BK Ca Channel Activity Research Articles

Commentaries on Viewpoint: A paradigm shift for local blood flow regulation

# COMMENT ON “A PARADIGM SHIFT FOR LOCAL BLOOD FLOW REGULATION” {#article-title-2} to the editor: The proposed role of O2− produced by NAD(P)H oxidase acting in concert with changes in mitochondrial O2 on regulating local blood flow hypothesized by Golub and Pittman ([5][1]) could very well

Effects of Parathyroid Hormone–Related Peptide on the Large Conductance Calcium–Activated Potassium Channel and Calcium Homeostasis in Vascular Smooth Muscle Cells

Aim: To demonstrate the impact of the parathyroid hormone–related peptide (PTHrP) on the large conductance calcium–activated potassium (BKCa) channels in vascular smooth muscle cells (VSMC) and hyperpolarization of the cell membrane and its dependence on calcium. Materials and Methods: VSMC were isolated from rat aorta and further subcultured. Four experiments were conducted in calcium–release measurements and each of them consisted of a control group, PTHrP, chemical substance, and PTHrP + chemical substance. Chemical substances used were: iberiotoxin, xestospongin C, xestospongin D, and thapsigargin, respectively. Fura–2 imaging was used to determine changes in calcium release of VSMC. In membrane–potential experiments, groups were designed similarly to the Fura–2 imaging experiments: iberiotoxin, BAPTA, and xestospongin D were added, in respective order. Changes in the membrane potential were examined using the fluorescence dye (DiBAC). Results: Given in a dose between 0.01 and 1.0 μmol/L, PTHrP caused a concentration–dependent decrease in fluorescence intensity, with a maximum effect at 0.5 μmol/L. The decrease, therefore, demonstrated a PTHrP–induced hyperpolarization of the VSMC. The effect was blocked by use of iberiotoxin (100 nmol/L), a highly selective inhibitor of BKCa. Furthermore, when the calcium chelator BAPTA (10 μmol/L) was added, there was a significant reduction in PTHrP–induced hyperpolarization. Use of PTHrP (0.5 μmol/L) also decreased the fluorescence intensity of the indicator for intracellular calcium, Fura–2AM (a membrane–permeable derivative of Fura 2). This effect was re–blocked by use of iberiotoxin. Xestospongin C (3 μmol/L) and xestospongin D (6 μmol/L), both inhibitors of the inositol 1,4,5 trisphosphate–triggered calcium release, inhibited the effects of PTHrP. Additionally, thapsigargin (1 μmol/L), a sarcoplasmic/endoplasmic reticulum Ca2+–ATPase inhibitor, inhibited the effect of PTHrP. Conclusion: The results of our study show that PTHrP induces hyperpolarization and activates BKCa in VSMC. The activation of BKCa channels is calcium dependent; activation is linked to the inositol 1,4,5 trisphosphate–triggered calcium release and is also dependent on the endo/sarcoplasmic reticulum calcium pump.

Structure–activity relationship of Baifuzi-cerebrosides on BKCa channel activation

Our previous study reported that a mixture of cerebrosides from traditional Chinese medicine Baifuzi could activate BKCa channel. It is curious to know the effect of each single cerebroside on the channel. Here we isolated 5 pure cerebrosides from the mixture and determined their chemical structures. The most potent one increased the single channel open probability 6 folds with EC50 value of 1.0 μM. The structure–activity relationship revealed that acyl chain length of cerebrosides has potent effect, while configuration of double bond at C8–C9 on their long chain base has weak effect on the channel activity. Thus, this research provides a guide for design and synthesis of a lead cerebroside with more potent effect on the BKCa channel.

Functional activity of BK(Ca) channels in coronary vascular smooth muscle cells during combined exposure to hyperglycemia and stress.

The effect of tetraethylammonium in a dose of 1mM (inhibiting functional activity and preventing opening of BKCa channels) was least pronounced during restraint stress. The influence of this agent was more significant in animals exposed to 14-day hyperglycemia alone or in combination with restraint stress. Therefore, hyperglycemia and stress (individual or combined exposure) significantly inhibit functional activity of BKCa channels in smooth muscles of the coronary vessels. Our results suggest that the development of hyperglycemia is realized via pathogenetic mechanisms of vascular injury in the heart (similarly to stress conditions). Permanent increase in blood glucose level and 6-h immobilization probably induces nonspecific post-stress abnormalities in channel function.

Stochastic Modeling of Ca2+-Channel / BKCa-Channel Complexes

Over the last several years evidence has accumulated that indicates that in the brain BKCa-channel activity often affects the firing properties of neurons and in some cases the extent of neurotransmitter release. And it has become clear that BKCa channels often form molecular complexes with voltage-gated Ca2+ channels (CaV channels) such that, when a CaV channel is activated, the ensuing influx of Ca2+ activates its closely associated BKCa channel. Thus, in modeling the electrical properties of neurons it would be advantageous to have quantitative models of CaV/BKCa complexes. Furthermore, in a population of CaV/BKCa complexes, because all CaV channels are not open at the same time, all BKCa channels are not exposed to the same Ca2+ concentration at the same time. Thus stochastic rather than deterministic models are required to simulate the behavior of populations of CaV/BKCa complexes. To date however no such stochastic models have been described. To address this need we have recently developed a stochastic CaV/BKCa model that faithfully reproduces the behaviors of both channels in vitro. Here we describe this model and demonstrate its response to various action-potential-like stimuli. The predicted relationship between the activation of a CaV channel and the subsequent activation of its associated BK channel has been examined. We view our model as an important building block for the development of more complex models of neural function.

Arachidonic acid activation of BKCa (Slo1) channels associated to the β1-subunit in human vascular smooth muscle cells

Arachidonic acid (AA) is a polyunsaturated fatty acid involved in a complex network of cell signaling. It is well known that this fatty acid can directly modulate several cellular target structures, among them, ion channels. We explored the effects of AA on high conductance Ca(2+)- and voltage-dependent K(+) channel (BKCa) in vascular smooth muscle cells (VSMCs) where the presence of β1-subunit was functionally demonstrated by lithocholic acid activation. Using patch-clamp technique, we show at the single channel level that 10μM AA increases the open probability (Po) of BKCa channels tenfold, mainly by a reduction of closed dwell times. AA also induces a left-shift in Po versus voltage curves without modifying their steepness. Furthermore, AA accelerates the kinetics of the voltage channel activation by a fourfold reduction in latencies to first channel opening. When AA was tested on BKCa channel expressed in HEK cells with or without the β1-subunit, activation only occurs in presence of the modulatory subunit. These results contribute to highlight the molecular mechanism of AA-dependent BKCa activation. We conclude that AA itself selectively activates the β1-associated BKCa channel, destabilizing its closed state probably by interacting with the β1-subunit, without modifying the channel voltage sensitivity. Since BKCa channels physiologically contribute to regulation of VSMCs contractility and blood pressure, we used the whole-cell configuration to show that AA is able to activate these channels, inducing significant cell hyperpolarization that can lead to VSMCs relaxation.

Apelin-13 Inhibits Large-Conductance Ca2+-Activated K+ Channels in Cerebral Artery Smooth Muscle Cells via a PI3-Kinase Dependent Mechanism

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca2+-activated K+ (BKCa) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BKCa channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM) caused concentration-dependent inhibition of BKCa in VSM cells. Apelin-13 (0.1 µM) significantly decreased BKCa current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NPo) of BKCa channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1µM) did not alter the NPo of BKCa channels, suggesting that the inhibitory effect of apelin-13 on BKCa is not mediated by a direct action on BKCa. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BKCa current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BKCa channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.

BKCa channels expressed in sensory neurons modulate inflammatory pain in mice

Large conductance calcium-activated potassium (BKCa) channels are important regulators of neuronal excitability. Although there is electrophysiological evidence for BKCa channel expression in sensory neurons, their in vivo functions in pain processing have not been fully defined. Using a specific antibody, we demonstrate here that BKCa channels are expressed in subpopulations of peptidergic and nonpeptidergic nociceptors. To test a functional association of BKCa channel activity in sensory neurons with particular pain modalities, we generated mice in which BKCa channels are ablated specifically from sensory neurons and analyzed their behavior in various models of pain. Mutant mice showed increased nociceptive behavior in models of persistent inflammatory pain. However, their behavior in models of neuropathic or acute nociceptive pain was normal. Moreover, systemic administration of the BKCa channel opener, NS1619, inhibited persistent inflammatory pain. Our investigations provide in vivo evidence that BKCa channels expressed in sensory neurons exert inhibitory control on sensory input in inflammatory pain states.

Underlying mechanisms involved in progesterone-induced relaxation to the pig bladder neck

Progesterone increases bladder capacity and improves the bladder compliance by its relaxant action on the detrusor. A poor information, however, exists concerning to the role of this steroid hormone on the bladder outflow region contractility. This study investigates the progesterone-induced action on the smooth muscle tension of the pig bladder neck. To this aim, urothelium-denuded bladder neck strips were mounted in myographs for isometric force recordings and for simultaneous measurements of intracellular Ca2+ concentration ([Ca2+]i) and tension. On phenylephrine (PhE)-precontracted strips, progesterone produced concentration-dependent relaxations only at high pharmacological concentrations. The blockade of progesterone receptors, nitric oxide (NO) synthase, guanylyl cyclase, large conductance Ca2+-activated K+ (BKCa) or ATP-dependent K+ (KATP) channels reduced the progesterone relaxations. The presence of the urothelium and the inhibition of cyclooxygenase (COX), intermediate- and small-conductance Ca2+-activated K+ channels failed to modify these responses. In Ca2+-free potassium rich physiological saline solution, progesterone inhibited the contraction to CaCl2 and to the L-type voltage-operated Ca2+ (VOC) channel activator BAY-K 8644. Relaxation induced by progesterone was accompanied by simultaneous decreases in smooth muscle [Ca2+]i. These results suggest that progesterone promotes relaxation of pig bladder neck through smooth muscle progesterone receptors via cGMP/NO pathway and involving the activation of BKCa and KATP channels and inhibition of the extracellular Ca2+ entry through L-type VOC channels.

Abstract 18514: Rottlerin Greatly Improves Cardiac Functional Recovery Following Ischemic Cardioplegic Arrest (CP) in Wild-Type but Not BKCa Channel Knockout Mice

Introduction: Mitochondrial BKCa channels are thought to provide protection during ischemic insults in the heart. Rottlerin, has been implicated as a potent BKCa activator, however this has not been firmly established. The purpose of this study was twofold: 1) investigate the efficacy of BKCa channel activation as a cardioprotective agent during cardioplegia/reperfusion (CP/R) and 2) assess the specificity of rottlerin for BKCa channels. Methods: Wt and BKCa KO mice were subjected to an isolated heart model of CP/R. Hearts were perfused for 30 min at 37C (baseline), followed by intermittent cold hypoxic CP with or without rottlerin (St Thomas II, 10C), and normoxic 37C reperfusion for 30 min. Mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential (TMRE) and ROS (DCFDA) production. Results: CP/R decreased LVDP (61.6 +/- 6 % baseline, n=6 ), +/- dP/dt (76.6+/- 7.3, 63.4 +/-7.7, % baseline respectively) and coronary flow (CF) (69.6+/-6.0 % baseline) in wt mice. Rottlerin, (100 nM (n=3), 500 nM (n=6)) dose dependently increased the recovery of LV function and CF to near baseline levels (% baseline 500 nM rottlerin, LVDP: 87.6+/- 4.0, +/-dPdt: 97.6 +/- 5.9, 90.9+/- 6.6, and CF: 125.3 +/-22.8, all p&lt;.01 vs CP ). BKCa KO hearts treated with (n=7) or without (n=7) 500nM rottlerin, were similar to wt CP hearts (% baseline BKCa KO + 500 nM rott: LVDP: 66.9+/- 6.0, +/-dP/dt 73.0 +/-3.6, 65.2+/- 4.2, and CF: 94.1 +/- 9.0). H9c2 cells subjected to in vitro CP/R, displayed reduced mitochondrial membrane potential (39 +/-.09 % decrease) and increased ROS generation (50+/-.04% increase), both of which were significantly (p&lt;.01) normalized by rottlerin (100 nM-1 uM) (n=8). Conclusions: Activation of BKCa channels profoundly rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.

RETRACTED: Docosahexaenoic acid attenuates hypoxic pulmonary vasoconstriction by activating the large conductance Ca2+-activated K+ currents in pulmonary artery smooth muscle cells

The inhibition of potassium (K(+)) channels plays an important role in pulmonary circulation for its close relationship with hypoxic pulmonary vasoconstriction (HPV). Docosahexaenoic acid (DHA), a n-3 polyunsaturated fatty acid, is well known for its prevention and treatment of cardiovascular diseases. However the role which DHA plays in HPV remains unclear. Here, we tested the hypothesis that DHA contributes to pulmonary vascular tone by activating the large conductance Ca(2+)-activated K(+) (BKCa) channels via calcium sparks. Isolated resistance pulmonary artery preparation was used to study the vasomotor response to DHA. Pulmonary artery smooth muscle cells (PASMCs) were isolated from third- to fourth order branches of pulmonary arteries by collagenase digestion method. BKCa and the voltage-dependent potassium channel (Kv) currents in PASMCs were measured by the whole-cell patch-clamp technique. Fluo-8 was used as a fluorescence indicator for the real-time measurement of calcium dynamics in PASMCs. DHA dilated resistance pulmonary arteries in a dose-dependent manner in hypoxic or normoxic solution, and the effects of DHA were abolished after pre-treatment with heparin (100 μg/ml), a 1,4,5-triphosphate (IP3) receptor (IP3R) inhibitor or iberiotoxin (100 nmol/L), a specific inhibitor of BKCa channel. DHA activated BKCa channels in a dose-dependent manner, however, the activation induced by DHA was not seen in PASMCs pre-incubated with heparin. While the Kv currents decreased from 102.6 ± 5.4 to 36.5 ± 6.7 pA/pF by addition of 10 μmol/L DHA. DHA also caused calcium sparks in PASMCs. Moreover, hypoxia inhibited BKCa currents in PASMCs, but this inhibition was reversed by DHA. Our findings suggest that DHA is a novel BKCa opener in PASMCs, which may indicate a potential therapeutic role in HPV.

Brief Daily Episode of Normoxia Inhibits Cardioprotection Conferred by Chronic Continuous Hypoxia. Role of Oxidative Stress and BK&lt;sub&gt;Ca&lt;/sub&gt; Channels

The purpose of the present study was to assess the impact of brief daily reoxygenation during adaptation to chronic continuous hypoxia (CCH) on protective cardiac phenotype. Adult male Wistar rats were kept at CCH (10% oxygen) for 5, 15 or 30 days; a subgroup of animals was exposed to room air daily for a single 60-min period. While 5 days of CCH did not affect myocardial infarction induced by 20-min coronary artery occlusion and 3-h reperfusion, 15 days reduced infarct size from 62% of the area at risk in normoxic controls to 52%, and this protective effect was more pronounced after 30 days (41%). Susceptibility to ischemic ventricular arrhythmias exhibited reciprocal development. CCH increased myocardial abundance of mitochondrial superoxide dismutase (MnSOD) without affecting malondialdehyde concentration. Daily reoxygenation abolished both the infarct size-limiting effect of CCH and MnSOD upregulation, and increased malondialdehyde (by 53%). Ventricular cardiomyocytes isolated from CCH rats exhibited better survival and lower lactate dehydrogenase release caused by simulated ischemia/reperfusion than cells from normoxic and daily reoxygenated groups. The cytoprotective effects of CCH were attenuated by the large-conductance Ca2+-activated K+ (BKCa) channel blocker paxilline, while the opener NS1619 reduced cell injury in the normoxic group but not in the CCH group. Daily reoxygenation restored the NS1619- induced protection, whereas paxilline had no effect, resembling the pattern observed in the normoxic group. The results suggest that CCH is cardioprotective and brief daily reoxygenation blunts its salutary effects, possibly by a mechanism involving oxidative stress and attenuation of the activation of mitochondrial BKCa channels.

Chronic hypoxia during gestation enhances uterine arterial myogenic tone via heightened oxidative stress.

Chronic hypoxia during gestation has profound adverse effects on the adaptation of uteroplacental circulation in pregnancy. Yet, the underlying mechanisms are not fully understood. The present study tested the hypothesis that enhanced production of reactive oxygen species (ROS) in uterine arteries plays a critical role in the maladaptation of uterine circulation associated with chronic hypoxia. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (∼300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia significantly increased ROS production in uterine arteries of pregnant, but not nonpregnant, sheep. This was associated with a significant increase in NADPH oxidase (Nox) 2, but not Nox1 or Nox4, protein abundance and total Nox activity in uterine arteries of pregnant animals. Chronic hypoxia significantly increased pressure-dependent uterine arterial myogenic tone in pregnant sheep, which was abrogated by a Nox inhibitor apocynin. Additionally, the hypoxia-induced increase in myogenic reactivity of uterine arteries to phorbol 12,13-dibutyrate in pregnant sheep was blocked by apocynin and tempol. In consistence with the myogenic responses, the hypoxia-mediated down-regulation of BKCa channel activity in uterine arteries of pregnant animals was reversed by apocynin. The findings suggest that heightened oxidative stress in uterine arteries plays a key role in suppressing the BKCa channel activity, resulting in increased myogenic reactivity and maladaptation of uteroplacental circulation caused by chronic hypoxia during gestation.

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca2+-activated K+ channel (BKCa) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BKCa channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K+ currents (STOC, a component pattern of BKCa currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BKCa currents) via an IP3 receptor- and sarcoplasmic Ca2+ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BKCa channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BKCa channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BKCa channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca2+ sensitivity of the channels.

Abstract 335: Activation of BKCa Channels With Rottlerin Greatly Improves Cardiac Functional Improvement Following Surgical Ischemia Associated With Cardioplegic Arrest.

Introduction: BKCa channels are thought to provide protection during ischemic insults in the heart. Rottlerin, has been implicated as a potent BKCa activator, however this has not been firmly established. The purpose of this study was two fold: 1) assess the specificity of rottlerin for BKCa channels and 2) investigate the efficacy of BKCa channel activation as a cardioprotective agent during cardioplegia/reperfusion (CP/R). Methods: Wild type and BKCa knock-out mice with and without rottlerin (only administered in CP) were subjected to an isolated heart model of CP/R. Hearts were perfused for 30 min at 37C (baseline), followed by intermittent cold crystalloid cardioplegia (intermittent St Thomas II, 10C), and normothermic, normoxic, reperfusion for 30 min. In additional studies, mechanism of rottlerin-induced cardioprotection was investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential (TMRE) and ROS (DCFDA) production. Results: CP and 30 min reperfusion decreased LVDP (58.7 +/- 5 % baseline, n=8 ), +/- dP/dt ( 71.1+/- 6.4, 59.2 +/-6.6, % baseline respectively) and coronary flow (CF) (66.8+/-7.4, %baseline) in wild type mice. Rottlerin, (100 (n=3), 500 nM (n=5)) delivered in the CP solution dose dependently increased the recovery of LV function and CF to near baseline levels (500 nM rottlerin, LVDP: 94.1+/- 2.3, +/-dPdt: 99.6 +/- 7.2, 111.0+/- 15.0, % baseline, and CF: 128.9 +/-30.0 %baseline ). BKCa KO hearts treated with (n=4)or without (n=3) 500nM rottlerin, were similar to wt CP hearts, showing no improved cardioprotection (BKCa KO + 500 nM rott: LVDP: 57.7+/- 4.0, +/-dP/dt 69.5 +/-12.0, 59.8+/- 4.9, and CF 73 +/- 6.7). H9c2 cells subjected to in vitro CP/R, displayed reduced mitochondrial membrane potential (39% +/-.09 % decrease relative to control, p&lt;.01) and increased ROS generation (50%+/-.04 increase, p&lt;.01), both of which were dose dependently normalized by rottlerin (100 nM-1 uM) (minimum n=8). Conclusions: Activation of BKCa channels profoundly rescues ischemic damage associated with CP, likely via mitochondrial effects on improved mitochondrial membrane potential and reduced ROS generation.

Oxygen Sensing by the Carotid Body: Is It All Just Rotten Eggs?

Ventilatory responses to hypoxia are initiated by the carotid body, where inhibition of specific K(+) channels causes cell depolarization, voltage-gated Ca(2+) influx, and neurotransmitter release. The identity of the upstream oxygen (O2) sensor is still controversial. The activity of BKCa channels is regulated by O2, carbon monoxide (CO), and hydrogen sulfide (H2S), suggesting that integration of these signals may be crucial to the physiological response of this tissue. BKCa is colocalized with hemeoxygenase-2, an enzyme that generates CO in the presence of O2, and CO is a BKCa channel opener. Reduced CO during hypoxia results in channel closure, conferring a degree of O2 sensitivity to the BKCa channel. Conversely, H2S is a potent BKCa inhibitor. H2S is produced endogenously by cystathionine-β-synthase and cystathionine-γ-lyase in the rat carotid body, and its intracellular concentration is dependent upon the balance between its enzymatic generation and its mitochondrial breakdown. During hypoxia, mitochondrial oxidation of H2S in many tissues is reduced, leading to hypoxia-evoked rises in its concentration. This may be sufficient to inhibit K(+) channels and lead to carotid body excitation. Carotid body function is heavily dependent upon regulated production and breakdown of CO and H2S and integration of signals from these newly emerging gasotransmitters, in combination with several other proposed mechanisms, may refine, or even define, responses of this tissue to hypoxia. Since several other sensors have been postulated, the challenge of future research is to begin to integrate each in a unifying mechanism, as has been attempted for the first time herein.

Effects of aerobic exercise training on large-conductance Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

Large-conductance Ca(2+)-activated K(+) (BKCa) channels provide a negative feedback that regulates vascular tone in the brain circulation. This study investigated the effects of aerobic exercise on gating properties of BKCa channels in rat cerebral artery. Rats were subjected to moderate-intensity exercise at low (EX-3d/w) and high (EX-5d/w) training volume on a motor-driven treadmill, and compared with age-matched sedentary animals (SED). Inside-out (I/O) patch clamp recording was performed to measure gating properties of the BKCa channel. Aerobic exercise induced a reduction in heart rate and body weight in both training groups. Exercise increased the channel activity, which was more pronounced in EX-5d/w than that in EX-3d/w group. Kinetic analysis revealed that (1) the contribution of short open states was elevated and the duration of both short and long open states were extended by exercising in EX-3d/w; (2) Ex-3d/w had no significant change on conformation of close states; (3) EX-3d/w increased the mean open time without changing mean closed time; (4) EX-5d/w increased both the contribution and duration of long open states; (5) EX-5d/w increased channel mean open time while decreased mean closed time. The results suggest that regular aerobic exercise may enhance BKCa channel activity in cerebral arterial myocytes by changing its biophysical properties, and the electrical remolding induced by exercise may be training volume-dependent.

Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BKCa channel modulators

Development of a cell-based functional assay for large-conductance calcium-activated potassium (BKCa) channels is challenging because of the unique requirement of both voltage and high concentrations of Ca2+ for activation of these channels. Here, we describe a new cell-based assay system that utilizes a hyperactive mutant BKCa channel. The hyperactive mutant was generated by introducing two point-mutations into the cytosolic flexible interface between the two RCK domains of the wild-type BKCa channel. The mutant channel exhibited a large negative shift in its conductance–voltage relationship, which indicates activation by modest depolarization at resting concentrations of intracellular Ca2+. Unlike the wild-type BKCa channel, the hyperactive mutant did not require a concomitant increase of intracellular Ca2+ for activation. Despite the observed shift in its voltage activation profile, activity of the mutant channel was further potentiated by a known BKCa channel activator. When tested in a commercially available cell-based K+ channel assay, cell-lines stably expressing the hyperactive BKCa channel generated a strong fluorescence signal under conditions that are typical for voltage-gated K+ channels. In summary, cell-lines expressing the hyperactive mutant BKCa channel represent a new cell-based assay system for investigation of BKCa channels that can be used to screen for novel modulators of these channels.

Smooth muscle relaxation and activation of the large conductance Ca++ – activated K+ (BKCa) channel by novel oestrogens

Oestrogens can interact directly with membrane receptors and channels and can activate vascular BK(Ca) channels. We hypothesized that novel oestrogen derivatives could relax smooth muscle by an extracllular effect on the α and β1 subunits of the BK(Ca) channel, rather than at an intracellular site. We studied the effects of novel oestrogens on the tension of pre-contracted isolated rat aortic rings, and on the electrophysiological properties of HEK 293 cells expressing the hSloα or hSloα+β1 subunits. Two of the derivatives incorporated a quaternary ammonium side-chain making them membrane impermeable. Oestrone, oestrone oxime and Quat DME-oestradiol relaxed pre-contracted rat aorta, but only Quat DME-oestradiol-induced relaxation was iberiotoxin sensitive. However, only potassium currents recorded in HEK 293 cells over-expressing both hSloα and hSloβ1 were activated by oestrone, oestrone oxime and Quat DME-oestradiol. The novel oestrogens were able to relax smooth muscle, but through different mechanisms. In particular, oestrone oxime required the presence of the endothelium to exert much of its effect, whilst Quat DME-oestradiol depended both on NO and BK(Ca) channel activation. The activation of BK(Ca) currents in HEK 293 cells expressing hSloα+β1 by Quat DME-oestradiol is consistent with an extracellular binding site between the two subunits. The binding site resides between the extracellular N terminal of the α subunit and the extracellular loop between TM1 and 2 of the β1 subunit. Membrane-impermeant Quat DME-oestradiol lacks an exchangeable hydrogen on the A ring obviating antioxidant activity.

N‐arachidonoyl glycine suppresses Na+/Ca2+ exchanger‐mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and PurposeN-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly's action on unstimulated and agonist-stimulated endothelial cells.Experimental ApproachThe effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.Key ResultsIn EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na+/Ca2+ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs+-based Na+-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na+ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca2+]i elevation evoked by Na+ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca2+-activated K+ (BKCa) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BKCa channel activity.Conclusion and ImplicationsOur data showed that NCX is a Ca2+ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca2+ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BKCa channels.