Large-conductance Potassium Channels Research Articles

Structural bases for blockade and activation of BK channels by Ba2+ ions.

We studied the impact of Ba2+ ions on the function and structure of large conductance potassium (BK) channels. Ion composition has played a crucial role in the physiological studies of BK channels due to their ability to couple ion composition and membrane voltage signaling. Unlike Ca2+, which activates BK channels through all Regulator of K + Conductance (RCK) domains, Ba2+ has been described as specifically interacting with the RCK2 domain. It has been shown that Ba2+ also blocks potassium permeation by binding to the channel's selectivity filter. The Cryo-EM structure of the Aplysia BK channel in the presence of high concentration Ba2+ here presented (PDBID: 7RJT) revealed that Ba2+ occupies the K+ S3 site in the selectivity filter. Densities attributed to K+ ions were observed at sites S2 and S4. Ba2+ ions were also found bound to the high-affinity Ca2+ binding sites RCK1 and RCK2, which agrees with functional work suggesting that the Ba2+ increases open probability through the Ca2+ bowl site (RCK2). A comparative analysis with a second structure here presented (PDBID: 7RK6), obtained without additional Ba2+, shows localized changes between the RCK1 and RCK2 domains, suggestive of coordinated dynamics between the RCK ion binding sites with possible relevance for the activation/blockade of the channel. The observed densities attributed to Ba2+ at RCK1 and RCK2 sites and the selectivity filter contribute to a deeper understanding of the structural basis for Ba2+'s dual role in BK channel modulation, adding to the existing knowledge in this field.

Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm.

Mucosa-associated biofilms are associated with many human disease states, but the host mechanisms promoting biofilm remain unclear. In chronic respiratory diseases like cystic fibrosis (CF), Pseudomonas aeruginosa establishes chronic infection through biofilm formation. P. aeruginosa can be attracted to interspecies biofilms through potassium currents emanating from the biofilms. We hypothesized that P. aeruginosa could, similarly, sense and respond to the potassium efflux from human airway epithelial cells (AECs) to promote biofilm. Using respiratory epithelial co-culture biofilm imaging assays of P. aeruginosa grown in association with CF bronchial epithelial cells (CFBE41o-), we found that P. aeruginosa biofilm was increased by potassium efflux from AECs, as examined by potentiating large conductance potassium channel, BKCa (NS19504) potassium efflux. This phenotype is driven by increased bacterial attachment and increased coalescence of bacteria into aggregates. Conversely, biofilm formation was reduced when AECs were treated with a BKCa blocker (paxilline). Using an agar-based macroscopic chemotaxis assay, we determined that P. aeruginosa chemotaxes toward potassium and screened transposon mutants to discover that disruption of the high-sensitivity potassium transporter, KdpFABC, and the two-component potassium sensing system, KdpDE, reduces P. aeruginosa potassium chemotaxis. In respiratory epithelial co-culture biofilm imaging assays, a KdpFABCDE deficient P. aeruginosa strain demonstrated reduced biofilm growth in association with AECs while maintaining biofilm formation on abiotic surfaces. Furthermore, we determined that the Kdp operon is expressed in vivo in people with CF and the genes are conserved in CF isolates. Collectively, these data suggest that P. aeruginosa biofilm formation can be increased by attracting bacteria to the mucosal surface and enhancing coalescence into microcolonies through aberrant AEC potassium efflux sensed by the KdpFABCDE system. These findings suggest host electrochemical signaling can enhance biofilm, a novel host-pathogen interaction, and potassium flux could be a therapeutic target to prevent chronic infections in diseases with mucosa-associated biofilms, like CF.

Kcnma1 alternative splicing in mouse kidney: regulation during development and by dietary K+ intake.

The pore-forming α-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channel-mediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney.NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+.

#1621 Quantifying ureter smooth muscle electrophysiology from calcium transient images to understand abnormal peristaltic contraction

Abstract Background and Aims Abnormal peristaltic contraction of the ureter smooth muscle (USM) causes acute kidney stone episodes. Intracellular electrical activities like membrane depolarization and action potentials play important roles in modulating the USM contraction by releasing intracellular calcium from the sarcoplasmic reticulum. Therefore, an electrophysiological study will help to assess the USM cell's electrical activities and in diagnosing abnormal USM contraction. The objective of this study is to quantify the contribution of ionic currents in shaping experimental calcium transient profiles using in-silico electrophysiological modeling. Method The simultaneous experimental recording of action potential (AP) and intracellular calcium transient images from the mouse ureter is obtained. The single isolated USM cell model comprises several voltage-gated ion channels, such as two voltage-gated calcium (T—type, and L—type) channels, one voltage-gated fast potassium (KA) channel, one calcium-dependent large conductance potassium channel, and an HCN channel. To describe the calcium-dependent gating of Ca2+-dependent potassium channels and to update the equilibrium potential of the Ca2+ ion, the intracellular Ca2+ concentration is updated during the simulation period. Results Simulation of simultaneous recordings of AP and cytosolic calcium [Ca2+]i are done on a single isolated cell. The model shows [Ca2+]i as a function of synaptic input-induced AP to simulate extracted experimental data, where Ca2+ transient is recorded simultaneously during AP in mouse USM cells. Fig. 1 shows both experimental and simulation of AP (A) and Calcium transient (B) in the USM cell. In our model, the radius “r” and time constant τ of the shell influence the Ca2+ transient profile. In the USM cell model, the submembrane calcium transient occurs from a depth of 0.1 μm to a depth of 0.6 μm. We have investigated whether Ca2+ current via the L-type Ca2+ channel is responsible for the firing of APs with fast upstroke generation. The AP and calcium transients are demolished with the absence of the L-type Ca2+ channel. Conclusion From this study, it is found that inhibition of the L-type Ca2+ channel not only prevented AP generation, it also reduced the cytosolic Ca2+ transient. This study supports the application of L-type Ca2+ channel inhibitor as a potential drug for abnormal peristaltic contraction of the ureter smooth muscle.

Ethanol’s interaction with BK channel α subunit residue K361 does not mediate behavioral responses to alcohol in mice

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol’s direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary β subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

Geometric slow–fast analysis of a hybrid pituitary cell model with stochastic ion channel dynamics

To obtain explicit understanding of the behavior of dynamical systems, geometrical methods and slow–fast analysis have proved to be highly useful. Such methods are standard for smooth dynamical systems and increasingly used for continuous, non-smooth dynamical systems. However, they are much less used for random dynamical systems, in particular for hybrid models with discrete, random dynamics. Here we propose a geometrical method that works directly with the hybrid system. We illustrate our approach through an application to a hybrid pituitary cell model in which the stochastic dynamics of very few active large-conductance potassium (BK) channels is coupled to a deterministic model of the other ion channels and calcium dynamics. To employ our geometric approach, we exploit the slow–fast structure of the model. The random fast subsystem is analyzed by considering discrete phase planes, corresponding to the discrete number of open BK channels, and stochastic events correspond to jumps between these planes. The evolution within each plane can be understood from nullclines and limit cycles, and the overall dynamics, e.g., whether the model produces a spike or a burst, is determined by the location at which the system jumps from one plane to another. Our approach is generally applicable to other scenarios to study discrete random dynamical systems defined by hybrid stochastic–deterministic models.

BK Channels Function in Nematocyst Discharge from Vibration-Sensitive Cnidocyte Supporting Cell Complexes of the Sea Anemone Diadumene lineata.

AbstractIntegrated chemo- and mechanosensory pathways, along with activated calcium influxes, regulate nematocyst discharge from sea anemone tentacles. Discharge from vibration-sensitive Type A cnidocyte supporting cell complexes use calcium-conducting transient receptor potential V4-like channels. Because calcium influxes often couple with calcium-activated, large-conductance potassium (BK) channels, we hypothesized that BK channels function in nematocyst discharge. To verify this hypothesis, we first tested five selective BK channel blockers on nematocyst-mediated prey killing in Diadumene lineata (aka Haliplanella luciae). All tested BK channel blockers inhibited prey killing at concentrations comparable to their inhibition of vertebrate BK channels. In addition, the BK channel blocker paxilline selectively inhibited prey killing mediated by vibration-sensitive Type A cnidocyte supporting cell complexes. We queried a mammalian BKα amino acid sequence to the Exaiptasia diaphena database, from which we identified a putative anemone, pore-forming BKα subunit sequence. Using the E. diaphena BKα sequence as a template, we assembled a BKα transcript from our assembled D. lineata transcriptome. In addition, the hydra homolog of D. lineata BKα localizes to nematocytes on the hydra single-cell RNA sequencing map. Our findings suggest that D. lineata expresses BK channels that play a role in vibration-sensitive nematocyst discharge from Type A cnidocyte supporting cell complexes. We believe this is the first functional demonstration of BK channels in nonbilaterians. Because stimulated chemoreceptors frequency tune Type A cnidocyte supporting cell complexes to frequencies matching swimming movements of prey via a protein kinase A signaling pathway and protein kinase A generally activates BK channels, we suggest that D. lineata BK channels may participate in protein kinase A-mediated frequency tuning.

#2696 IN SILICO ELECTROPHYSIOLOGICAL STUDY REVEALS TAMSULOSIN MEDIATE URETER SMOOTH MUSCLE CONTRACTION BY INCREASING POTASSIUM CURRENT

Abstract Background and Aims Tamsulosin is a common α1-AR antagonist that is prescribed to patients with acute kidney stone episodes [Scotland et al., 2021]. According to accumulated experimental data, abnormal peristaltic contraction of the ureter smooth muscle (USM) causes acute kidney stone episodes [Lang et al., 2006]. Intracellular electrical activities like membrane depolarization and action potentials play important roles in modulating the USM contraction by releasing intracellular calcium from the sarcoplasmic reticulum. Therefore, an electrophysiological study will be helpful to assess the USM cell's electrical activities and in diagnosing abnormal USM contraction. Transient potassium channels (KA) are prevalent in different systems and hold immense importance for maintaining/performing selective electrophysiological functions. The present study investigates the altered electrophysiological characteristics of the USM cell because of the induction of Tamsulosin doses. Method The single isolated USM cell comprises several voltage-gated ion channels, such as two voltage-gated calcium (T-type, and L-type) channels, one voltage-gated fast potassium (KA) channel, one calcium-dependent large conductance potassium channel, and an HCN channel. The individual ion channel currents and the membrane potential are recorded by utilizing both the voltage clamp and current clamp protocols. A Tamsulosin drug (0.5 mg) model for the KA channel is introduced by multiplying the maximal conductance of the KA channel with a scaling factor between 0 and 1 to mimic the drug concentration. Statistical analysis protocols are evaluated for analytical purposes. Results In this in silico electrophysiological model, the resting membrane potential is maintained at −75 mV, as this value is cited by various experimental studies. The ionic concentrations and biophysical parameters for all ion channels are varied in the physiological ranges. Under the voltage clamp protocol, the voltage steps are increased from a holding potential of −80 mV to −10 mV with a 10 mV of step voltage. In Figure 1, the red and blue solid lines represent recorded outward KA current from the isolated USM cell under both control and applied Tamsulosin dose conditions. Figure 1 reveals that the KA current is elevated after the application of the Tamsulosin drug regarding all step voltages. As a result, the total outward current is elevated and the USM action potential duration is shortened. It is also observed that the steady-state activation curve of the KA channel is shifted to more negative after applying Tamsulosin. The USM cell is electrically less excitable with the Tamsulosin. Conclusion This first in-silico study reveals the KA ionic current, which plays a major role in coordinating ureteral contractions, is altered due to Tamsulosin. It implies that the pharmacological maneuver of Tamsulosin is substantially beneficial to treat dysfunctional peristalsis by reducing the symptoms and installation complexities from the ureteral stents procedure.

Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

Ca2+- and Voltage-Activated K+ (BK) Channels in the Nervous System: One Gene, a Myriad of Physiological Functions.

BK channels are large conductance potassium channels characterized by four pore-forming α subunits, often co-assembled with auxiliary β and γ subunits to regulate Ca2+ sensitivity, voltage dependence and gating properties. BK channels are abundantly expressed throughout the brain and in different compartments within a single neuron, including axons, synaptic terminals, dendritic arbors, and spines. Their activation produces a massive efflux of K+ ions that hyperpolarizes the cellular membrane. Together with their ability to detect changes in intracellular Ca2+ concentration, BK channels control neuronal excitability and synaptic communication through diverse mechanisms. Moreover, increasing evidence indicates that dysfunction of BK channel-mediated effects on neuronal excitability and synaptic function has been implicated in several neurological disorders, including epilepsy, fragile X syndrome, mental retardation, and autism, as well as in motor and cognitive behavior. Here, we discuss current evidence highlighting the physiological importance of this ubiquitous channel in regulating brain function and its role in the pathophysiology of different neurological disorders.

Structural bases for blockade and activation of BK channels by Ba2+ ions.

Calcium ions increase the open probability of the large conductance potassium (BK) channels by binding to high affinity sites within the large cytosolic Ca2+ sensing structure called the gating ring. This Ca2+ sensing structure is formed by eight Regulator of K+ Conductance (RCK) domains, two from each subunit of the channel, termed RCK1 and RCK2. Other divalent species, like Ba2+ ions, are also able to activate BK channels by interacting with the Ca2+ sensor. Unlike Ca2+ that activate BK channel through all RCK domains, Ba2+ does it selectively through the RCK2 domain. In addition to activation, Ba2+ can also block K+ permeation by binding to the selectivity filter of BK channels. Here we present and discuss Cryo-EM structures of BK channels with Ba2+. Structurally, blockade arises from one Ba2+ occupying the traditional K+ site S3 in the selectivity filter of the channel. In addition, electronic densities attributed to K+ ions are detected at K+ sites S2 and S4. In the gating ring, eight Ba2+ ions are bound to the corresponding high affinity Ca2+ binding sites. Thereby, the lack of functional significance of the RCK1 on the activation by Ba2+ should arise because the architectures of the gating rings in our structures represent intermediate transitions between the close and the open configurations. Finally, these Ba2+ structures reveal an intricate series of concerted changes of the RCK 1 and RCK2 from the same subunit, suggesting coordinated intra-subunit dynamics.

Cerebrovascular Effects of Alcohol Combined with Tetrahydrocannabinol.

Introduction: Alcohol (ethanol) and cannabis are among the most widely used recreational drugs in the world. With increased efforts toward legalization of cannabis, there is an alarming trend toward the concomitant (including simultaneous) use of cannabis products with alcohol for recreational purpose. While each drug possesses a distinct effect on cerebral circulation, the consequences of their simultaneous use on cerebral artery diameter have never been studied. Thus, we set to address the effect of simultaneous application of alcohol and (-)-trans-Δ-9-tetrahydrocannabinol (THC) on cerebral artery diameter. Materials and Methods: We used Sprague-Dawley rats because rat cerebral circulation closely mimics morphology, ultrastructure, and function of cerebral circulation of humans. We focused on the middle cerebral artery (MCA) because it supplies blood to the largest brain territory when compared to any other cerebral artery stemming from the circle of Willis. Experiments were performed on pressurized MCA ex vivo, and in cranial windows in vivo. Ethanol and THC were probed at physiologically relevant concentrations. Researchers were "blind" to experimental group identity during data analysis to avoid bias. Results: In males, ethanol mixed with THC resulted in greater constriction of ex vivo pressurized MCA when compared to the effects exerted by separate application of each drug. In females, THC, ethanol, or their mixture failed to elicit measurable effect. Vasoconstriction by ethanol/THC mixture was ablated by either endothelium removal or pharmacological block of calcium- and voltage-gated potassium channels of large conductance (BK type) and cannabinoid receptors. Block of prostaglandin production and of endothelin receptors also blunted constriction by ethanol/THC. In males, the in vivo constriction of MCA by ethanol/THC did not differ from ethanol alone. In females, the in vivo constriction of this artery by ethanol was significantly smaller than in males. However, artery constriction by ethanol/THC did not differ from the constriction in males. Conclusions: Our data point at the complex nature of the cerebrovascular effects elicited by simultaneous use of ethanol and THC. These effects include both local and systemic components.

The role of ВK<sub>Са</sub> and IK<sub>Са</sub> channels in H<sub>2</sub>S-induced dilatation of pial arteries in rats after nephrectomy

BACKGROUND. Chronic kidney disease (CKD) is accompanied by the development of endothelial dysfunction, leading to a decrease in arterial reactivity to vasoactive agents. Uremia causes a change in the dilatation of arteries in various vascular regions, incl. and arteries of the pial membrane of the brain. The action of hydrogen sulfide (H2S), which can induce relaxation of smooth muscle cells of blood vessels, is currently considered a possible route of vasoprotection in various diseases, particularly, in CKD. THE AIM. To evaluate the role of calcium-activated potassium channels of large (BKCa) and intermediate (IKCa) conductance in H2S-induced dilatation of pial arteries in nephrectomized (NE) rats. MATERIAL AND METHODS. In Wistar rats nephrectomy (NE) was performed by resection of 5/6 of the renal tissue mass. Sham-operated (LO) animals served as control. The reaction of the pial arteries of the sensomotor cortex of NE and control SO rats to the application of H2S under physiological conditions and against the background of the use of BKCa channel blockers – tetraethylammonium (TEA) and IKCa – channels – TRAM-34. RESULTS. 4 months after NE, the application of H2S led to the dilatation of a smaller number of pial arteries (1.4 – 1.7 times) compared with SO rats. The preliminary exposure to TEA led to a decrease in the number of pial arteries responding by dilatation to the action of H2S in NE and SO rats. Against the background of the action of TRAM-34, the number of dilated arteries decreased under the action of H2S in SO rats, while in NE rats it practically did not change. CONCLUSION. Under physiological conditions, dilatation of the pial arteries in rats under the action of H2S is realized (at least in part) through the activation of the BKCa and IKCa channels of the membrane of endothelial and smooth muscle cells. Uremia, caused by nephrectomy, leads to impairment of the mechanism of dilatation of pial arteries, mediated by activation of calcium-activated potassium channels intermediate conductance apparently due to dysfunction of endothelial cells.

Role of Cholesterol in the Regulation of Hydrogen Sulfide Signaling within the Vascular Endothelium.

H2S is a gaseous signaling molecule enzymatically produced in mammals and H2S-producing enzymes are expressed throughout the vascular wall. We previously reported that H2S-induced vasodilation is mediated through transient receptor potential cation channel subfamily V member 4 (TRPV4) and large conductance (BKCa) potassium channels; however, regulators of this pathway have not been defined. Previous reports have shown that membrane cholesterol limits activity of TRPV4 and BKCa potassium channels. The current study examined the ability of endothelial cell (EC) plasma membrane (PM) cholesterol to regulate H2S-induced vasodilation. We hypothesized that EC PM cholesterol hinders H2S-mediated vasodilation in large mesenteric arteries. In pressurized, U46619 pre-constricted mesenteric arteries, decreasing EC PM cholesterol in large arteries using methyl-β-cyclodextrin (MBCD, 100 µM) increased H2S-induced dilation (NaHS 10, 100 µM) but MBCD treatment had no effect in small arteries. Enface fluorescence showed EC PM cholesterol content is higher in large mesenteric arteries than in smaller arteries. The NaHS-induced vasodilation following MBCD treatment in large arteries was blocked by TRPV4 and BKCa channel inhibitors (GSK219384A, 300 nM and iberiotoxin, 100 nM, respectively). Immunohistochemistry of mesenteric artery cross-sections show that TRPV4 and BKCa are both present in EC of large and small arteries. Cholesterol supplementation into EC PM of small arteries abolished NaHS-induced vasodilation but the cholesterol enantiomer, epicholesterol, had no effect. Proximity ligation assay studies did not show a correlation between EC PM cholesterol content and the association of TRPV4 and BK. Collectively, these results demonstrate that EC PM cholesterol limits H2S-induced vasodilation through effects on EC TRPV4 and BKCa channels.

Vibegron inhibits enhanced spontaneous contractions induced by anoxia/reoxygenation in isolated whole bladder from rats

It has been recently proposed that repeated bladder ischemia/reperfusion induced by chronic pelvic ischemia may lead to detrusor overactivity, followed by lower urinary tract symptoms. Vibegron is a selective β3-adrenoceptor agonist approved for the treatment of overactive bladder. Several studies have tested β3-adrenoceptor agonists using animal models with detrusor overactivity related to bladder ischemia/reperfusion. However, whether β3-adrenoceptor agonists directly affect ischemia/reperfusion-evoked detrusor overactivity is unclear. Therefore, we examined whether bladder anoxia/reoxygenation could enhance spontaneous bladder contractions (SBCs) and investigated the effect of vibegron on enhanced SBCs. Isolated whole bladders from rats were incubated with Krebs solution aerated with 95% N2 + 5% CO2 for 5 h (anoxia). Subsequently, the bathing solution was replaced with an oxygen-saturated solution (reoxygenation). Anoxia/reoxygenation caused enhancement of the amplitude but not the frequency of SBC compared with that before reoxygenation. Vibegron (0.3–30 μM) inhibited this increase in SBC amplitude, but not the frequency, in a dose-dependent manner. The inhibitory effect of vibegron was not affected by pretreatment with the adenylyl cyclase inhibitor SQ22536 (100 μM) or protein kinase A inhibitor KT5720 (1 μM) and was not accompanied by considerable changes in cyclic adenosine monophosphate (cAMP) content in the bladder. In contrast, the large conductance potassium channel inhibitor iberiotoxin (100 nM) suppressed the inhibitory effect of vibegron. These results suggest that bladder ischemia/reperfusion induces SBC enhancement and vibegron directly inhibits detrusor overactivity via the large conductance potassium channel, which involves β3-adrenoceptor, rather than the cAMP signaling pathway.

Inhibition of BKCa channels protects neonatal hearts against myocardial ischemia and reperfusion injury

BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channel’s activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes, which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL-positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.

Title: β3 Adrenergic Receptor Signaling in the Human Myometrium

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. Although β2 adrenergic agonists fail to provide adequate tocolysis, the expression of the β3 adrenergic receptor in myometrium and its unique signaling suggest a role for β3 agonist in the management of preterm labor. Western blot analysis showed that the β3 adrenergic receptor expression increased in human pregnancy myometrium compared to nonpregnant tissues (p < 0.0001). There was no difference in β3 adrenergic receptor expression throughout pregnancy (p > 0.05). The addition of the β3 agonist mirabegron in the tissue bath relaxed oxytocin contracted myometrium with an EC50 of 41.5 µM. Relaxation was partially blocked by the addition of the eNOS blocker Nω-nitro-L-arginine, or the large conductance potassium channel blocker paxilline. Combination of Nω-nitro-L-arginine and paxilline prevented mirabegron-mediated relaxation. Imaging revealed that the β3 adrenergic receptors are expressed by both myocyte and microvascular endothelial cells isolated from human myometrium. Nitric oxide production measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate revealed that mirabegron stimulated nitric oxide production in myometrial endothelial cells. These data suggest that both endothelial and smooth muscle cells contribute to relaxation through disparate signaling pathways. Repurposing of approved medications tested in human myometrium as uterine tocolytics can advance prevention of preterm birth. These data argue that further examination of β3 adrenergic receptor signaling in myometrium may reveal mirabegron as a useful tocolytic in combination tocolysis regimens.

Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa)

IntroductionPeripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC.MethodsFemoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique.ResultsR/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening.ConclusionR/NIR vasodilation requires indirect activation of the BKca channel.

Single channel properties of mitochondrial large conductance potassium channel formed by BK-VEDEC splice variant

The activation of mitochondrial large conductance calcium-activated potassium (mitoBKCa) channels increases cell survival during ischemia/reperfusion injury of cardiac cells. The basic biophysical and pharmacological properties of mitoBKCa correspond to the properties of the BKCa channels from the plasma membrane. It has been suggested that the VEDEC splice variant of the KCNMA1 gene product encoding plasma membrane BKCa is targeted toward mitochondria. However there has been no direct evidence that this protein forms a functional channel in mitochondria. In our study, we used HEK293T cells to express the VEDEC splice variant and observed channel activity in mitochondria using the mitoplast patch-clamp technique. For the first time, we found that transient expression with the VEDEC isoform resulted in channel activity with the conductance of 290 ± 3 pS. The channel was voltage-dependent and activated by calcium ions. Moreover, the activity of the channel was stimulated by the potassium channel opener NS11021 and inhibited by hemin and paxilline, which are known BKCa channel blockers. Immunofluorescence experiments confirmed the partial colocalization of the channel within the mitochondria. From these results, we conclude that the VEDEC isoform of the BKCa channel forms a functional channel in the inner mitochondrial membrane. Additionally, our data show that HEK293T cells are a promising experimental model for expression and electrophysiological studies of mitochondrial potassium channels.

Preliminary Studies Towards the Examination of Hypoxia‐related Transcriptional Regulation of Ryanodine Receptor Activity in Pulmonary Arteries of Fetal and Newborn Sheep

Before birth the pulmonary vasculature does not receive much blood supply and is relatively constricted. However, with birth the vasculature dilates and blood flow and oxygenation increase. Evidence indicates that pulmonary arterial dilation during birth is related to an increase in the activation of large conductance potassium channels that are activated by Ca2+ spark events after activation of ryanodine receptors (RyR) on the sarcoplasmic reticulum. This coupled mechanism is largely driven through activation of L-type Ca2+ channels (CaL), which stimulate RyRs. Depolarizing cells with 30 mM potassium activates CaL and drives RyR mediated Ca2+ events. Previous evidence from our group illustrates that long term maternal hypoxia disrupts Ca2+ sparks and causes pulmonary hypertension in newborn lambs. We intend to modulate key transcriptional processes to better understand the molecular mechanisms that disrupt Ca2+ spark, vasoreactivity, and pulmonary pressure following long term hypoxia. The current studies were therefore performed as a foundation for our upcoming studies that will examine transcriptional control of RyRs and potassium channels. We tested the hypothesis that we could replicate earlier work that showed membrane depolarization with 30 mM K increases Ca2+ spark activity through activation of RyRs. To address this hypothesis, pulmonary arterial myocytes of fetal and newborn sheep were examined in the presence and absence of 30K and 10 mM ryanodine, which is a RyR antagonist. The intracellular Ca2+ was recorded in myocytes of isolated pulmonary arteries that were loaded with Fluo-4 using line-scan techniques on a confocal microscope. Spatial and temporal characteristics of the events were analyzed using customized software. As predicted, 30K increased Ca2+ spark activity while ryanodine treatment decreased activity. This confirmatory evidence in fetus and newborns provides a platform for upcoming studies to evaluate the role of transcriptional regulation to cell excitability in response to maternal hypoxia.