BK Channel Openers Research Articles

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.

Pulmonary Circulation Under Pressure: Pathophysiological and Therapeutic Implications of BK Channel.

The large-conductance Ca2+-activated K+ (BK) channel is widely expressed in the pulmonary blood vessels and plays a significant role in regulating pulmonary vascular tonus. It opens under membrane depolarization, increased intracellular Ca+2 concentration, and chronic hypoxia, resulting in massive K+ efflux, membrane hyperpolarization, decreased L-type Ca+2 channel opening, and smooth muscle relaxation. Several reports have demonstrated an association between BK channel dysfunction and pulmonary hypertension (PH) development. Decreased BK channel subunit expression and impaired regulation by paracrine hormones result in decreased BK channel opening, increased pulmonary vascular resistance, and pulmonary arterial pressure being the cornerstone of PH. The resulting right ventricular pressure overload ultimately leads to ventricular remodeling and failure. Therefore, it is unsurprising that the BK channel has arisen as a potential target for treating PH. Recently, a series of selective, synthetic BK channel agonists have proven effective in attenuating the pathophysiological progression of PH without adverse effects in animal models.

Small molecule modulation of the large-conductance calcium-activated potassium channel suppresses salicylate-induced tinnitus in mice

Tinnitus is the phantom perception of sound that has no external source. A neurological signature of tinnitus, and the frequently associated hyperacusis, is an imbalance between excitatory and inhibitory activity in the central auditory system (CAS), leading to dysregulated network excitability. The large conductance, calcium-activated potassium (BK) channel is a key player in pre- and post-synaptic excitability through its mediation of K+ currents. Changes in BK channel activity are associated with aberrant network activity in sensory regions of the CNS, raising the possibility that BK channel modulation could regulate activity associated with tinnitus and hyperacusis. To test whether BK channel openers are able to suppress biomarkers of drug-induced tinnitus and hyperacusis, the 1,3,4 oxadiazole BMS-191011 was given to young adult CBA mice that had been administered 250 mg/kg sodium salicylate (SS). Systemic treatment with BMS-191011 reduced behavioral manifestations of SS-induced tinnitus, but not hyperacusis, probed via the gap-in-noise startle response method. Systemic BMS-191011 treatment did not influence SS-induced increases in auditory brainstem response functions, but local application at the inferior colliculus did reverse SS-suppressed spontaneous activity, particularly in the frequency region of the tinnitus percept. Thus, action of BMS-191011 in the inferior colliculus may contribute to the reduction in behaviorally measured tinnitus. Together, these findings support the utility of BK channel openers in reducing central auditory processing changes associated with the formation of the tinnitus percept.

Effect of an autism-associated KCNMB2 variant, G124R, on BK channel properties.

BK K+ channels are critical regulators of neuron and muscle excitability, comprised of a tetramer of pore-forming αsubunits from the KCNMA1 gene and cell- and tissue-selective β subunits (KCNMB1-4). Mutations in KCNMA1 are associated with neurological disorders, including autism. However, little is known about the role of neuronal BK channel β subunits in human neuropathology. The β2 subunit is expressed in central neurons and imparts inactivation to BK channels, as well as altering activation and deactivation gating. In this study, we report the functional effect of G124R, a novel KCNMB2 mutation obtained from whole-exome sequencing of a patient diagnosed with autism spectrum disorder. Residue G124, located in the extracellular loop between TM1 and TM2, is conserved across species, and the G124R missense mutation is predicted deleterious with computational tools. To investigate the pathogenicity potential, BK channels were co-expressed with β2WT and β2G124R subunits in HEK293T cells. BK/β2 currents were assessed from inside-out patches under physiological K+ conditions (140/6 mM K+ and 10 μM Ca2+) during activation and inactivation (voltage-dependence and kinetics). Using β2 subunits lacking inactivation (β2IR) revealed that currents from BK/β2IRG124R channels activated 2-fold faster and deactivated 2-fold slower compared with currents from BK/β2IRWT channels, with no change in the voltage-dependence of activation (V1/2). Despite the changes in the BK channel opening and closing, BK/β2G124R inactivation rates (τinact and τrecovery), and the V1/2 of inactivation, were unaltered compared with BK/β2WT channels under standard steady-state voltage protocols. Action potential-evoked current was also unchanged. Thus, the mutant phenotype suggests the β2G124R TM1-TM2 extracellular loop could regulate BK channel activation and deactivation kinetics. However, additional evidence is needed to validate pathogenicity for this patient-associated variant in KCNMB2.

NS-11021 Modulates Cancer-Associated Processes Independently of BK Channels in Melanoma and Pancreatic Duct Adenocarcinoma Cell Lines.

Simple SummaryPotassium channels permit the selective passage of K+ ions across the cell me brane and are important for setting the membrane potential and for the transmission of electrical signals in all cells. Ca2+ activated K+ channels provide a means to couple intracellular calcium signaling to changes of the membrane potential. Among these, the large-conductance Ca2+-activated K+ channel, known as BK, has been proposed to be involved in several cancer-associated processes. In the present work, we tested various BK channel activators for anti-cancer effects in melanoma and pancreatic duct carcinoma cell lines. Only one of the activators (NS-11021) had effects on cancer-associated processes. However, the compound, as a side-effect, also increased the intracellular Ca2+ concentration independently of BK channel activation. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects.Potassium channels have emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large-conductance Ca2+-activated K+ channel, often referred to as BK channel, is involved in several cancer-associated processes. Here, we investigated the effects of different BK activators, NS-11021, NS-19504, and BMS-191011, in IGR39 (primary melanoma cell line) and Panc-1 (primary pancreatic duct carcinoma cell line), highly expressing the channel, and in IGR37 (metastatic melanoma cell line) that barely express BK. Our data showed that NS-11021 and NS-19504 potently activated BK channels in IGR39 and Panc-1 cells, while no effect on channel activation was detected in IGR37 cells. On the contrary, BK channel activator BMS-191011 was less effective. However, only NS-11021 showed significant effects in cancer-associated processes, such as cell survival, migration, and proliferation in these cancer cell lines. Moreover, NS-11021 led to an increase of intracellular Ca2+ concentration, independent of BK channel activation, thus complicating any interpretation of its role in the regulation of cancer-associated mechanisms. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects in the melanoma and PDAC cell lines examined. Importantly, our results raise an alarm flag regarding the use of presumably specific BK channel openers as anti-cancer agents.

Synthesis and BK channel-opening activity of 2-amino-1,3-thiazole derivatives

A series of 2-amino-5-arylmethyl- or 5-heteroarylmethyl-1,3-thiazole derivatives were synthesized and evaluated for BK channel-opening activities in cell-based fluorescence assay and electrophysiological recording. The assay results indicated that the activities of the investigated compounds were influenced by the physicochemical properties of the substituent at benzene ring.

Opening large-conductance potassium channels selectively induced cell death of triple-negative breast cancer

BackgroundUnlike other breast cancer subtypes that may be treated with a variety of hormonal or targeted therapies, there is a need to identify new, effective targets for triple-negative breast cancer (TNBC). It has recently been recognized that membrane potential is depolarized in breast cancer cells. The primary objective of the study is to explore whether hyperpolarization induced by opening potassium channels may provide a new strategy for treatment of TNBC.MethodsBreast cancer datasets in cBioPortal for cancer genomics was used to search for ion channel gene expression. Immunoblots and immunohistochemistry were used for protein expression in culture cells and in the patient tissues. Electrophysiological patch clamp techniques were used to study properties of BK channels in culture cells. Flow cytometry and fluorescence microscope were used for cell viability and cell cycle studies. Ultrasound imaging was used to study xenograft in female NSG mice.ResultsIn large datasets of breast cancer patients, we identified a gene, KCNMA1 (encoding for a voltage- and calcium-dependent large-conductance potassium channel, called BK channel), overexpressed in triple-negative breast cancer patients. Although overexpressed, 99% of channels are closed in TNBC cells. Opening BK channels hyperpolarized membrane potential, which induced cell cycle arrest in G2 phase and apoptosis via caspase-3 activation. In a TNBC cell induced xenograft model, treatment with a BK channel opener significantly slowed tumor growth without cardiac toxicity.ConclusionsOur results support the idea that hyperpolarization induced by opening BK channel in TNBC cells can become a new strategy for development of a targeted therapy in TNBC.

BK channel openers NS1619 and NS11021 reverse hydrogen peroxide-induced membrane potential changes in skeletal muscle

Large conductance calcium-activated potassium (BK) channels play a crucial role in the repolarization and after-hyperpolarization phases of the cell membrane. The channel openers are also used in treatment of some diseases, including hypo/hyperkalemic periodic paralysis. However, little is known about the effects of BK channels and the channel activators on membrane potentials in skeletal muscle. In addition, the effects of reactive oxygen species (ROS) on BK channels in skeletal muscle are also unknown. Therefore, the aim of this study was to determine the effects of BK channel openers and ROS on membrane potentials in skeletal muscle fibers. For this purpose, resting membrane potentials and action potentials (AP) of frog gastrocnemius muscles were recorded in the presence of commonly used BK channel openers NS1619 and NS11021, H2O2 (a type of ROS), and both using intracellular microelectrode technique. The channel activators significantly and dose-dependently decreased amplitude and increased rise time of AP but did not impact repolarization. The presence of H2O2 plus NS1619 or NS11021 resulted in significant change because the channel openers completely reversed the deleterious effects of hydrogen peroxide on the repolarization phase of AP in skeletal muscle fibers. In the present study, the contributions of BK channel activation and the modulatory role of H2O2 on membrane potentials was demonstrated in skeletal muscle fibers, for the first time. Moreover, it should be noted that BK channel openers should be used in the treatment of reactive oxygen species-induced skeletal muscle diseases.

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

Vasodilation of rat skeletal muscle arteries by the novel BK channel opener GoSlo is mediated by the simultaneous activation of BK and Kv 7 channels.

Background and PurposeBK channels play important roles in various physiological and pathophysiological processes and thus have been the target of several drug development programmes focused on creating new efficacious BK channel openers, such as the GoSlo‐SR compounds. However, the effect of GoSlo‐SR compounds on vascular smooth muscle has not been studied. Therefore, we tested the hypothesis that GoSlo‐SR compounds dilate arteries exclusively by activating BK channels.Experimental ApproachExperiments were performed on rat Gracilis muscle, saphenous, mesenteric and tail arteries using isobaric and isometric myography, sharp microelectrodes, digital droplet PCR and the patch‐clamp technique.Key ResultsGoSlo‐SR compounds dilated isobaric and relaxed and hyperpolarised isometric vessel preparations and their effects were abolished after (a) functionally eliminating K+ channels by pre‐constriction with 50 mM KCl or (b) blocking all K+ channels known to be expressed in vascular smooth muscle. However, these effects were not blocked when BK channels were inhibited. Surprisingly, the Kv7 channel inhibitor XE991 reduced their effects considerably, but neither Kv1 nor Kv2 channel blockers altered the inhibitory effects of GoSlo‐SR. However, the combined blockade of BK and Kv7 channels abolished the GoSlo‐SR‐induced relaxation. GoSlo‐SR compounds also activated Kv7.4 and Kv7.5 channels expressed in HEK 293 cells.Conclusion and ImplicationsThis study shows that GoSlo‐SR compounds are effective relaxants in vascular smooth muscle and mediate their effects by a combined activation of BK and Kv7.4/Kv7.5 channels. Activation of Kv1, Kv2 or Kv7.1 channels or other vasodilator pathways seems not to be involved.

A computational model of large conductance voltage and calcium activated potassium channels: implications for calcium dynamics and electrophysiology in detrusor smooth muscle cells.

The large conductance voltage and calcium activated potassium (BK) channels play a crucial role in regulating the excitability of detrusor smooth muscle, which lines the wall of the urinary bladder. These channels have been widely characterized in terms of their molecular structure, pharmacology and electrophysiology. They control the repolarising and hyperpolarising phases of the action potential, thereby regulating the firing frequency and contraction profiles of the smooth muscle. Several groups have reported varied profiles of BK currents and I-V curves under similar experimental conditions. However, no single computational model has been able to reconcile these apparent discrepancies. In view of the channels' physiological importance, it is imperative to understand their mechanistic underpinnings so that a realistic model can be created. This paper presents a computational model of the BK channel, based on the Hodgkin-Huxley formalism, constructed by utilising three activation processes - membrane potential, calcium inflow from voltage-gated calcium channels on the membrane and calcium released from the ryanodine receptors present on the sarcoplasmic reticulum. In our model, we attribute the discrepant profiles to the underlying cytosolic calcium received by the channel during its activation. The model enables us to make heuristic predictions regarding the nature of the sub-membrane calcium dynamics underlying the BK channel's activation. We have employed the model to reproduce various physiological characteristics of the channel and found the simulated responses to be in accordance with the experimental findings. Additionally, we have used the model to investigate the role of this channel in electrophysiological signals, such as the action potential and spontaneous transient hyperpolarisations. Furthermore, the clinical effects of BK channel openers, mallotoxin and NS19504, were simulated for the detrusor smooth muscle cells. Our findings support the proposed application of these drugs for amelioration of the condition of overactive bladder. We thus propose a physiologically realistic BK channel model which can be integrated with other biophysical mechanisms such as ion channels, pumps and exchangers to further elucidate its micro-domain interaction with the intracellular calcium environment.

Forskolin (cAMP) Promotes Insertion of Large Conductance Potassium (KCa1.1) Channels into Apical Membranes of Rat Distal Colon

Mammalian colon contributes significantly to overall K+ homeostasis. Under normal conditions, the proximal colon secretes and distal colon absorbs K+. Distal colon also has a secretory capacity, which becomes important to K+ homeostasis under pathological conditions, such as end‐stage renal failure. K+ secretion in the distal colon is primarily mediated through apical large conductance (BK) channels. We hypothesized that the BK opener BMS‐204352 (BMS) and the adenylate cyclase activator forskolin (FSK) induce K+ secretion through separate mechanisms. K+ flux studies were performed in distal colonic mucosal tissues obtained from male Sprague‐Dawley rats (200–225g). Unidirectional 86Rb fluxes (K+ surrogate), and short‐circuit current (ISC) were measured under voltage‐clamped conditions in an Ussing chamber setup. Fluxes were performed in the presence of amiloride (10 mM) to inhibit ENaC‐mediated ISC, as well as VO4 (1mM) to inhibit apical H+/K+ ATPase and unmask BK‐mediated K+ secretion. Results indicate that BMS (10mM) and FSK (10mM) induced a net K+ secretion of 0.083 ± 0.04 and 0.274 ± 0.05 mEq/cm2·hr, respectively (n=4, p<0.05). FSK, but not BMS, increased ISC compared to baseline (220.5 ± 30.5 vs 132.2 ± 18.7 uA/cm2). Immunofluorescence studies performed on the same tissues used in flux experiments showed markedly increased apical BK staining in tissues exposed to FSK, but not in those exposed to only BMS. Taken together, these data suggest that while BMS may only open BK channels already present on apical membranes, FSK may cause further K+ secretion by triggering the insertion of more BK channels into the apical membrane.Support or Funding Information• R01 DK104791/DK/NIDDK NIH HHS/United States

BK channels in rat and human pulmonary smooth muscle cells are BKα-β1 functional complexes lacking the oxygen-sensitive stress axis regulated exon insert.

A loss of K+ efflux in pulmonary arterial smooth muscle cells (PASMCs) contributes to abnormal vasoconstriction and PASMC proliferation during pulmonary hypertension (PH). Activation of high-conductance Ca2+-activated (BK) channels represents a therapeutic strategy to restore K+ efflux to the affected PASMCs. However, the properties of BK channels in PASMCs-including sensitivity to BK channel openers (BKCOs)-are poorly defined. The goal of this study was to compare the properties of BK channels between PASMCs of normoxic (N) and chronic hypoxic (CH) rats and then explore key findings in human PASMCs. Polymerase chain reaction results revealed that 94.3% of transcripts encoding BKα pore proteins in PASMCs from N rats represent splice variants lacking the stress axis regulated exon insert, which confers oxygen sensitivity. Subsequent patch-clamp recordings from inside-out (I-O) patches confirmed a dense population of BK channels insensitive to hypoxia. The BK channels were highly activated by intracellular Ca2+ and the BKCO lithocholate; these responses require BKα-β1 subunit coupling. PASMCs of CH rats with established PH exhibited a profound overabundance of the dominant oxygen-insensitive BKα variant. Importantly, human BK (hBK) channels in PASMCs from human donor lungs also represented the oxygen-insensitive BKα variant activated by BKCOs. The hBK channels showed significantly enhanced Ca2+ sensitivity compared with rat BK channels. We conclude that rat BK and hBK channels in PASMCs are oxygen-insensitive BKα-β1 complexes highly sensitive to Ca2+ and the BKCO lithocholate. BK channels are overexpressed in PASMCs of a rat model of PH and may provide an abundant target for BKCOs designed to restore K+ efflux.

Differential efficacy of GoSlo-SR compounds on BKα and BKαγ1–4 channels

ABSTRACTLarge conductance, voltage and Ca2+ activated K+ channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases. GoSlo-SR compounds were designed to be potent and efficacious BK channel openers. Although their structural activity relationships, activation in both BKα and BKαβ channels and the hypothetical mode of action of these compounds has been studied in detail in recent years, their effectiveness to open the BKαγ channels still remains unexplored. In this study, we have examined the efficacy of 3 closely related GoSlo-SR openers, GoSlo-SR-5-6 (SR-5-6), GoSlo-SR-5-44 (SR-5-44) and GoSlo-SR-5-130 (SR-5-130) using inside out patches on BKα channels coexpressed with 4 different LRRC (γ1–4) subunits in HEK293 cells. Our data suggests that the activation effects due to SR-5-6 were not significantly affected in the presence of γ1–4 subunits. Interestingly, the effects of more efficacious BK channel opener SR-5-44 were altered by different γ subunits. In cells expressing BKα channels, the shift in V1/2 (ΔV1/2) induced by SR-5-44 (3 μM) was −76 ± 3 mV, whereas it was significantly reduced by ∼70 % in BKαγ1 channels (ΔV1/2= −23 ± 3, p < 0.001, ANOVA). In BKαγ2 channels the ΔV1/2 was −36 ± 1 mV, which was less than that observed in BKαγ3 and BKαγ4 channels where the ΔV1/2 was −47 ± 5 mV, and −82 ± 5 mV, respectively. Additionally, the excitatory effects of a ‘β specific’ BK channel opener, SR-5-130 were only partially restored in the patches containing BKαγ1–4 channels. Together this data highlights that subtle modifications in GoSlo-SR structures alter their effectiveness on BK channels with accessory γ subunits and this study might provide a scaffold for the development of more tissue specific BK channel openers.

Presynaptic BK channels control transmitter release: physiological relevance and potential therapeutic implications.

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 Ca(2+) sensitivity, voltage dependence and gating properties. Abundantly expressed in the CNS, they have the peculiar characteristic of being activated by both voltage and intracellular calcium rise. The increase in intracellular calcium via voltage-dependent calcium channels (Cav ) during spiking triggers conformational changes and BK channel opening. This narrows the action potential and induces a fast after-hyperpolarization that shuts calcium channels. The tight coupling between BK and Cav channels at presynaptic active zones makes them particularly suitable for regulating calcium entry and neurotransmitter release. While in most synapses, BK channels exert a negative control on transmitter release under basal conditions, in others they do so only under pathological conditions, serving as an emergency brake to protect against hyperactivity. In particular cases, by interacting with other channels (i.e. limiting the activation of the delayed rectifier and the inactivation of Na(+) channels), BK channels induce spike shortening, increase in firing rate and transmitter release. Changes in transmitter release following BK channel dysfunction have been implicated in several neurological disorders including epilepsy, schizophrenia, fragile X syndrome, mental retardation and autism. In particular, two mutations, one in the αand one in the β3 subunit, resulting in a gain of function have been associated with epilepsy. Hence, these discoveries have allowed identification of BK channels as new drug targets for therapeutic intervention.

The synthesis and BK channel-opening activity of N-acylaminoalkyloxime derivatives of dehydroabietic acid.

A series of N-acylaminoalkyloxime derivatives of dehydroabietic acid were synthesized and evaluated for BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKα channels. The structure-activity relationship study revealed that a non-covalent interaction between the S atom of the 2-thiophene and the carbonyl O atom may contribute to conformation restriction for interaction with the ion channel. This research could guide the design and synthesis of novel abietane-based BK channel opener.

Dual action of leptin on rest-firing and stimulated catecholamine release via phosphoinositide 3-kinase-driven BK channel up-regulation in mouse chromaffin cells.

Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect and, as such, exerts a relevant action on the adipo-adrenal axis. Leptin has a dual action on adrenal mouse chromaffin cells both at rest and during stimulation. At rest, the adipokine inhibits the spontaneous firing of most cells by enhancing the probability of BK channel opening through the phosphoinositide 3-kinase signalling cascade. This inhibitory effect is absent in db(-) /db(-) mice deprived of Ob receptors. During sustained stimulation, leptin preserves cell excitability by generating well-adapted action potential (AP) trains of lower frequency and broader width and increases catecholamine secretion by increasing the size of the ready-releasable pool and the rate of vesicle release. In conclusion, leptin dampens AP firing at rest but preserves AP firing and enhances catecholamine release during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release. Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect. Besides being expressed in the hypothalamus and hippocampus, leptin receptors (ObRs) are also present in chromaffin cells of the adrenal medulla. In the present study, we report the effect of leptin on mouse chromaffin cell (MCC) functionality, focusing on cell excitability and catecholamine secretion. Acute application of leptin (1nm) on spontaneously firing MCCs caused a slowly developing membrane hyperpolarization followed by complete blockade of action potential (AP) firing. This inhibitory effect at rest was abolished by the BK channel blocker paxilline (1μm), suggesting the involvement of BK potassium channels. Single-channel recordings in 'perforated microvesicles' confirmed that leptin increased BK channel open probability without altering its unitary conductance. BK channel up-regulation was associated with the phosphoinositide 3-kinase (PI3K) signalling cascade because the PI3K specific inhibitor wortmannin (100nm) fully prevented BK current increase. We also tested the effect of leptin on evoked AP firing and Ca(2+) -driven exocytosis. Although leptin preserves well-adapted AP trains of lower frequency, APs are broader and depolarization-evoked exocytosis is increased as a result of the larger size of the ready-releasable pool and higher frequency of vesicle release. The kinetics and quantal size of single secretory events remained unaltered. Leptin had no effect on firing and secretion in db(-) /db(-) mice lacking the ObR gene, confirming its specificity. In conclusion, leptin exhibits a dual action on MCC activity. It dampens AP firing at rest but preserves AP firing and increases catecholamine secretion during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release.

Properties of the high‐conductance Ca2+‐activated K+ (BK) channel in pulmonary arterial smooth muscle cells

Pulmonary hypertension (PH) is associated with a loss of vasodilator influences in the pulmonary circulation, which results in pathogenic vasoconstriction. However, we observed an over‐expression of high‐conductance, Ca2+‐activated K+ (BK) channels in pulmonary arteries (PA) during chronic hypoxia (CH)‐induced PH, providing a potential vasodilator target. Here, we report that BK channels of PA smooth muscle cells (PASMCs) from CH rats retain high Ca2+‐sensitivity, α‐β1 subunit coupling that confers sensitivity to BK channel openers, and hypoxia‐insensitivity as compared to normal (N) rats (n=4‐11). Importantly, these properties may permit pharmacological activation of the BK channel even in a hypoxic environment. In inside‐out membrane patches used to record unitary BK currents in PASMCs from CH rats (n= 4‐7), the BK channel log(Ca2+ EC50(mol/L)) was ‐5.9 in both N and CH rats. Lithocholate (LCA), a BKβ1 subunit‐dependent opener, caused a 26‐fold and 19‐fold increase in BK channel open‐state probability in PASMC from CH and N rats, respectively. Using lung samples from organ donors without diagnosed PH, we also defined BK channel properties in human PASMCs. Similar to rat, the hBK channel exhibited high Ca2+‐sensitivity (log(Ca2+ EC50(mol/L)) = ‐6.1), was activated by LCA, and was not inhibited by acute hypoxia (n=4‐7). Thus, PASMCs from N and CH rats and humans without diagnosed lung disease express a β1 subunit‐coupled, hypoxia‐insensitive BK channel, which is responsive to BK channel openers.Supported by AHA 13PRE17240055 (NDD), NIH UL1RR029884 (NDD), and the UAMS College of Medicine Dean's Society.

Effects of the novel BK (KCa 1.1) channel opener GoSlo-SR-5-130 are dependent on the presence of BKβ subunits.

GoSlo-SR compounds are efficacious BK (KCa 1.1) channel openers, but little is known about their mechanism of action or effect on bladder contractility. We examined the effects of two closely related compounds on BK currents and bladder contractions. A combination of electrophysiology, molecular biology and synthetic chemistry was used to examine the effects of two novel channel agonists on BK channels from bladder smooth muscle cells and in HEK cells expressing BKα alone or in combination with either β1 or β4 subunits. GoSlo-SR-5-6 shifted the voltage required for half maximal activation (V1/2 ) of BK channels approximately -100 mV, irrespective of the presence of regulatory β subunits. The deaminated derivative, GoSlo-SR-5-130, also shifted the activation V1/2 in smooth muscle cells by approximately -100 mV; however, this was reduced by ∼80% in HEK cells expressing only BKα subunits. When β1 or β4 subunits were co-expressed with BKα, efficacy was restored. GoSlo-SR-5-130 caused a concentration-dependent reduction in spontaneous bladder contraction amplitude and this was abolished by iberiotoxin, consistent with an effect on BK channels. GoSlo-SR-5-130 required β1 or β4 subunits to mediate its full effects, whereas GoSlo-SR-5-6 worked equally well in the absence or presence of β subunits. GoSlo-SR-5-130 inhibited spontaneous bladder contractions by activating BK channels. The novel BK channel opener, GoSlo-SR-5-130, is approximately fivefold more efficacious on BK channels with regulatory β subunits and may be a useful scaffold in the development of drugs to treat diseases such as overactive bladder.

Molecular mechanisms underlying the effect of the novel BK channel opener GoSlo: Involvement of the S4/S5 linker and the S6 segment

GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 μM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan-Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.