BK Channel Activity Research Articles

Docking-based analysis and modeling of the activity of bile acids and their synthetic analogues on large conductance Ca2+ activated K channels in smooth muscle cells.

The objective of this study was to perform docking-based analysis of bile acid binding on the protein complex of channels and to derive neural network that predicts the influence of bile acids and their synthetic analogues on the activity of BK(Ca) channels in smooth muscle cells based on descriptors for bile acids and their synthetic analogues and on their already published activities using patch-clamp techniques. Ligands for molecular docking were optimized using computer routine for minimization of energy by using the force field MMFF94 via Chem3D 15.0 and ligands and protein channel complex were prepared in AutoDockTools 1.5.6. AutoDock Vina 4.0 software was used for blind docking; processing and verification of the obtained results was performed via Discovery Studio 4.0. Neural network was derived using descriptors for bile acids and their synthetic analogues and their already published activities on calcium-activated K+ channels in smooth muscle cells (ChemDraw Professional 15.0, Dragon 6 software). Molecular docking was performed for: lithocholic acid, deoxycholic acid, 5β-cholanoic acid, 3β-hydroxi-5β-cholanoic acid, henodeoxycholic acid, ursocholic acid and α-muricholic acid. Neural network model Multiple layer perceptron is derived, having 0.9259 training performances and 0.3673 test performances, training error 0.0073 and test error 0,1607. Model was tested for henodeoxycholic, ursocholic and α-muricholic acid, and internal validation of the model is performed. Molecular docking suggested that the pharmacophore for maximizing the activity of BK(Ca) channels in the steroid skeleton of bile acids is the C3 quasi-axial α-OH group and the C24 carboxyl function. Derived neural network model successfully predicted activities of tested bile acids on Ca2+ activated K+ channels in smooth muscle cells.

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.

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.

IP3R-mediated activation of BK channels contributes to mGluR5-induced protection against spinal cord ischemia-reperfusion injury

Spinal cord ischemia-reperfusion injury (SCIRI) can cause dramatic neuron loss and lead to paraplegia in patients. In this research, the role of mGluR5, a member of the metabotropic glutamate receptors (mGluRs) family, was investigated both in vitro and in vivo to explore a possible method to treat this complication. In vitro experiment, after activating mGluR5 via pretreating cells with (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG) and 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), excitotoxicity induced by glutamate (Glu) was attenuated in primary spinal cord neurons, evidenced by higher neuron viability, decreased lactate dehydrogenase (LDH) release and less detected TUNEL-positive cells. According to Western Blot (WB) results, Glu treatment resulted in a high level of large-conductance Ca2+- and voltage-activated K+ (BK) channels, with activation relying on the mGluR5-IP3R (inositol triphosphate) pathway. In vivo part, a rat model of SCIRI was built to further investigate the role of mGluR5. After pretreating them with CHPG and CDPPB, the rats showed markedly lower spinal water content, attenuated motor neuron injury in the spinal cord of L4 segments, and better neurological function. This effect could be partially reversed by paxilline, a blocker of BK channels. In addition, activating BK channels alone using specific openers: NS1619 or NS11021 can protect spinal cord neurons from injury induced by either SCIRI or Glu. In conclusion, in this research, we proved that mGluR5 exerts a protective role in SCIRI, and this effect partially works via IP3R-mediated activation of BK channels.

Role for calcium-activated potassium channels (BK) in migration control of human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a leading cause of cancer‐related death worldwide. Its high metastasis rate is significantly correlated with poor patient prognosis. Elucidating the molecular mechanism underlying HCC metastasis is essential for HCC treatment. Owing to their high conductance, large‐conductance calcium‐activated potassium channels (BK channels) play a critical role in the control of membrane potential and have repeatedly been proposed as potential targets for cancer therapy. Emerging evidence suggests that BK channels are involved in the progression of cancer malignancies. The present study investigated the role of BK channels in mediating the hypoxia‐stimulated migration of HCC cells both in vitro and in vivo in the absence and presence of various BK channels modulators. We found that BK channels were functionally expressed on the membranes of the SMMC‐7721 and Huh7 HCC cell lines. Furthermore, blockage or activation of BK channels on the surface of HCC cells correspondingly inhibited or promoted HCC cell proliferation, migration and invasion in hypoxia conditions, with altered expression and distribution of cell‐cell adhesion molecule E‐cadherin and typical marker of mesenchymal cells, Vimentin, but not N‐cadherin. Hypoxia conditions did not alter BK channels expression but increased its open probability. Moreover, BK channels blocker IbTX significantly inhibited HCC cell remote colonization in HCC cell xenografted mice. In conclusion, the results of this study suggest that blocking BK channels offers an attractive strategy for treating HCC.

The Control of Rat Hippocampal Gamma Oscillation Strength by BK Channel Activity

Neuronal network oscillations in the gamma frequency band (30–80 Hz, γ oscillations) are associated with the higher brain functions such as perception, attention, learning and memory. BK channels mediate rapid repolarization and fast afterhyperpolarization in neurons and control neuronal excitability, and potentially control hippocampal γ oscillations. In this study, we examined the effects of modulating BK channels on hippocampal γ oscillations in the absence or presence of Ca2+ influx through voltage-gated Ca2+ channels (VGCC) or Ca2+-permeable AMPA receptors (CP-AMPAR). We found that blocking BK channels enhanced γ power, without affecting oscillation frequency or regularity, suggesting that BK channel activity suppresses γ oscillations. Blocking either VGCC or CP-AMPAR itself enhanced γ power, and completely occluded the effect of BK channel blockers on γ oscillations, whereas blocking BK channels first could not prevent a further γ power increase upon blockade of either CP-AMPAR or VGCC. We propose that Ca2+ influx through VGCC or CP-AMPAR during γ oscillations, cause membrane BK channel activation and regulate hippocampal γ oscillation strength by negative feedback.

Large-conductance calcium-activated K+ channels, rather than KATP channels, mediate the inhibitory effects of nitric oxide on mouse lymphatic pumping.

KATP channels are negative regulators of lymphatic vessel excitability and contractility and are proposed to be targets for immune cell products that inhibit lymph transport. Previous studies in rat and guinea pig mesenteric lymphatics found that NO-mediated inhibition of lymphatic contraction was prevented or reversed by the KATP channel inhibitor, glibenclamide. We revisited this hypothesis using mouse lymphatic vessels and KATP channel knockout mice. Mouse popliteal lymphatics were isolated, and contractility was assessed using pressure myography. K+ channel expression was determined by PCR analysis of FACS-purified lymphatic smooth muscle cells. The NO-producing agonist, ACh, and the NO donor, NONOate, both produced dose-dependent inhibition of spontaneous lymphatic contractions that were blocked by the soluble GC inhibitor, ODQ, or the PKG inhibitor, Rp-8-Br-PET-cGMPS. Surprisingly, the inhibitory effects of both were preserved in Kir 6.1-/- vessels, suggesting that KATP channels did not mediate NO-induced responses. We hypothesized a role for BK channels, given their prominence in arterial smooth muscle. Indeed, BK channels were expressed in mouse lymphatic smooth muscle and NS11021 (a BK channel activator) caused dilation and reduced contraction frequency, whereas iberiotoxin and penitrem A (BK channel inhibitors) produced right-ward shifts in NONOate concentration-response curves. Inhibition of mouse lymphatic contractions by NO primarily involves activation of BK channels, rather than KATP channels. Thus, BK channels are a potential target for therapeutic reversal of lymph pump inhibition by NO generated by immune cell activation of iNOS in chronic lymphoedema.

State-dependent inhibition of BK channels by the opioid agonist loperamide.

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase.

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline—a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc—a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole—two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Deletion of BK channels decreased skeletal and cardiac muscle function but increased smooth muscle contraction in rats

Large conductance calcium-activated potassium channel (BK channel) is widely expressed in skeletal muscle, myocardium, smooth muscle and other muscle tissues. Mutation, abnormal expression and altered activity of BK channel are linked to muscle-related diseases such as dyskinesia, epilepsy and erectile dysfunction. In order to compare the effects of BK channel on different muscle tissues, we constructed BK channel gene knockout rats‌‌‌‌‍‍‍ (BK-/- rats). HE staining, open field and grip strength tests, ultrasound, blood pressure measurement and vascular tension test were utilized to explore the effects of BK channel deletion on the structure and function changes in skeletal muscle, myocardium, and vascular smooth muscle (VSM). It was found that compared with wild-type rats, the BK-/- rats showed decreased skeletal muscle fiber area, grip, movement distance and speed at 2 and 12 months of ages. At heart, the muscle fiber area, cardiac systolic/diastolic function and heart rate decreased in BK-/- rats. The wall of the left ventricle became thin. However, the vascular wall of BK-/- rats thickened, the pulse wave velocity was increased, and the VSM contraction was enhanced. Unexpectedly, both systolic and diastolic blood pressure were reduced in BK-/- rats, while pulse pressure difference was increased. These results suggest that BK channel may have different effects on different types of muscle tissue, and it should be noted that different parts of muscle tissue may have different effects when BK channel-related drugs are used.

L-WNK1 is required for BK channel activation in intercalated cells.

Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet.NEW & NOTEWORTHY When mice are placed on a high-K+ diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K+ channel, blunted large-conductance K+ channel currents in intercalated cells, and increased blood K+ concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K+ diet.

BK Channel-Mediated Microglial Phagocytosis Alleviates Neurological Deficit After Ischemic Stroke.

Microglial phagocytosis benefits neurological recovery after stroke. Large-conductance Ca2+-activated K+ currents are expressed in activated microglia, and BK channel knockout aggravates cerebral ischemic injury. However, the effect of BK channels on microglial phagocytosis after ischemic stroke remains unknown. Here, we explored whether BK channel activation is beneficial for neurological outcomes through microglial phagocytosis after ischemic stroke. ICR mice after transient middle cerebral artery occlusion (tMCAO) were treated with dimethyl sulfoxide (DMSO), BK channel activator NS19504, and inhibitor Paxilline. The results showed a decrease in BK channel expression after tMCAO. BK channel activator NS19504 alleviates neurological deficit after experimental modeling of tMCAO in mice compared to the control. Furthermore, we treated primary microglia with DMSO, NS19504, and Paxilline after oxygen glucose deprivation (OGD). NS19504 promoted primary microglial phagocytosing fluorescent beads and neuronal debris, which reduced neuronal apoptosis after stroke. These effects could be reversed by BK channel inhibitor Paxilline. Finally, NS19504 increased relative phosphorylated extracellular signal-regulated kinase 1/2 expression compared to the Paxilline group at the third day after stroke. Our findings indicate that microglial BK channels are a potential target for acute stage of ischemic stroke therapy.

BK channel activation by L-type Ca2+ channels CaV1.2 and CaV1.3 during the subthreshold phase of an action potential.

Mammalian circadian (24 h) rhythms are timed by the pattern of spontaneous action potential firing in the suprachiasmatic nucleus (SCN). This oscillation in firing is produced through circadian regulation of several membrane currents, including large-conductance Ca2+- and voltage-activated K+ (BK) and L-type Ca2+ channel (LTCC) currents. During the day steady-state BK currents depend mostly on LTCCs for activation, whereas at night they depend predominantly on ryanodine receptors (RyRs). However, the contribution of these Ca2+ channels to BK channel activation during action potential firing has not been thoroughly investigated. In this study, we used a pharmacological approach to determine that both LTCCs and RyRs contribute to the baseline membrane potential of SCN action potential waveforms, as well as action potential-evoked BK current, during the day and night, respectively. Since the baseline membrane potential is a major determinant of circadian firing rate, we focused on the LTCCs contributing to low voltage activation of BK channels during the subthreshold phase. For these experiments, two LTCC subtypes found in SCN (CaV1.2 and CaV1.3) were coexpressed with BK channels in heterologous cells, where their differential contributions could be separately measured. CaV1.3 channels produced currents that were shifted to more hyperpolarized potentials compared with CaV1.2, resulting in increased subthreshold Ca2+ and BK currents during an action potential command. These results show that although multiple Ca2+ sources in SCN can contribute to the activation of BK current during an action potential, specific BK-CaV1.3 partnerships may optimize the subthreshold BK current activation that is critical for firing rate regulation.NEW & NOTEWORTHY BK K+ channels are important regulators of firing. Although Ca2+ channels are required for their activation in excitable cells, it is not well understood how BK channels activate using these Ca2+ sources during an action potential. This study demonstrates the differences in BK current activated by CaV1.2 and CaV1.3 channels in clock neurons and heterologous cells. The results define how specific ion channel partnerships can be engaged during distinct phases of the action potential.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by β-glycerophosphate (β-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in β-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In β-GP-treated VSMCs, BK channel opener NS1619 (20 μM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 μM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 μM) or FoxO1 inhibitor AS1842856 (3 μM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

The Membrane Cholesterol Modulates the Interaction Between 17-βEstradiol and the BK Channel.

BK channels are composed by the pore forming α subunit and, in some tissues, is associated with different accessory β subunits. These proteins modify the biophysical properties of the channel, amplifying the range of BK channel activation according to the physiological context. In the vascular cells, the pore forming BKα subunit is expressed with the β1 subunit, where they play an essential role in the modulation of arterial tone and blood pressure. In eukaryotes, cholesterol is a structural lipid of the cellular membrane. Changes in the ratio of cholesterol content in the plasma membrane (PM) regulates the BK channel activation altering its open probability, and hence, vascular contraction. It has been shown that the estrogen 17β-Estradiol (E2) causes a vasodilator effect in vascular cells, inducing a leftward shift in the V0.5 of the GV curve. Here, we evaluate whether changes in the membrane cholesterol concentration modify the effect that E2 induces on the BKα/β1 channel activity. Using binding and electrophysiology assays after cholesterol depletion or enrichment, we show that the cholesterol enrichment significantly decreases the expression of the α subunit, while cholesterol depletion increased the expression of that α subunit. Additionally, we demonstrated that changes in the membrane cholesterol cause the loss of the modulatory effect of E2 on the BKα/β1 channel activity, without affecting the E2 binding to the complex. Our data suggest that changes in membrane cholesterol content could affect channel properties related to the E2 effect on BKα/β1 channel activity. Finally, the results suggest that an optimal membrane cholesterol content is essential for the activation of BK channels through the β1 subunit.

Amyloid β and Amyloid Precursor Protein Synergistically Suppress Large-Conductance Calcium-Activated Potassium Channel in Cortical Neurons.

Intracellular amyloid β (Aβ) injection suppresses the large-conductance calcium-dependent potassium (BK) channel in cortical pyramidal cells from wild-type (WT) mice. In 3xTg Alzheimer’s disease (AD) model mice, intraneuronal Aβ is genetically programed to accumulate, which suppresses the BK channel. However, the mode of BK channel suppression remained unclarified. The present report revealed that only one (11A1) of the three anti-Aβ-oligomer antibodies that we examined, but not anti-monomer-Aβ-antibodies, was effective in recovering BK channel activity in 3xTg neurons. Antibodies against amyloid precursor protein (APP) were also found to be effective, suggesting that APP plays an essential part in this Aβ-oligomer-induced BK channel suppression in 3xTg neurons. In WT neurons, by contrast, APP suppressed BK channels by itself, which suggests that either APP or Aβ is sufficient to block BK channels, thus pointing to a different co-operativity of Aβ and APP in WT and 3xTg neurons. To clarify this difference, we relied on our previous finding that the scaffold protein Homer1a reverses the BK channel blockade in both WT and 3xTg neurons. In cortical neurons from 3xTg mice that bear Homer1a knockout (4xTg mice), neither anti-APP antibodies nor 11A1, but only the 6E10 antibody that binds both APP and Aβ, rescued the BK channel suppression. Given that Homer1a expression is activity dependent and 3xTg neurons are hyperexcitable, Homer1a is likely to be expressed sufficiently in 3xTg neurons, thereby alleviating the suppressive influence of APP and Aβ on BK channel. A unique way that APP modifies Aβ toxicity is thus proposed.

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2- cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.

Glycosylation of β1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels

Background:The accessory β1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by β1 subunits. Methods:Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in β1 subunits.Results: The results show that deglycosylation of β1 subunits through double-site mutations (β1 N80A/N142A or β1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+β1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation.Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of β1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of β1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.

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 (β1) 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.

Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes.

Podocyte injury is a common hallmark in various glomerular diseases. The level of LRRC55 was increased in podocytes of patients with focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN), and membranous nephropathy (MN). Upregulated LRRC55 and increased intracellular Ca2+ led to BK channel activation and the loss of intracellular potassium, resulting in apoptosome formation and caspase-3 activation in angiotensin II (Ang II)-treated podocytes. Knockout of Lrrc55 or the BK channel prevented the BK current and ameliorated podocyte injury in Ang II-treated mice. Upstream, NFATc3 regulated the expression of LRRC55. Increased LRRC55 expression in podocytes was also evident in animal models of FSGS, DN, and MN. Treatment with losartan or LRRC55 siRNA suppressed LRRC55 expression, prevented BK channel activation, and attenuated podocyte injury in animal models of FSGS, DN, and MN. In conclusion, upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes in FSGS, DN, and MN. LRRC55 inhibition may represent a new therapeutic approach for podocyte injury.