function is activity-dependent, but the mechanisms are unknown. In this study, we found that blockade of BK channels with paxilline (10 μM) decreased IAHP amplitude and increased spike half-width and instantaneous frequency in response to a +100 pA depolarization. In addition, induction of long term potentiation (LTP) by theta burst stimulation (TBS) in CA1 pyramidal neurons reduced BK channel’s contribution to IAHP, spike repolarization, and instantaneous frequency. This result indicates that BK channel activity is decreased following synaptic potentiation. Interestingly, blockade of mammalian target of rapamycin (MTORC1) with rapamycin (400 nM) following synaptic potentiation restored BK channel function, suggesting a role for protein translation in signaling events which decreased postsynaptic BK channel activity following synaptic potentiation.

The Subproteome of mitoBKCa from Cardiomyocytes Reveals Novel Insights into BK Channel Function and Pathology

The mitochondrial large-conductance Ca2+ -activated K+ channel (mitoBKCa) of adult cardiomyocytes contains a C-terminal 50 amino acid insert (DEC) as essential for mitochondrial targeting. The activation of mitochondrial BK channels (mitoBKCa) protects the heart from ischemia and reperfusion injury, highlighting its significant role in cardiac function. Here, we characterized the subproteome of mitoBKCa in cardiomyocytes in the quest to better understand its physiological and pathological relevance in the heart. To this end, we used GST-DEC and GST proteins to pull down proteins interacting with DEC using cardiomyocyte lysates and mitochondrial lysates. We also used monoclonal BK antibody and IgG to immunoprecipitate proteins interacting with mitoBKCa using Percoll purified mitochondrial lysates from whole heart. In all, we identified 212 cellular proteins and 157 mitochondrial proteins interacting with DEC; and 60 mitochondrial proteins interacting with mitoBKCa. Gene oncology analysis revealed mitoBKCa potential contribution to multiple cellular functions. It could contribute to oxidative respiration through cytochrome c oxidase (5 subunits) and ADP/ATP translocase (AAC), including AAC1 (10 peptides, n=2) and AAC2 (13 peptides, n=2), and regulate mitochondrial metabolism through acetyl-Coenzyme transferase 2 (7 peptides, n=2). In addition, our data revealed that mitoBKCa has a broader impact. It might facilitate the pathological process of Parkinson's disease and multiple types of cancer by interacting with critical enzymes, such as NADH dehydrogenase (7 subunits, 3-7 peptides each) and integrin linked kinase (2 peptides, n =1). Excitingly, mitoBKCa interacted with 15 different subunits of the ATP synthase, which forms the permeability transition pore, underscoring a new mechanism for mitoBKCa to protect the heart. In summary, proteomic analysis identified a wide variety of cellular and mitochondrial proteins forming complex with mitoBKCa channel, revealing new paradigms for BK channel function.

Regulation of BK channels by auxiliary γ subunits.

The large-conductance, calcium- and voltage-activated potassium (BK) channel has the largest single-channel conductance among potassium channels and can be activated by both membrane depolarization and increases in intracellular calcium concentration. BK channels consist of pore-forming, voltage- and calcium-sensing α subunits, either alone or in association with regulatory subunits. BK channels are widely expressed in various tissues and cells including both excitable and non-excitable cells and display diverse biophysical and pharmacological characteristics. This diversity can be explained in part by posttranslational modifications and alternative splicing of the α subunit, which is encoded by a single gene, KCNMA1, as well as by tissue-specific β subunit modulation. Recently, a leucine-rich repeat-containing membrane protein, LRRC26, was found to interact with BK channels and cause an unprecedented large negative shift (~-140 mV) in the voltage dependence of the BK channel activation. LRRC26 allows BK channels to open even at near-physiological calcium concentration and membrane voltage in non-excitable cells. Three LRRC26-related proteins, LRRC52, LRRC55, and LRRC38, were subsequently identified as BK channel modulators. These LRRC proteins are structurally and functionally distinct from the BK channel β subunits and were designated as γ subunits. The discovery of the γ subunits adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. Unlike BK channel β subunits, which have been intensively investigated both mechanistically and physiologically, our understanding of the γ subunits is very limited at this stage. This article reviews the structure, modulatory mechanisms, physiological relevance, and potential therapeutic implications of γ subunits as they are currently understood.

BK channel activators and their therapeutic perspectives

The large conductance calcium- and voltage-activated K+ channel (KCa1.1, BK, MaxiK) is ubiquitously expressed in the body, and holds the ability to integrate changes in intracellular calcium and membrane potential. This makes the BK channel an important negative feedback system linking increases in intracellular calcium to outward hyperpolarizing potassium currents. Consequently, the channel has many important physiological roles including regulation of smooth muscle tone, neurotransmitter release and neuronal excitability. Additionally, cardioprotective roles have been revealed in recent years. After a short introduction to the structure, function and regulation of BK channels, we review the small organic molecules activating BK channels and how these tool compounds have helped delineate the roles of BK channels in health and disease.

Lipid regulation of BK channel function.

This mini-review focuses on lipid modulation of BK (MaxiK, BKCa) current by a direct interaction between lipid and the BK subunits and/or their immediate lipid environment. Direct lipid-BK protein interactions have been proposed for fatty and epoxyeicosatrienoic acids, phosphoinositides and cholesterol, evidence for such action being less clear for other lipids. BK α (slo1) subunits are sufficient to support current perturbation by fatty and epoxyeicosatrienoic acids, glycerophospholipids and cholesterol, while distinct BK β subunits seem necessary for current modulation by most steroids. Subunit domains or amino acids that participate in lipid action have been identified in a few cases: hslo1 Y318, cerebral artery smooth muscle (cbv1) R334,K335,K336, cbv1 seven cytosolic CRAC domains, slo1 STREX and β1 T169,L172,L173 for docosahexaenoic acid, PIP2, cholesterol, sulfatides, and cholane steroids, respectively. Whether these protein motifs directly bind lipids or rather transmit the energy of lipid binding to other areas and trigger protein conformation change remains unresolved. The impact of direct lipid-BK interaction on physiology is briefly discussed.

WNK1 activates large-conductance Ca2+-activated K+ channels through modulation of ERK1/2 signaling.

With no lysine (WNK) kinases are members of the serine/threonine kinase family. We previously showed that WNK4 inhibits renal large-conductance Ca(2+)-activated K(+) (BK) channel activity by enhancing its degradation through a lysosomal pathway. In this study, we investigated the effect of WNK1 on BK channel activity. In HEK293 cells stably expressing the α subunit of BK (HEK-BKα cells), siRNA-mediated knockdown of WNK1 expression significantly inhibited both BKα channel activity and open probability. Knockdown of WNK1 expression also significantly inhibited BKα protein expression and increased ERK1/2 phosphorylation, whereas overexpression of WNK1 significantly enhanced BKα expression and decreased ERK1/2 phosphorylation in a dose-dependent manner in HEK293 cells. Knockdown of ERK1/2 prevented WNK1 siRNA-mediated inhibition of BKα expression. Similarly, pretreatment of HEK-BKα cells with the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of WNK1 siRNA on BKα expression in a dose-dependent manner. Knockdown of WNK1 expression also increased the ubiquitination of BKα channels. Notably, mice fed a high-K(+) diet for 10 days had significantly higher renal protein expression levels of BKα and WNK1 and lower levels of ERK1/2 phosphorylation compared with mice fed a normal-K(+) diet. These data suggest that WNK1 enhances BK channel function by reducing ERK1/2 signaling-mediated lysosomal degradation of the channel.

An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel.

Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol-BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel's physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function.

LRRC26 is a functional BK channel auxiliary γ subunit in arterial smooth muscle cells.

Smooth muscle cell (myocyte) large-conductance calcium (Ca)(2+)-activated potassium (BK) channels are functionally significant modulators of arterial contractility. Arterial myocytes express both pore-forming BKα and auxiliary β1 subunits, which increase channel Ca(2+) sensitivity. Recently, several leucine-rich repeat containing (LRRC) proteins have been identified as auxiliary γ subunits that elevate the voltage sensitivity of recombinant and prostate adenocarcinoma BK channels. LRRC expression and physiological functions in native cell types are unclear. Investigate the expression and physiological functions of leucine-rich repeat containing protein 26 (LRRC26) in arterial myocytes. Reverse transcription polymerase chain reaction and Western blotting detected LRRC26 mRNA and protein in cerebral artery myocytes. Biotinylation, immunofluorescence resonance energy transfer microscopy, and coimmunoprecipitation indicated that LRRC26 was located in close spatial proximity to, and associated with, plasma membrane BKα subunits. LRRC26 knockdown (RNAi) reduced total and surface LRRC26, but did not alter BKα or β1, proteins in arteries. LRRC26 knockdown did not alter Ca(2+) sparks but reduced BK channel voltage sensitivity, which decreased channel apparent Ca(2+) sensitivity and transient BK current frequency and amplitude in myocytes. LRRC26 knockdown also increased myogenic tone over a range (40-100 mm Hg) of intravascular pressures, and reduced vasoconstriction to iberiotoxin and vasodilation to NS1619, BK channel inhibitors and activators, respectively. In contrast, LRRC26 knockdown did not alter depolarization (60 mmol/L K(+))-induced vasoconstriction. LRRC26 is expressed, associates with BKα subunits, and elevates channel voltage- and apparent Ca(2+) sensitivity in arterial myocytes to induce vasodilation. This study indicates that arterial myocytes express a functional BK channel γ subunit.