BK Channel Function Research Articles

Traumatic Brain Injury Alters BK Channel Subunit Expression, Localization, and Function in Cerebral Arterial Myocytes

Traumatic brain injury (TBI) induces acute changes in cerebrovascular function, which include alterations to cerebral artery myogenic tone and autoregulation. In arterial smooth muscle cells (myocytes), large‐conductance calcium‐activated potassium (BK) channels play a vital role in modulating plasma membrane potential and myogenic tone. Here, we examined BK channel expression and function in cerebral resistance size arteries one week following moderately‐severe TBI induced by controlled cortical impact with craniotomy. Total BKα (pore‐forming) and β1 (regulatory) subunit expression were reduced one week post‐TBI. Biotinylation of plasma membrane‐bound proteins revealed a compensatory increase of β1 subunits to the plasma membrane following TBI, which normalized single BK channel activation as assessed using patch‐clamp electrophysiology. However, the loss of total BKα subunits resulted in diminished whole cell, transient BK currents. Pressurized cerebral arteries from TBI rats exhibited greater myogenic tone at 60 mmHg and a diminished response to iberiotoxin, a BK channel pore blocker. In conclusion, moderately‐severe TBI enhances cerebral artery contractility, in part, through reductions in arterial myocyte BK channel subunit expression and function. Moreover, these TBI‐induced alterations in cerebral artery function can persist well beyond the initial 24–48 hour period post‐injury.Support or Funding InformationFlorida State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long Term Hypoxia Negatively Influences Ca2+ Signaling in Basilar Arterial Myocytes of Fetal and Adult Sheep

Gestational long term hypoxia (LTH) is a common prenatal stress caused by maternal anemia, high altitude living, smoking, and other disorders. Such gestational LTH can lead to a variety of cerebrovascular disorders in the neonate that compromise brain blood flow. Local and whole‐cell Ca2+ signals are important to the regulation of cerebrovascular tone. Rapid and localized Ca2+ transients, often referred to as sparks, activate large‐conductance K+ channels (BK), which dilate vessels. Whole‐cell Ca2+ oscillations, in comparison, are important to arterial contraction. Previous work shows that LTH and post‐natal maturity influence BK channel function in basilar arteries, arterial wall Ca2+ signals, cerebrovascular tone, as well as brain blood flow. We hypothesize that LTH‐dependent changes in spontaneous and depolarization mediated Ca2+ sparks and whole‐cell oscillations are important to BK channel activity, arterial wall Ca2+ signals, and vascular reactivity changes our group has previously observed. To begin evaluating the role of individual myocyte Ca2+ signals to the observed changes in cerebrovascular function, we isolated basilar arteries from adult and near term fetal (~141 gestational days) normoxic and hypoxic sheep that were raised at 3,800 m. for 110+ days. Cytosolic Ca2+ signals were examined in individual myocytes of intact arterial preparations loaded with fluo‐4 using laser scanning confocal microscopy techniques. Ca2+ spark activity as well as whole‐cell oscillations were enhanced by depolarizing myocytes with 30 mM K+. Maturation increased Ca2+ spark activity and depolarization‐dependent oscillations. LTH decreased oscillatory activity in fetal and adult animals but only spark frequency in adult myocytes. LTH decreased the area under the curve for Ca2+ oscillations independent of age as well as suppressed the ability of adult myocytes to respond to membrane depolarization. Depolarization, maturation, and LTH also influenced the spatial and temporal relationships of the Ca2+ oscillations. Overall, these observations illustrate that maturation and gestation at high‐altitude modify local and whole‐cell Ca2+ signaling, which likely contribute to adjustments in BK channel function, arterial wall Ca2+ signals, vasoreactivity, and cerebral blood flow that our group has examined in the past.Support or Funding InformationThis material is based upon work supported by NIH grants P01HD083132 (LZ). Imaging was performed in the LLUSM Advanced Imaging and Microscopy Core with support of NSF Grant MRI‐DBI 0923559 and the Loma Linda University School of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional Coupling of Cav2.3 and BK Potassium Channels Regulates Action Potential Repolarization and Short-Term Plasticity in the Mouse Hippocampus.

Voltage-gated ion channels are essential for signal generation and propagation in neurons and other excitable cells. The high-voltage activated calcium-channel Cav2.3 is expressed throughout the central and peripheral nervous system, and within CA1 hippocampal pyramidal neurons it is localized throughout the somato-dendritic region and dendritic spines. Cav2.3 has been shown to provide calcium for other calcium-dependent potassium channels including small-conductance calcium-activated potassium channels (SK), but big-conductance calcium-activated potassium channels (BK) have been thought to be activated by calcium from all known voltage-gated calcium channels, except Cav2.3. Here we show for the first time that CA1 pyramidal cells which lack Cav2.3 show altered action potential (AP) waveforms, which can be traced back to reduced SK- and BK-channel function. This change in AP waveform leads to strengthened synaptic transmission between CA1 and the subiculum, resulting in increased short-term plasticity. Our results demonstrate that Cav2.3 impacts cellular excitability through functional interaction with BK channels, impacting communication between hippocampal subregions.

Regulation of BK Channels by Beta and Gamma Subunits.

Ca2+- and voltage-gated K+ channels of large conductance (BK channels) are expressed in a diverse variety of both excitable and inexcitable cells, with functional properties presumably uniquely calibrated for the cells in which they are found. Although some diversity in BK channel function, localization, and regulation apparently arises from cell-specific alternative splice variants of the single pore-forming α subunit ( KCa1.1, Kcnma1, Slo1) gene, two families of regulatory subunits, β and γ, define BK channels that span a diverse range of functional properties. We are just beginning to unravel the cell-specific, physiological roles served by BK channels of different subunit composition.

13 DEFICIENT ACTIVITY OF A GOBLET CELL-SPECIFIC BK-TYPE POTASSIUM CHANNEL ENHANCES SUSCEPTIBILITY TO DSS-INDUCED COLITIS IN MICE

Goblet cells (GC) contribute critically to intestinal barrier function and mucosal immune homeostasis. GC dysfunction is linked to human inflammatory bowel disease (IBD). However, the cellular mechanisms that regulate GC function are incompletely understood. Using a novel mouse model generated in our lab, we have recently identified that an important regulatory subunit of the BK-type potassium channel, called LRRC26 (leucine-rich-repeat containing protein 26), specifically localizes to intestinal GCs. BK channels are membrane proteins enabling K+-efflux from cells in response to an increase in intracellular calcium concentration and/or membrane depolarization. The presence of LRRC26 in GC-BK channels is remarkable since this subunit drastically shifts the activation range such that LRRC26-associated BK channels can contribute to K+ efflux near normal physiological resting conditions. Although upregulation of the gene encoding for the BK channel (the pore-forming subunit) has been associated with increased K+ losses in Ulcerative Colitis patients, the specific role played by BK channels in the IBD pathophysiology is unknown. In this study, we have found that the genetic ablation of LRRC26 or BK-pore forming subunit in mice results in a dramatically enhanced susceptibility to DSS-induced colitis. By using a fluorescence-tagged GC mouse, we have confirmed that murine colonic GCs have functional LRRC26-associated BK channels which become inoperant in the absence of LRRC26. These results suggest that a normal potassium flux though LRRC26-associated BK channels in GCs has protective effects against colitis and perhaps reveals a new potential therapeutic target for IBD. (Supported by LC Pakula IBD Education & Innovation Fund IA-2018-10-IBD-2 to VGP and GM118114 to CJL)

Regulation of BK Channel Activity by Cholesterol and Its Derivatives.

Cholesterol (CLR) is an essential structural lipid in the plasma membrane of animal cells. In addition, CLR has been widely recognized as a critical modulator of protein function, including ion channels. Voltage- and Ca2+-gated K+ (BK) channels control a wide variety of physiological processes, including cell excitability, smooth muscle contractility, sensory perception, neurotransmitter release, and hormone secretion. Thus, disruption of BK currents has been implicated in the pathophysiology of prevalent human diseases. The current chapter reviews the literature documenting CLR modulation of BK channel function at a variety of levels ranging from organ systems to artificial lipid bilayers. We discuss the use of CLR isomers and structural analogs as a tool to help in discerning the mechanisms underlying CLR-driven modification of BK current. The chapter is finalized with an overview of the phenomenology and potential mechanisms that govern CLR control over the alcohol (ethyl alcohol, ethanol) sensitivity of BK channels. Studies on CLR regulation of BK currents may ultimately pave the way for novel therapeutic approaches to combat prevalent pathophysiological and morbid conditions.

Colonic Potassium Absorption and Secretion in Health and Disease.

The colon has large capacities for K+ absorption and K+ secretion, but its role in maintaining K+ homeostasis is often overlooked. For many years, passive diffusion and/or solvent drag were thought to be the primary mechanisms for K+ absorption in human and animal colon. However, it is now clear that apical H+ ,K+ -ATPase, in coordination with basolateral K+ -Cl- cotransport and/or K+ and Cl- channels operating in parallel, mediate electroneutral K+ absorption in animal colon. We now know that K+ absorption in rat colon reflects ouabain-sensitive and ouabain-insensitive apical H+ ,K+ -ATPase activities. Ouabain-insensitive and ouabain-sensitive H+ ,K+ -ATPases are localized in surface and crypt cells, respectively. Colonic H+ ,K+ -ATPase consists of α- (HKCα ) and β- (HKCβ ) subunits which, when coexpressed, exhibit ouabain-insensitive H+ ,K+ -ATPase activity in HEK293 cells, while HKCα coexpressed with the gastric β-subunit exhibits ouabain-sensitive H+ ,K+ -ATPase activity in Xenopus oocytes. Aldosterone enhances apical H+ ,K+ -ATPase activity, HKCα specific mRNA and protein expression, and K+ absorption. Active K+ secretion, on the other hand, is mediated by apical K+ channels operating in a coordinated way with the basolateral Na+ -K+ -2Cl- cotransporter. Both Ca2+ -activated intermediate conductance K+ (IK) and large conductance K+ (BK) channels are located in the apical membrane of colonic epithelia. IK channel-mediated K+ efflux provides the driving force for Cl- secretion, while BK channels mediate active (e.g., cAMP-activated) K+ secretion. BK channel expression and activity are increased in patients with end-stage renal disease and ulcerative colitis. This review summarizes the role of apical H+ ,K+ -ATPase in K+ absorption, and apical BK channel function in K+ secretion in health and disease. © 2018 American Physiological Society. Compr Physiol 8:1513-1536, 2018.

Glutamate-activated BK channel complexes formed with NMDA receptors

The large-conductance calcium- and voltage-activated K+ (BK) channel has a requirement of high intracellular free Ca2+ concentrations for its activation in neurons under physiological conditions. The Ca2+ sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK-NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former's intracellular S0-S1 loop with the latter's cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca2+ influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0-S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK-NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons.

Lysosomal Potassium Channels: Potential Roles in Lysosomal Function and Neurodegenerative Diseases.

The lysosome is a membrane-enclosed organelle widely found in every eukaryotic cell. It has been deemed as the stomach of the cells. Recent studies revealed that it also functions as an intracellular calcium store and is a platform for nutrient-dependent signal transduction. Similar with the plasma membrane, the lysosome membrane is furnished with various proteins, including pumps, ion channels and transporters. So far, two types of lysosomal potassium channels have been identified: large-conductance and Ca2+-activated potassium channel (BK) and TMEM175. TMEM175 has been linked to several neurodegeneration diseases, such as the Alzheimer and Parkinson disease. Recent studies showed that TMEM175 is a lysosomal potassium channel with novel architecture and plays important roles in setting the lysosomal membrane potential and maintaining pH stability. TMEM175 deficiency leads to compromised lysosomal function, which might be responsible for the pathogenesis of related diseases. BK is a well-known potassium channel for its function on the plasma membrane. Studies from two independent groups revealed that functional BK channels are also expressed on the lysosomal plasma membrane. Dysfunction of BK causes impaired lysosomal calcium signaling and abnormal lipid accumulation, a featured phenotype of most lysosomal storage diseases (LSDs). Boosting BK activity could rescue the lipid accumulation in several LSD cell models. Overall, the lysosomal potassium channels are essential for the lysosome physiological function, including lysosomal calcium signaling and autophagy. The dysfunction of lysosomal potassium channels is related to some neurodegeneration disorders. Therefore, lysosomal potassium channels are suggested as potential targets for the intervention of lysosomal disorders.

The G protein-coupled estrogen receptor agonist, G-1, attenuates BK channel activation in cerebral arterial smooth muscle cells.

The G protein–coupled estrogen receptor (GPER) is a significant modulator of arterial contractility and blood flow. The GPER‐specific activator, G‐1, has been widely used to characterize GPER function in a variety of tissue types. Large conductance, calcium (Ca2+)‐activated K+ (BK) channels are sensitive to 17β‐estradiol (17β‐E2) and estrogenic compounds (e.g., tamoxifen, ICI 182 780) that target estrogen receptors. The purpose of this study was to investigate the effects of G‐1 on BK channel activation and function in cerebral arterial myocytes. Inside‐out and perforated patch clamp were utilized to assess the effects of G‐1 (50 nmol·L−1‐5 μmol·L−1) on BK channel activation and currents in cerebral arterial myocytes. Pressurized artery myography was used to investigate the effects of G‐1 on vasodilatory response and BK channel function of cerebral resistance size arteries. G‐1 reduced BK channel activation in cerebral arterial myocytes through elevations in BK channel mean close times. Depressed BK channel activation following G‐1 application resulted in attenuated physiological BK currents (transient BK currents). G‐1 elicited vasodilation, but reduced BK channel function, in pressurized, endothelium‐denuded cerebral arteries. These data suggest that G‐1 directly suppresses BK channel activation and currents in cerebral arterial myocytes, BK channels being critically important in the regulation of myocyte membrane potential and arterial contractility. Thus, GPER‐mediated vasodilation using G‐1 to activate the receptor may underestimate the physiological function and relevance of GPER in the cardiovascular system.

Downregulation of BK channel function and protein expression in coronary arteriolar smooth muscle cells of type 2 diabetic patients.

Type 2 diabetes (T2D) is strongly associated with cardiovascular morbidity and mortality in patients. Vascular large conductance Ca2+-activated potassium (BK) channels, composed of four pore-forming α subunits (BK-α), and four regulatory β1 subunits (BK-β1), are densely expressed in coronary arterial smooth muscle cells (SMCs) and play an important role in regulating vascular tone and myocardial perfusion. However, the role of BK channels in coronary microvascular dysfunction of human subjects with diabetes is unclear. In this study, we examined BK channel function and protein expression, and BK channel-mediated vasodilation in freshly isolated coronary arterioles from T2D patients. Atrial tissues were obtained from 16 patients with T2D and 25 matched non-diabetic subjects during cardiopulmonary bypass procedure. Microvessel videomicroscopy and immunoblot analysis were performed in freshly dissected coronary arterioles and inside-out single BK channel currents was recorded in enzymatically isolated coronary arteriolar SMCs. We found that BK channel sensitivity to physiological Ca2+ concentration and voltage was downregulated in the coronary arteriolar SMCs of diabetic patients, compared with non-diabetic controls. BK channel kinetics analysis revealed that there was significant shortening of the mean open time and prolongation of the mean closed time in diabetic patients, resulting in a remarkable reduction of the channel open probability. Functional studies showed that BK channel activation by dehydrosoyasaponin-1 was diminished and that BK channel-mediated vasodilation in response to shear stress was impaired in diabetic coronary arterioles. Immunoblot experiments confirmed that the protein expressions of BK-α and BK-β1 subunits were significantly downregulated, but the ratio of BK-α/BK-β1 was unchanged in the coronary arterioles of T2D patients. Our results demonstrated for the first time that BK channel function and BK channel-mediated vasodilation were abnormal in the coronary microvasculature of diabetic patients, due to decreased protein expression and altered intrinsic properties of BK channels.

Estrous Cycle-Mediated Regional Diversity in BK Channel Expression and Function in Arterial Myocytes

Estrous Cycle-Mediated Regional Diversity in BK Channel Expression and Function in Arterial Myocytes

Regulation of BK Channel Auxiliary Gamma Subunits by N-Glycosylation

BK channels consist of pore-forming, voltage- and Ca2+-sensing α subunits and auxiliary β and γ subunits. The auxiliary γ1-3 subunits potently modulate the BK channel's function by shifting voltage-dependence of channel activation by 50 - 150 mV toward the negative voltage direction. All BK channel γ subunits contain single or multiple consensus N-linked glycosylation sites in their extracellular LRR domains. We found that inhibition of protein N-glycosylation in HEK-293 cells by tunicamycin caused a great reduction (γ1) or full loss (γ2 and γ3) of the γ subunit's effectiveness in modulating BK channels. We identified the glycosylation sites of N147 in the γ1 subunit, N112, N148 and N211 in the γ2 subunit, and N82, N111 and N137 in the γ3 subunit to be all necessary for the γ subunits’ channel-modulation function. Blockade of glycosylation at single or multiple sites by mutation (N→Q) caused a decrease in protein size and a conversion of either all or a significant portion of BK channels to be unmodulated by the auxiliary γ subunit. Overexpression of the γ1-N147Q mutant can partially compensate its deficiency in BK channel modulation. We conclude that N-glycosylation plays a critical role in maintaining the modulatory function of the BK channel auxiliary γ subunits, presumably through facilitating the protein's surface expression.

+mRNA expression of LRRC55 protein (leucine-rich repeat-containing protein 55) in the adult mouse brain.

LRRC55 (leucine-rich repeat-containing protein 55) protein is an auxiliary γ subunit of BK (Big conductance potassium channel) channels, which leftward shifts GVs of BK channels around 50 mV in the absence of cytosolic Ca2+. LRRC55 protein is also the only γ subunit of BK channels that is expressed in mammalian nervous system. However, the expression pattern of LRRC55 gene in adult mammalian brain remains elusive. In this study, we investigated the distribution of LRRC55 mRNA in the adult mouse brain by using in situ hybridization. We found that LRRC55 mRNA is richly expressed in the adult mouse medial habenula nucleus (MHb), cerebellum and pons. However, the potential role of LRRC55 in MHb and cerebellum could be different based on the function of BK channels in these brain regions.

Chronic Prenatal Hypoxia Down-Regulated BK Channel Β1 Subunits in Mesenteric Artery Smooth Muscle Cells of the Offspring

Background/Aims: Chronic hypoxia in utero could impair vascular functions in the offspring, underlying mechanisms are unclear. This study investigated functional alteration in large-conductance Ca²⁺-activated K⁺ (BK) channels in offspring mesenteric arteries following prenatal hypoxia. Methods: Pregnant rats were exposed to normoxic control (21% O₂, Con) or hypoxic (10.5% O₂, Hy) conditions from gestational day 5 to 21, their 7-month-old adult male offspring were tested for blood pressure, vascular BK channel functions and expression using patch clamp and wire myograh technique, western blotting, and qRT-PCR. Results: Prenatal hypoxia increased pressor responses and vasoconstrictions to phenylephrine in the offspring. Whole-cell currents density of BK channels and amplitude of spontaneous transient outward currents (STOCs), not the frequency, were significantly reduced in Hy vascular myocytes. The sensitivity of BK channels to voltage, Ca²⁺, and tamoxifen were reduced in Hy myocytes, whereas the number of channels per patch and the single-channel conductance were unchanged. Prenatal hypoxia impaired NS1102- and tamoxifen-mediated relaxation in mesenteric arteries precontracted with phenylephrine in the presence of N^ω-nitro-L-arginine methyl ester. The mRNA and protein expression of BK channel β1, not the α-subunit, was decreased in Hy mesenteric arteries. Conclusions: Impaired BK channel β1-subunits in vascular smooth muscle cells contributed to vascular dysfunction in the offspring exposed to prenatal hypoxia.

Downregulation of KCNMB4 expression and changes in BK channel subtype in hippocampal granule neurons following seizure activity.

A major challenge is to understand maladaptive changes in ion channels that sets neurons on a course towards epilepsy development. Voltage- and calcium-activated K+ (BK) channels contribute to early spike timing in neurons, and studies indicate that the BK channel plays a pathological role in increasing excitability early after a seizure. Here, we have investigated changes in BK channels and their accessory β4 subunit (KCNMB4) in dentate gyrus (DG) granule neurons of the hippocampus, key neurons that regulate excitability of the hippocampus circuit. Two days after pilocarpine-induced seizures, we found that the predominant effect is a downregulation of the β4 accessory subunit mRNA. Consistent with reduced expression, single channel recording and pharmacology indicate a switch in the subtype of channels expressed; from iberiotoxin-resistant, type II BK channels (BK α/β4) that have higher channel open probability and slow gating, to iberiotoxin-sensitive type I channels (BK α alone) with low open probability and faster gating. The switch to a majority of type I channel expression following seizure activity is correlated with a loss of BK channel function on spike threshold while maintaining the channel’s contribution to increased early spike frequency. Using heterozygous β4 knockout mice, we find reduced expression is sufficient to increase seizure sensitivity. We conclude that seizure-induced downregulation of KCNMB4 is an activity dependent mechanism that increases the excitability of DG neurons. These novel findings indicate that BK channel subtypes are not only defined by cell-specific expression, but can also be plastic depending on the recent history of neuronal excitability.

Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum.

BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking.

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

Aim: The objective of this study was to examine the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on coronary arterial large conductance Ca²⁺-activated K⁺ (BK) channel function in coronary smooth muscle cells (SMCs) of streptozotocin-induced diabetic rats. Methods: The effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on coronary BK channel open probabilities were determined using the patch clamp technique. The mRNA and protein expressions of BK channel subunits were measured using qRT-PCR and Western blots. The coronary artery tension and coronary SMC Ca²⁺ concentrations were measured using a myograph system and fluorescence Ca²⁺ indicator. Results: Compared to nondiabetic control rats, the BK channel function was impaired with a reduced response to EPA and DHA in freshly isolated SMCs of diabetic rats. Oral administration of n-3 PUFAs had no effects on protein expressions of BK channel subunits in nondiabetic rats, but significantly enhanced those of BK-β₁ in diabetic rats without altering BK-α protein levels. Moreover, coronary ring tension induced by iberiotoxin (a specific BK channel blocker) was increased and cytosolic Ca²⁺ concentrations in coronary SMCs were decreased in diabetic rats, but no changes were found in nondiabetic rats. Conclusions: n-3 PUFAs protect the coronary BK channel function and coronary vasoreactivity in diabetic rats as a result of not only increasing BK-β₁ protein expressions, but also decreasing coronary artery tension and coronary smooth muscle cytosolic Ca²⁺ concentrations.

Role of Smad3 and p38 Signalling in Cigarette Smoke-induced CFTR and BK dysfunction in Primary Human Bronchial Airway Epithelial Cells

Mucociliary clearance (MCC) is a major airway host defence system that is impaired in patients with smoking-associated chronic bronchitis. This dysfunction is partially related to a decrease of airway surface liquid (ASL) volume that is in part regulated by apically expressed cystic fibrosis transmembrane conductance regulator (CFTR) and large-conductance, Ca2+-activated, and voltage dependent K+ (BK) channels. Here, data from human bronchial epithelial cells (HBEC) confirm that cigarette smoke not only downregulates CFTR activity but also inhibits BK channel function, thereby causing ASL depletion. Inhibition of signalling pathways involved in cigarette smoke-induced channel dysfunction reveals that CFTR activity is downregulated via Smad3 signalling whereas BK activity is decreased via the p38 cascade. In addition, pre-treatment with pirfenidone, a drug presently used to inhibit TGF-β signalling in idiopathic pulmonary fibrosis, ameliorated BK dysfunction and ASL volume loss. Taken together, our results highlight the importance of not only CFTR but also BK channel function in maintaining ASL homeostasis and emphasize the possibility that pirfenidone could be employed as a novel therapeutic regimen to help improve MCC in smoking-related chronic bronchitis.

Proximal clustering between BK and CaV1.3 channels promotes functional coupling and BK channel activation at low voltage.

CaV-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and CaV1.3 channels, unique CaV1 channels that activate at low voltages. We found that when BK and CaV1.3 channels were co-expressed in the same cell, BK channels started activating near -50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated CaV2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of CaV1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of CaV1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials.