BK Channel Function Research Articles

Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

BK channel dysfunction disrupts attention-controlled behaviors and altered perseverative responses in murine instrumental learning

This study examined the effect of knockout of KCNMA1 gene, coding for the BK channel, on cognitive and attentional functions in mice, with an aim to better understand its implications for human neurodevelopmental disorders. The study used the 3-choice serial reaction time task (3-CSRTT) to assess the learning performance, attentional abilities, and repetitive behaviors in mice lacking the KCNMA1 gene (KCNMA1-/-) compared to wild-type (WT) controls. Results showed no significant differences in learning accuracy between the two groups. However, KCNMA1-/- mice were more prone to omitting responses to stimuli. In addition, when the timing of cue presentation was randomized, the KCNMA1-/- showed premature responses. Notably, these mice also demonstrated a marked reduction in perseverative responses, which include repeated nose-poke behaviors following decisions. These findings highlight the involvement of the KCNMA1 gene in managing attention, impulsivity, and potentially moderating repetitive actions.

Large conductance voltage-and calcium-activated K+ (BK) channel in health and disease.

Large Conductance Voltage- and Calcium-activated K+ (BK) channels are transmembrane pore-forming proteins that regulate cell excitability and are also expressed in non-excitable cells. They play a role in regulating vascular tone, neuronal excitability, neurotransmitter release, and muscle contraction. Dysfunction of the BK channel can lead to arterial hypertension, hearing disorders, epilepsy, and ataxia. Here, we provide an overview of BK channel functioning and the implications of its abnormal functioning in various diseases. Understanding the function of BK channels is crucial for comprehending the mechanisms involved in regulating vital physiological processes, both in normal and pathological conditions, controlled by BK. This understanding may lead to the development of therapeutic interventions to address BK channelopathies.

Sorbs2 Deficiency and Vascular BK Channelopathy in Diabetes.

Vascular large conductance Ca2+-activated K+ (BK) channel, composed of the α-subunit (BK-α) and the β1-subunit (BK-β1), 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. The role of Sorbs2 in regulating vascular BK channel activity was determined using patch-clamp recordings, molecular biological techniques, and in silico analysis. 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-β1 subunits. Deletion of the SH3 or SoHo domains abolishes the Sorbs2 effect on the BK-α/BK-β1 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. 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.

Lysosomal BK channels facilitate silica-induced inflammation in macrophages

Background Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1β release. Methods Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1β release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. Results PAX pretreatment significantly attenuated silica-induced cell death and IL-1β release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1β release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. Conclusion Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1β release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.

Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway.

Background: Advanced glycation end products (AGEs) impair vascular physiology in Diabetes mellitus (DM). However, the underlying mechanisms remain unclear. Vascular large conductance calcium-activated potassium (BK) channels play important roles in coronary arterial function.Purpose: Our study aimed to investigate the regulatory role of AGEs in BK channels.Research Design: Using gavage of vehicle (V, normal saline) or aminoguanidine (A) for 8 weeks, normal and diabetic rats were divided into four groups: C+V group, DM+V group, C+A group, and DM+A group.Study Sample: Coronary arteries from different groups of rats and human coronary smooth muscle cells were used in this study.Data Collection and Analysis: Data were presented as mean ± SEM (standard error of mean). Student's t-test was used to compare data between two groups. One-way ANOVA with post-hoc LSD analysis was used to compare data between multiple groups.Results: Compared to the C+V group, vascular contraction induced by iberiotoxin (IBTX), a BK channel inhibitor, was impaired, and BK channel densities decreased in the DM+V group. However, aminoguanidine administration reduced the impairment. Protein expression of BK-β1, phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein kinase B (PKB or Akt) were down-regulated, while F-box protein 32 (FBXO32) expression increased in the DM+V group and in high glucose (HG) cultured human coronary smooth muscle cells. Treatment with aminoguanidine in vitro and in vivo could reverse the above protein expression. The effect of aminoguanidine on the improvement of BK channel function by inhibiting the generation of AGEs was reversed by adding MK2206 (Akt inhibitor) or Compound C (AMPK inhibitor) in HG conditions in vitro.Conclusions: AGEs aggravate BK channel dysfunction via the AMPK/Akt/FBXO32 signaling pathway.

Functional characterization of KCNMA1 mutation associated with dyskinesia, seizure, developmental delay, and cerebellar atrophy

KCNMA1 located on chromosome 10q22.3, encodes the pore-forming α subunit of the ‘Big K+’ (BK) large conductance calcium and voltage-activated K + channel. Numerous evidence suggests the functional BK channel alterations produced by different KCNMA1 alleles may associate with different symptoms, such as paroxysmal non kinesigenic dyskinesia with gain of function and ataxia with loss of function. Functional classifications revealed two major patterns, gain of function and loss of function effects on channel properties in different cell lines. In the literature, two mutations have been shown to confer gain of function properties to BK channels: D434G and N995S. In this study, we report the functional characterization of a variant which was previously reported the whole exome sequencing revealed bi-allelic nonsense variation of the cytoplasmic domain of calcium-activated potassium channel subunit alpha-1 protein. To detect functional consequences of the variation, we parallely conducted two independent approaches. One is immunostaining using and the other one is electrophysiological recording using patch-clamp on wild-type and R458X mutant cells to detect the differences between wild-type and the mutant cells. We detected the gain of function effect for the mutation (NM_001161352.1 (ENST00000286628.8):c.1372C > T;Arg458*) using two parallel approaches. According to the result we found, the reported mutation causes the loss of function in the cell. It should be noted that in future studies, it can be thought that the functions of genes associated with channelopathies may have a dual effect such as loss and gain.

BK channel dysfunction underlies small vessel disease of the brain in both hypertension and Alzheimer's disease

Alzheimer's disease (AD) and hypertension-related vascular dementia share many common clinical and radiological features including cerebral small vessel disease (cSVD) of the brain, thought to be the mechanism behind the reduction in cerebral blood flow (CBF). The aim of this study was to determine if there are common mechanisms underlying a reduced CBF in dementia. CBF is determined by depolarisation induced contraction of arterial smooth muscle cells (SMCs). This contraction is buffered by the activation of the large-conductance Ca 2+ -activated K + (BK) channels on the plasma membrane (PM), by transient localised Ca 2+ release from ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR), known as Ca 2+ sparks.Hypothesis: Defects in BK channel activation leads to the cSVD phenotype in these diseases.8-month old male spontaneously hypertensive (BPH/2) mice and normotensive controls (BPN/3) were used to study hypertension-induced cSVD. To study AD related cSVD we used 18–20 month old male APP23 mice. We used a range of physiological techniques to investigate vascular ion channel function within the cerebral microvasculature. Laser Doppler flowmetry and behavioural assessments were performed on the BPH/2 and BPN/3 mice to initially determine if they are a model of hypertension-related vascular dementia.BPH/2 mice displayed reduced CBF and cognitive impairment. Pressure-induced constriction from cerebral pial arteries was significantly greater in both the BPH/2 and APP23 animals compared to controls. This was the result of reduced BK channel activity, as confirmed by reduced constriction to the BK channel blocker paxilline. Electrophysiology experiments showed a decrease in spontaneous transient outward currents, which represent BK channel activation by Ca 2+ sparks. In the APP23 model, this attenuated BK channel activation was due to reduced Ca 2+ spark frequency. However, in the BPH/2 mice, Ca 2+ spark frequency was equal between hypertensive and normotensive mice. Furthermore, there were no differences in single channel BK channel activity over a range of [Ca 2+ ]. Live cell imaging of isolated SMCs labelled with membrane dyes, showed a reduced interaction between the SR and the PM. Proximity ligation assay was used on fixed SMCs to determine if BK channel and RyR are within 40 nm of each other. SMCs from BPH/2 cerebral arteries has significantly less puncta suggesting a physical separation between Ca 2+ sparks and BK channel, rendering the [Ca 2+ ] local to the BK channel insufficient for activation.Our data indicate that vascular BK channel dysfunction is a common mechanism underpinning reduced CBF in SVD. Therapies aimed at improving vascular BK channel function could provide new therapeutic targets for both AD and hypertension-induced vascular dementia. British Heart Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ca2+-Activated K+ Channels in Progenitor Cells of Musculoskeletal Tissues: A Narrative Review.

Musculoskeletal disorders represent one of the main causes of disability worldwide, and their prevalence is predicted to increase in the coming decades. Stem cell therapy may be a promising option for the treatment of some of the musculoskeletal diseases. Although significant progress has been made in musculoskeletal stem cell research, osteoarthritis, the most-common musculoskeletal disorder, still lacks curative treatment. To fine-tune stem-cell-based therapy, it is necessary to focus on the underlying biological mechanisms. Ion channels and the bioelectric signals they generate control the proliferation, differentiation, and migration of musculoskeletal progenitor cells. Calcium- and voltage-activated potassium (KCa) channels are key players in cell physiology in cells of the musculoskeletal system. This review article focused on the big conductance (BK) KCa channels. The regulatory function of BK channels requires interactions with diverse sets of proteins that have different functions in tissue-resident stem cells. In this narrative review article, we discuss the main ion channels of musculoskeletal stem cells, with a focus on calcium-dependent potassium channels, especially on the large conductance BK channel. We review their expression and function in progenitor cell proliferation, differentiation, and migration and highlight gaps in current knowledge on their involvement in musculoskeletal diseases.

BK channels of five different subunit combinations underlie the de novo KCNMA1 G375R channelopathy.

The molecular basis of a severe developmental and neurological disorder associated with a de novo G375R variant of the tetrameric BK channel is unknown. Here, we address this question by recording from single BK channels expressed to mimic a G375R mutation heterozygous with a WT allele. Five different types of functional BK channels were expressed: 3% were consistent with WT, 12% with homotetrameric mutant, and 85% with three different types of hybrid (heterotetrameric) channels assembled from both mutant and WT subunits. All channel types except WT showed a marked gain-of-function in voltage activation and a smaller decrease-of-function in single-channel conductance, with both changes in function becoming more pronounced as the number of mutant subunits per tetrameric channel increased. The net cellular response from the five different types of channels comprising the molecular phenotype was a shift of -120 mV in the voltage required to activate half of the maximal current through BK channels, giving a net gain-of-function. The WT and homotetrameric mutant channels in the molecular phenotype were consistent with genetic codominance as each displayed properties of a channel arising from only one of the two alleles. The three types of hybrid channels in the molecular phenotype were consistent with partial dominance as their properties were intermediate between those of mutant and WT channels. A model in which BK channels randomly assemble from mutant and WT subunits, with each subunit contributing increments of activation and conductance, approximated the molecular phenotype of the heterozygous G375R mutation.

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.

PS-B10-7: A VASCULAR SMOOTH MUSCLE POTASSIUM CHANNEL DYSFUNCTION UNDERLIES SMALL VESSEL DISEASE OF THE BRAIN IN BOTH HYPERTENSION AND ALZHEIMER'S DISEASE

Alzheimer's disease (AD) and hypertension-related vascular dementia share many common clinical and radiological features. Indeed, at post-mortem the majority of patients diagnosed with either disorder show extensive small vessel disease (SVD) of the brain. One phenotypic character of SVD is a reduction in cerebral blood flow (CBF). The large-conductance calcium-activated potassium (BK) channel reduces arterial contractility through hyperpolarisation of the vascular smooth muscle cell membrane. The BK channel is activated via small, localised calcium release events from ryanodine receptors (RyR), known as calcium sparks. We wished to examine whether defects in calcium spark to BK channel coupling contributes to the SVD phenotype in these diseases, which could suggest new avenues for therapy based on improvement of brain vascular health. Male spontaneously hypertensive (BPH/2) mice and normotensive controls (BPN/3) were used at 8 months of age to study hypertension-induced cSVD. To study AD we used 18–20 month old male APP23 mice that have a 7-fold increased expression of amyloid precursor protein, which results in significant amyloid-beta accumulation as is seen in human AD. During this study we used a range of physiological techniques; mainly pressure myography, patch clamp electrophysiology and high-speed spinning disc confocal microscopy, to investigate in depth, vascular ion channel function within the cerebral microvasculature of these mice. Pressure-induced constriction from cerebral pial arteries was significantly greater in both the BPH/2 and APP23 animals compared to controls. Pressure myography studies revealed that in both models there was a reduction in BK channel activity. This was confirmed with electrophysiology data that showed a decrease in spontaneous transient outward currents, which represent BK channel openings in response to calcium sparks. In the APP23 model, this reduction in BK channel function was due to a decrease in calcium spark frequency. However in the BPH/2 mice, spark frequency was equal between hypertensive individuals and controls. In this model separation of the BK channel and RyR resulted in BK channel dysfunction. Overall our data indicates that vascular BK channel dysfunction is a common mechanism underpinning reduced CBF in SVD. Therefore, therapies aimed at improving vascular BK channel function could provide new therapeutic targets for the treatment of both AD and hypertension-induced vascular dementia.

The function of BK channels extracted and purified within SMALPs.

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of β and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and β1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA–PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of β1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified β1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.

442-P: Sorbs2 Deficiency–Mediated Vascular BK Channel Dysfunction in Type 2 Diabetes

Vascular BK channels, composed of the pore-forming α-subunits (BK-α) and the regulatory β1 subunits (BK-β1) , is a key determinant of vasoreactivity, and its function is impaired in diabetes. However, our understanding of the mechanisms causing BK channel dysfunction in diabetic vessels is incomplete. The Sorbin and SH3 domain-containing protein 2 (Sorbs2) is abundantly expressed in vascular smooth muscle cells (SMCs) . We hypothesize that Sorbs2 is critical for vascular BK channel physiology. In this study, we compared vascular BK channel function between Sorbs2 KO and WT mice at 4 months of age. There was no difference in body weight and blood glucose between WT and Sorbs2 KO mice (body weight: 25.9±1.3 g of WT vs. 23.3±1.5 g of KO, n=14, p=N.S.; blood glucose: 166.4±7.7 mg/dL of WT vs. 152.2±12.2 mg/dL of KO, n=14, p=N.S.) . However, Sorbs2 KO mice have markedly reduced cell surface and cytoplasmic expression of BK-α and BK-β1, as well as BK channel-mediated vasodilation in carotid arteries with a maximal dilatation of 64.70±1.92% for WT mice (n=4) and 20.74±4.66% for KO mice (n=5, p<0.05) . Patch clamp experiments revealed that BK channel sensitivity to Ca2+ activation was impaired in the coronary SMCs of Sorbs2 KO mice (EC50: 2.87±0.68 µM, n=13, p<0.05) with decreased Hill coefficient (nH: 1.26) , compared to those of WT mice (EC50: 0.48±0.µM, nH: 2.70, n=13) . In vivo studies showed that Sorbs2 interacted with BK-α and BK-β1 protein in vascular SMCs. Silencing of Sorbs2 by shRNA decreased BK-α and BK-β1 expression, while adenoviral expression of Sorbs2 increased BK-α and BK-β1 expression in vascular SMCs. Most importantly, Sorbs2 expression was significantly reduced in the arteries of db/db type 2 diabetic mice and high fat diet-induced diabetic mice. Our results indicate that Sorbs2 plays an important role in regulating BK channel function and expression in vascular SMCs. Sorbs2 deficiency is a risk factor of BK channelopathy and vasculopathy in diabetes, independent of obesity and glucotoxicity. Disclosure T.Lu: None.

The Roles of BK Potassium Channels in Neuronal Excitability Differ in Excitatory and Inhibitory Hippocampal Neurons During Early Development

BK potassium channels are are activated by intracellular Ca+ and depolarization, opening during action potential firing and speeding repolarization. Because of this, they are key regulators of fast spiking activity and important determinants of neuronal excitability. Due to their role in regulating excitability as well as their broad expression in both excitable and non‐excitable tissues, BK channels are implicated in many pathological states including epilepsy. The BK channel antagonist, paxilline, has been shown to delay seizure induction in adult rodents that have had prior seizures, but not in seizure naïve rodents. History of seizures has been shown induce expression of more Ca2+ sensitive BK channel splice variants, which may underlie the difference in paxilline efficacy between the two states. Because expression of more Ca2+ sensitive isoforms predominates in neonatal rodents ‐similar to in the epileptic adults, we hypothesize that paxilline may be an effective inhibitor of seizure activity even in seizure‐naïve neonatal rodents. Our first goal is to understand how developmental shifts in BK channel expression and function map to changes in neuronal and circuit excitability. Our second goal is to test whether BK channel blockade can modulate seizure activity in neonates, as there is a need to identify pharmacological targets for seizures in neonates and children, which are often intractable to conventional treatments.To test how BK channel function changes with age and by cell type we measured the effect of paxilline on action potential (AP) firing ‐both on single spike kinetics and prolonged firing‐ in two classes of hippocampal neurons, CA1 pyramidal neurons and parvalbumin interneurons (PVIns), a key inhibitory component of local hippocampal circuits. We used P4‐5, P9‐10, and P14‐15 C57BL/6J mice with labelled PVIns. We also tested the efficacy of paxilline in reducing hyperexcitability evoked by application of 0 Mg2+ aCSF in brain slices, an established seizure model.We patched pyramidal neurons and PVIns in whole‐cell current‐clamp configuration and evoked APs using square depolarizing pulses. BK channel blockade with 1mM paxilline lengthens duration of APs in P4‐5 and P9‐10 pyramidal neurons and P4‐5 PVIns but does not affect P9‐10 PVIn AP duration (paired t‐tests, p<0.05). While BK blockade has the least effect on single APs in the P9‐10 PVIns, it has the most effect on the input‐output relationship and spike‐frequency accommodation in these neurons (3‐way ANOVA, p<0.001 for interaction of age and cell type in input‐output; 3‐way ANOVA p<0.05 for main effect of age in frequency adaptation).We measured the effect of BK channel blockade in controlling hyperexcitability in P4‐15 mice. We found that 0 Mg2+ aCSF application induced spontaneous AP firing in patched hippocampal pyramidal neurons within 3.86±0.87 minutes but when 0 Mg2+ aCSF was coapplied with 1 mM paxilline the average onset of spontaneous activity occurred at 8.43±1.40 minutes (t‐test p<0.05).For the first goal, we show that BK channels’ relative importance in regulating AP firing changes with age and by neuronal population. Toward the second goal we report that BK channel blockade can reduce hyperexcitability in a brain slice model of epilepsy.

A dual potassium channelopathy underlies small vessel disease of the brain in a mouse model of Alzheimer's Disease

Mounting clinical and basic evidence has suggested that cerebral microcirculatory dysfunction is key to the pathogenesis of Alzheimer’s disease (AD), thus posing a potential new therapeutic target for AD treatment. However, despite observations of altered cerebral blood flow and autoregulatory capacity in AD patients, the underlying pathogenic mechanisms behind these defects are currently unknown. Here we present the first detailed study in to vascular ion channel function in the APP23 mouse model of AD. This model has a seven‐fold increase in amyloid precursor protein leading to substantial amyloid‐β plaque accumulation within the brain, as is described in AD patients. Using a range of physiological techniques; mainly pressure myography, patch clamp electrophysiology and high‐speed spinning disc confocal microscopy, we show that attenuation of large conductance calcium–activated potassium (BK) channel function in this model, due to a reduced frequency of calcium sparks, is responsible for the reduction in cerebral blood flow described. Further investigation into the role of amyloid‐β 1‐40, in particular, showed that vessels exposed to the peptide for 30 minutes exhibited significantly reduced BK channel function at both the cellular and vascular level. However, disparate from the APP23 mouse model, acute exposure to amyloid‐β 1‐40 resulted in an increase in calcium waves. Both a decrease in calcium sparks (as seen in the APP23 model) and an increase in calcium waves (as seen with acute application of the amyloid‐β 1‐40 peptide) results in contraction of cerebral arteries and a subsequent reduction of cerebral blood flow. The data directs future research in to preventative strategies that may help to stop alterations in calcium event initiation, restore microvasculature function and promote a healthy brain environment to limit disease progression in AD patients.

Haloperidol blocks BK channels by a "foot in the door" mechanism

Large conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and gene mutations affecting BK channel function are linked to human neurological disease. We have recently found that the widely-used opioid agonist loperamide blocks BK channel current by binding preferentially in the open pore, while it apparently dissociates from the pore after channels have closed, consistent with the idea that BK channel gating does not occur by a “trap door” mechanism (Vouga et al.

Chronic stress facilitates bursting electrical activity in pituitary corticotrophs.

Coordination of an appropriate stress response is dependent upon anterior pituitary corticotroph excitability in response to hypothalamic secretagogues and glucocorticoid negative feedback. A key determinant of corticotroph excitability is large conductance calcium- and voltage-activated (BK) potassium channels that are critical for promoting corticotrophin-releasing hormone (CRH)-induced bursting that enhances adrenocorticotrophic hormone secretion. Previous studies revealed hypothalamic-pituitary-adrenal axis hyperexcitability following chronic stress (CS) is partly a function of increased corticotroph output. Thus, we hypothesise that chronic stress promotes corticotroph excitability through a BK-dependent mechanism. Corticotrophs from CS mice displayed significant increase in spontaneous bursting, which was suppressed by the BK blocker paxilline. Mathematical modelling reveals that the time constant of BK channel activation, plus properties and proportion of BK channels functionally coupled to L-type Ca2+ channels determines bursting activity. Surprisingly, CS corticotrophs (but not unstressed) display CRH-induced bursting even when the majority of BK channels are inhibited by paxilline, which modelling suggests is a consequence of the stochastic behaviour of a small number of BK channels coupled to L-type Ca2+ channels. Our data reveal that changes in the stochastic behaviour of a small number of BK channels can finely tune corticotroph excitability through stress-induced changes in BK channel properties. Importantly, regulation of BK channel function is highly context dependent allowing dynamic control of corticotroph excitability over a large range of time domains and physiological challenges in health and disease. This is likely to occur in other BK-expressing endocrine cells, with important implications for the physiological processes they regulate and the potential for therapy. KEY POINTS: Chronic stress (CS) is predicted to modify the electrical excitability of anterior pituitary corticotrophs. Electrophysiological recordings from isolated corticotrophs from CS male mice display spontaneous electrical bursting behaviour compared to the tonic spiking behaviour of unstressed corticotrophs. The increased spontaneous bursting from CS corticotrophs is BK-dependent and mathematical modelling reveals that the time constant of activation, properties and proportion of BK channels functionally coupled to L-type calcium channels determines the promotion of bursting activity. CS (but not unstressed) corticotrophs display corticotrophin-releasing hormone-induced bursting even when the majority of BK channels are pharmacologically inhibited, which can be explained by the stochastic behaviour of a small number of BK channels with distinct properties. Corticotroph excitability can be finely tuned by the stochastic behaviour of a small number of BK channels dependent on their properties and functional co-localisation with L-type calcium channels to control corticotroph excitability over diverse time domains and physiological challenges.

The Large-Conductance, Calcium-Activated Potassium Channel: A Big Key Regulator of Cell Physiology.

Large-conductance Ca2+-activated K+ channels facilitate the efflux of K+ ions from a variety of cells and tissues following channel activation. It is now recognized that BK channels undergo a wide range of pre- and post-translational modifications that can dramatically alter their properties and function. This has downstream consequences in affecting cell and tissue excitability, and therefore, function. While finding the “silver bullet” in terms of clinical therapy has remained elusive, ongoing research is providing an impressive range of viable candidate proteins and mechanisms that associate with and modulate BK channel activity, respectively. Here, we provide the hallmarks of BK channel structure and function generally, and discuss important milestones in the efforts to further elucidate the diverse properties of BK channels in its many forms.

BK Channel Gating Mechanisms: Progresses Toward a Better Understanding of Variants Linked Neurological Diseases.

The large conductance Ca2+-activated potassium (BK) channel is activated by both membrane potential depolarization and intracellular Ca2+ with distinct mechanisms. Neural physiology is sensitive to the function of BK channels, which is shown by the discoveries of neurological disorders that are associated with BK channel mutations. This article reviews the molecular mechanisms of BK channel activation in response to voltage and Ca2+ binding, including the recent progress since the publication of the atomistic structure of the whole BK channel protein, and the neurological disorders associated with BK channel mutations. These results demonstrate the unique mechanisms of BK channel activation and that these mechanisms are important factors in linking BK channel mutations to neurological disorders.