Deletion of large-conductance calcium-activated potassium channels promotes vascular remodelling through the CTRP7-mediated PI3K/Akt signaling pathway.

The large-conductance calcium-activated potassium (BK) channel is a critical regulator and potential therapeutic target of vascular tone and architecture, and abnormal expression or dysfunction of this channel is linked to many vascular diseases. Vascular remodelling is the early pathological basis of severe vascular diseases. Delaying the progression of vascular remodelling can reduce cardiovascular events, but the pathogenesis remains unclear. To clarify the role of BK channels in vascular remodelling, we use rats with BK channel α subunit knockout (BK α ‒/‒). The results show that BK α ‒/‒ rats have smaller inner and outer diameters, thickened aortic walls, increased fibrosis, and disordered elastic fibers of the aortas compared with WT rats. When the expression and function of BK α are inhibited in human umbilical arterial smooth muscle cells (HUASMCs), the expressions of matrix metalloproteinase 2 (MMP2), MMP9, and interleukin-6 are enhanced, while the expressions of smooth muscle cell contractile phenotype proteins are reduced. RNA sequencing, bioinformatics analysis and qPCR verification show that C1q/tumor necrosis factor-related protein 7 ( CTRP7) is the downstream target gene. Furthermore, except for that of MMPs, a similar pattern of IL-6, smooth muscle cell contractile phenotype proteins expression trend is observed after CTRP7 knockdown. Moreover, knockdown of both BK α and CTRP7 in HUASMCs activates PI3K/Akt signaling. Additionally, CTRP7 is expressed in vascular smooth muscle cells (VSMCs), and BK α deficiency activates the PI3K/Akt pathway by reducing CTRP7 level. Therefore, we first show that BK channel deficiency leads to vascular remodelling. The BK channel and CTRP7 may serve as potential targets for the treatment of cardiovascular diseases.

Glucose fluctuations promote vascular BK channels dysfunction via PKCα/NF-κB/MuRF1 signaling.

Glucose fluctuations may contribute to large conductance calcium activated potassium (BK) channel dysfunction. However, the underlying mechanisms remain elusive. The aim of this study was to investigate the molecular mechanisms involved in BK channel dysfunction as a result of glucose fluctuations. A rat diabetic model was established through the injection of streptozotocin. Glucose fluctuations in diabetic rats were induced via consumption and starvation. Rat coronary arteries were isolated and coronary vascular tensions were measured after three weeks. Rat coronary artery smooth muscle cells were isolated and whole-cell BK channel currents were recorded using a patch clamp technique. Human coronary artery smooth muscle cells in vitro were used to explore the underlying mechanisms. After incubation with iberiotoxin (IBTX), the Δ tensions (% Max) of rat coronary arteries in the controlled diabetes mellitus (C-DM), the uncontrolled DM (U-DM) and the DM with glucose fluctuation (GF-DM) groups were found to be 84.46 ± 5.75, 61.89 ± 10.20 and 14.77 ± 5.90, respectively (P < .05), while the current densities of the BK channels in the three groups were 43.09 ± 4.35 pA/pF, 34.23 ± 6.07 pA/pF and 17.87 ± 4.33 pA/pF, respectively (P < .05). The Δ tensions (% Max) of rat coronary arteries after applying IBTX in the GF-DM rats injected with 0.9% sodium chloride (NaCl) (GF-DM + NaCl) and the GF-DM rats injected with N-acetyl-L-cysteine (NAC) (GF-DM + NAC) groups were found to be 8.86 ± 1.09 and 48.90 ± 10.85, respectively (P < .05). Excessive oxidative stress and the activation of protein kinase C (PKC) α and nuclear factor (NF)-κB induced by glucose fluctuations promoted the decrease of BK-ß1 expression, while the inhibition of reactive oxygen species (ROS), PKCα, NF-κB and muscle ring finger protein 1 (MuRF1) reversed this effect. Glucose fluctuations aggravate BK channel dysfunction via the ROS overproduction and the PKCα/NF-κB/MuRF1 signaling pathway.

Downregulation of AT2R decreases the responsiveness of BKCa channels to angiotensin II in patients with hypertension.

Angiotensin II (Ang II) modulates blood pressure via Ang II type 1 receptor (AT1R) and type 2 receptor (AT2R). The activation of AT2R relaxes vascular tone through opening large-conductance Ca2+-activated potassium (BKCa) channels in vascular smooth muscle cells (SMCs). In the present study, we studied the role of the AT2R-BKCa pathway in patients with hypertension. The mesenteric arterial SMCs (MSMCs) were obtained from normotensive patients (NP) and hypertensive patients (HP). BKCa currents were recorded with patch clamp and the expressions of mRNAs and proteins of AT1R/AT2R were analyzed by RT-PCR and Western blotting, respectively. Ang II significantly increased the macroscopic BKCa currents at the whole cell level, while increased the open probability and decreased the mean close time of BKCa channels at the single channel level with AT1R blockade by valsartan in NP. However, Ang II had no effect on the BKCa currents at the same condition in HP. Furthermore, the expressions of mRNA and protein of AT2R but not AT1R were markedly decreased in the MSMCs of HP compared to that of NP. The data suggest that AT2R is well functioned in the MSMCs in NP but not in HP and deficiency in the AT2R-BKCa pathway may contribute to the development of hypertension.

Modulation of the mitochondrial large-conductance calcium-regulated potassium channel by polyunsaturated fatty acids.

Polyunsaturated fatty acids (PUFAs) and their metabolites can modulate several biochemical processes in the cell and thus prevent various diseases. PUFAs have a number of cellular targets, including membrane proteins. They can interact with plasma membrane and intracellular potassium channels. The goal of this work was to verify the interaction between PUFAs and the most common and intensively studied mitochondrial large conductance Ca(2+)-regulated potassium channel (mitoBKCa). For this purpose human astrocytoma U87 MG cell lines were investigated using a patch-clamp technique. We analyzed the effects of arachidonic acid (AA); eicosatetraynoic acid (ETYA), which is a non-metabolizable analog of AA; docosahexaenoic acid (DHA); and eicosapentaenoic acid (EPA). The open probability (Po) of the channel did not change significantly after application of 10µM ETYA. Po increased, however, after adding 10µM AA. The application of 30µM DHA or 10µM EPA also increased the Po of the channel. Additionally, the number of open channels in the patch increased in the presence of 30µM EPA. Collectively, our results indicate that PUFAs regulate the BKCa channel from the inner mitochondrial membrane.

Caveolin-1 facilitates the direct coupling between large conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ channels and their clustering to regulate membrane excitability in vascular myocytes.

L-type voltage-dependent Ca(2+) channels (LVDCC) and large conductance Ca(2+)-activated K(+) channels (BKCa) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca(2+) release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BKCa activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BKCa and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BKCa by Ca(2+) influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BKCa co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BKCa can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BKCa and Cav1.2 but also the functional coupling between BKCa and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K(+) revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BKCa-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca(2+) dynamics including the negative feedback, to control the arterial excitability and contractility.

Empagliflozin Induces Vascular Relaxation in Rat Coronary Artery Due to Activation of BK Channels.

Purpose: The aim of this study was to investigate the effects and mechanisms of SGLT2 inhibitor empagliflozin on diabetic coronary function. Methods: A rat diabetic model was established by injection of streptozotocin. Rats in the treated group were administered empagliflozin by gavage and rat coronary vascular tensions were measured after eight weeks. Large conductance calcium activated K+ channel currents were recorded using a patch clamp technique, while human coronary artery smooth muscle cells were used to explore the underlying mechanisms. Results: After incubation with empagliflozin (10, 30, 100, 300, 1000 µmol/L), the Δ relaxation % of rat coronary arteries were 2.459 ± 1.304, 3.251 ± 1.119, 6.946 ± 3.407, 28.36 ± 11.47, 86.90 ± 3.868, respectively. Without and with empagliflozin in the bath solution, BK channel opening probabilities at a membrane potential of +60 mV were 0.0458 ± 0.0517 and 0.3413 ± 0.2047, respectively (p < 0.05, n = 4 cells). After incubation with iberiotoxin, the Δ tensions of rat coronary arteries in the control (Ctrl), untreated (DM), low empagliflozin (10 mg/kg/d)-treated (DM+L-EMPA) and high empagliflozin (30mg/kg/d)-treated (DM+H-EMPA) group were 103.20 ± 5.85, 40.37 ± 22.12, 99.47 ± 28.51, 78.06 ± 40.98, respectively (p < 0.01 vs Ctrl, n = 3-7; p < 0.001 vs DM+L-EMPA, n = 5-7). Empagliflozin restored high glucose-induced downregulation of Sirt1, Nrf2, and BK-ß1, while the effect of empagliflozin disappeared in the presence of EX-527, a Sirt1 selective inhibitor. Conclusion: Empagliflozin has a vasodilation effect on the coronary arteries in a concentration-dependent manner and can activate BK channels via the Sirt1-Nrf2 mechanism.

Coenzyme Q deficiency in endothelial mitochondria caused by hypoxia; remodeling of the respiratory chain and sensitivity to anoxia/reoxygenation.

This study examined the effects of hypoxia on coenzyme Q (Q) levels and mitochondrial function in EA. hy926 endothelial cells, shedding light on their responses to changes in oxygen levels. Chronic hypoxia during endothelial cell culture reduced Q synthesis by reducing hydroxy-methylglutaryl-CoA reductase (HMGCR) levels via hypoxia-inducible factor 1α (HIF1α), leading to severe Q deficiency. In endothelial mitochondria, hypoxia led to reorganization of the respiratory chain through upregulation of supercomplexes (I+III2+IV), forming a complete mitochondrial Q (mQ)-mediated electron transfer pathway. Mitochondria of endothelial cells cultured under hypoxic conditions showed reduced respiratory rates and membrane potential, as well as increased production of mitochondrial reactive oxygen species (mROS) as a result of increased mQ reduction levels (mQH2/mQtot). Anoxia/reoxygenation (A/R) in vitro caused impairment of endothelial mitochondria, manifested by reduced maximal respiration, complex III activity, membrane potential, coupling parameters, and increased mQ reduction and mROS production. Weaker A/R-induced changes compared to control mitochondria indicated better tolerance of A/R stress by the mitochondria of hypoxic cells. Moreover, in endothelial mitochondria, hypoxia-induced increases in uncoupling protein 3 (UCP3) and mitochondrial large-conductance Ca2+-activated potassium channel (mitoBKCa) levels and activities appear to have alleviated reoxygenation injury after A/R. These results not only highlight hypoxia-induced changes in mQ redox homeostasis and related mitochondrial function, but also indicate that chronic hypoxia during endothelial cell culture leads to mitochondrial adaptations that help mitochondria better withstand subsequent oxygen fluctuations.

Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway.

Background: Advanced glycation end products (AGEs) impair vascular physiology in Diabetes mellitus (DM). However, the underlying mechanisms remain unclear. Vascular large conductance calcium-activated potassium (BK) channels play important roles in coronary arterial function.Purpose: Our study aimed to investigate the regulatory role of AGEs in BK channels.Research Design: Using gavage of vehicle (V, normal saline) or aminoguanidine (A) for 8 weeks, normal and diabetic rats were divided into four groups: C+V group, DM+V group, C+A group, and DM+A group.Study Sample: Coronary arteries from different groups of rats and human coronary smooth muscle cells were used in this study.Data Collection and Analysis: Data were presented as mean ± SEM (standard error of mean). Student's t-test was used to compare data between two groups. One-way ANOVA with post-hoc LSD analysis was used to compare data between multiple groups.Results: Compared to the C+V group, vascular contraction induced by iberiotoxin (IBTX), a BK channel inhibitor, was impaired, and BK channel densities decreased in the DM+V group. However, aminoguanidine administration reduced the impairment. Protein expression of BK-ß1, phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein kinase B (PKB or Akt) were down-regulated, while F-box protein 32 (FBXO32) expression increased in the DM+V group and in high glucose (HG) cultured human coronary smooth muscle cells. Treatment with aminoguanidine in vitro and in vivo could reverse the above protein expression. The effect of aminoguanidine on the improvement of BK channel function by inhibiting the generation of AGEs was reversed by adding MK2206 (Akt inhibitor) or Compound C (AMPK inhibitor) in HG conditions in vitro.Conclusions: AGEs aggravate BK channel dysfunction via the AMPK/Akt/FBXO32 signaling pathway.

Propofol-induced vasodilation of mesenteric arterioles via BKCa channel and gap junction.

The present study aimed to investigate the role of propofol in mediating the vasomotor activity of the mesenteric arteriole (MA) of Sprague Dawley (SD) rats, and to elucidate the underlying mechanisms. The pressure myograph technique was used to examine the effect of different concentrations of propofol on the relaxation of blood vessels in the 2-3 mm MA segments freshly separated from the SD rats. The whole-cell patch-clamp technique was applied to observe the outward current of single vascular smooth muscle cells (VSMCs) obtained from the MAs of the SD rats. Furthermore, immunofluorescence was utilized to assess the expression of connexin (Cx) in the MAs of SD rats. The results indicated the following: i) Propofol relaxed the MA of SD rats in a concentration-dependent manner from 1×10-7 to 3×10-4 mol/l; ii) in the acutely dissociated VSMCs, propofol (1×10-7 to 3×10-4 mol/l) enhanced the outward current of VSMCs in a concentration-dependent manner; iii) the enhanced outward currents induced by propofol (1×10-5 mol/l) may be reversed by tetraethylammonium (TEA; 1 mmol/l), a calcium-activated K+ channel inhibitor; iv) the effect of propofol on the relaxation of the vasculature wAS reduced after perfusion with 1 mmol/l TEA; v) Cx40, Cx43 and Cx45 were expressed on the MA; 6) 18ß-glycyrrhetintic acid and 2-aminoethoxydiphenyl borate, two types of gap junction blocker, inhibited the propofol-induced relaxation. The present study provides evidence that propofol relaxes the MA, which may be associated with its effect of enhancing the channel current of large-conductance calcium voltage-activated potassium channels, contributing to the K+ outflow and leading to VSMC hyperpolarization; the gap junction may facilitate the hyperpolarization, which may lead to vascular synchronized relaxation and thereby reduce the blood pressure.

Chronic intermittent hypobaric hypoxia antagonizes renal vascular hypertension by enhancement of vasorelaxation via activating BKCa.

AIM: The purpose of the present study was to explore anti-hypertensive effect of chronic intermittent hypobaric hypoxia (CIHH) in renovascular hypertension (RVH) rats, as well as the role of large-conductance calcium-activated potassium channel (BKCa) in anti-hypertensive effect of CIHH. MAIN METHODS: Male adult age- and body weight-matched Sprague-Dawley rats were divided into SHAM, CIHH, RVH and RVH+CIHH groups. Hypertension was induced by two-kidney-1-clip method (2K1C) in RVH rats. CIHH rats were exposed to 28-days hypobaric hypoxia simulating 5000m altitude, 6h daily. SHAM rats got an operation without 2K1C, and RVH+CIHH rats received CIHH treatment after 2K1C. The endothelium-dependent vasorelaxation induced by acetylcholine (ACh), BKCa currents in smooth muscle cells (VSMCs) of mesenteric arteries and the protein expression of BKCa in mesenteric arteries was examined. KEY FINDINGS: The systolic arterial blood pressure (SAP) in RVH rats was higher than that in SHAM rats and CIHH treatment significantly decreased SAP in RVH rats. The enhanced vasorelaxation of mesenteric artery in CIHH-treated RVH rats was cancelled by BKCa blocker IBTX. The vasorelaxation induced by BKCa activator was reduced in RVH rats and the decreased vasorelaxation was improved by CIHH treatment. The ß1 subunit of BKCa in mesenteric artery was upregulated and BKCa current in VSMCs was increased in CIHH-treated RVH rats compared with RVH rats. SIGNIFICANCE: In conclusion, CIHH treatment enhances the relaxation of mesenteric artery through activation of BKCavia up-regulating ß1 subunit of BKCa, which might be one of mechanisms for anti-hypertensive effect of CIHH in RVH rats.

Improvement of spatial learning by facilitating large-conductance calcium-activated potassium channel with transcranial magnetic stimulation in Alzheimer's disease model mice.

Transcranial magnetic stimulation (TMS) is fragmentarily reported to be beneficial to Alzheimer's patients. Its underlying mechanism was investigated. TMS was applied at 1, 10 or 15 Hz daily for 4 weeks to young Alzheimer's disease model mice (3xTg), in which intracellular soluble amyloid-ß is notably accumulated. Hippocampal long-term potentiation (LTP) was tested after behavior. TMS ameliorated spatial learning deficits and enhanced LTP in the same frequency-dependent manner. Activity of the large conductance calcium-activated potassium (Big-K; BK) channels was suppressed in 3xTg mice and recovered by TMS frequency-dependently. These suppression and recovery were accompanied by increase and decrease in cortical excitability, respectively. TMS frequency-dependently enhanced the expression of the activity-dependently expressed scaffold protein Homer1a, which turned out to enhance BK channel activity. Isopimaric acid, an activator of the BK channel, magnified LTP. Amyloid-ß lowering was detected after TMS in 3xTg mice. In 3xTg mice with Homer1a knocked out, amyloid-ß lowering was not detected, though the TMS effects on BK channel and LTP remained. We concluded that TMS facilitates BK channels both Homer1a-dependently and -independently, thereby enhancing hippocampal LTP and decreasing cortical excitability. Reduced excitability contributed to amyloid-ß lowering. A cascade of these correlated processes, triggered by TMS, was likely to improve learning in 3xTg mice.

Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BK(Ca) channel modulators.

Development of a cell-based functional assay for large-conductance calcium-activated potassium (BK(Ca)) channels is challenging because of the unique requirement of both voltage and high concentrations of Ca²âº for activation of these channels. Here, we describe a new cell-based assay system that utilizes a hyperactive mutant BK(Ca) 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 BK(Ca) 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 Ca²âº. Unlike the wild-type BK(Ca) channel, the hyperactive mutant did not require a concomitant increase of intracellular Ca²âº for activation. Despite the observed shift in its voltage activation profile, activity of the mutant channel was further potentiated by a known BK(Ca) channel activator. When tested in a commercially available cell-based K⁺ channel assay, cell-lines stably expressing the hyperactive BK(Ca) channel generated a strong fluorescence signal under conditions that are typical for voltage-gated K⁺ channels. In summary, cell-lines expressing the hyperactive mutant BK(Ca) channel represent a new cell-based assay system for investigation of BK(Ca) channels that can be used to screen for novel modulators of these channels.