The smooth muscle-type β1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels

Large-conductance Ca²⁺-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC₄(3) does not depend, as initially proposed, on the β₁ or β₄ subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the β₁ subunit is not essential to this activation but exerts a large potentiating effect. DiBAC₄(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca²⁺. K_d values for BK channel activation by DiBAC₄(3) in 0 mM Ca²⁺ are approximately 20 μM (α) and 5 μM (α+β₁), and G-V curves shift up to −40mV and −110 mV, respectively. β₁ to β₂ mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the β₁ subunit in cardiovascular pharmacology.     

Arachidonic acid effect on the allosteric gating mechanism of BK (Slo1) channels associated with the β1 subunit

Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca²⁺- and voltage-dependent K⁺ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary β1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with β1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca²⁺ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the β1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary β1 subunit.     

The BK channel accessory β1 subunit determines alcohol-induced cerebrovascular constriction

Ethanol-induced inhibition of myocyte large conductance, calcium- and voltage-gated potassium (BK) current causes cerebrovascular constriction, yet the molecular targets mediating EtOH action remain unknown. Using BK channel-forming (cbv1) subunits from cerebral artery myocytes, we demonstrate that EtOH potentiates and inhibits current at lower and higher than ∼15 μM, respectively. By increasing cbv1’s apparent -sensitivity, accessory BK β₁ subunits shift the activation-to-inhibition crossover of EtOH action to <3 μM , with consequent inhibition of current under conditions found during myocyte contraction. Knocking-down KCNMB1 suppresses EtOH-reduction of arterial myocyte BK current and vessel diameter. Therefore, BK β₁ is the molecular effector of alcohol-induced BK current inhibition and cerebrovascular constriction.     

The calcium channel β2 (CACNB2) subunit repertoire in teleosts

Background  

Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology [1]. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility [2]. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts.     

The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit.     

Are dipyridamole (sensitive) calcium channels present in esophageal smooth muscle?

Calcium (Ca²⁺) entry from the extra-cellular space into the cytoplasm through voltage-dependent Ca²⁺ channels, specifically dipyridamole (DHP) sensitive ones (L-type), control a variety of biological processes, including excitation-contraction coupling in vascular and GI muscle cells. It has also been proposed that these channels may control esophageal contractility. However, DHP-sensitive Ca²⁺ channels in esophagus have not been well characterized biochemically. Thus, it is not known if these channels are similar in number or affinity to those in vascular or neural tissues — organs for which clinical use of calcium channel blockers has been successful. Thus, the purpose of this study was to identify and characterize DHP-sensitive calcium channels in esophagus and compare them to vascular, neural, and other GI tissues. Methods — We carried out in vitro receptor binding assays on lower esophageal muscle homogenates, gastric and intestinal and colonic homogenates, and aortic muscle homogenates from ca; and on brain homogenates from rat. We used a radio-labeled dihydropyridine derivative [³H]nitrendipine, to label these sites and co-administration of unlabeled nimodipine to define specific binding. Results — As expected, ligand binding to L-type Ca²⁺ channels in aortic vascular smooth muscle and brain was readily detectable: brain, Bmax = 252 fmol/mg protein, Kd = 0.88 nM; aorta, Bmax = 326 fmol/mg protein, Kd = 0.84 nM. For esophagus (Bmax = 97; Kd = 0.73) and for other GI tissues, using the same assay conditions, we detected a smaller signal, suggesting that L-type Ca²⁺ channels are present in lower quantities. Conclusion — L-type Ca²⁺ channel are present in esophagus and in other GI muscles, their affinity is similar, but their density is relatively sparse. These findings are consistent with the relatively limited success that has been experienced clinically in the use of calcium channel blockers for treatment of esophageal dysmotility.     

IL-1β modulate the Ca2+-activated big-conductance K channels (BK) via reactive oxygen species in cultured rat aorta smooth muscle cells

The large conductance Ca²⁺-activated K⁺ (BK) channel, abundantly expressed in vascular smooth muscle cells, plays a critical role in controlling vascular tone. Activation of BK channels leads to membrane hyperpolarization and promotes vasorelaxation. BK channels are activated either by elevation of the intracellular Ca²⁺ concentration or by membrane depolarization. It is also regulated by a diversity of vasodilators and vasoconstrictors. Interleukin-1β (IL-1β) is one of the cytokines that play important roles in the development and progression of a variety of cardiovascular diseases. The effects of IL-1β on vascular reactivity are controversial, and little is known about the modulation of BK channel function by IL-1β. In this study, we investigated how IL-1β modulates BK channel function in cultured arterial smooth muscle cells (ASMCs), and examined the role of H₂O₂ in the process. We demonstrated that IL-1β had biphasic effects on BK channel function and membrane potential of ASMCs, that is both concentration and time dependent. IL-1β increased BK channel-dependent K⁺ current and hyperpolarized ASMCs when applied for 30 min. While long-term (24–48 h) treatment of IL-1β resulted in decreased expression of α-subunit of BK channel, suppressed BK channel activity, decreased BK channel-dependent K⁺ current and depolarization of the cells. H₂O₂ scavenger catalase completely abolished the early effect of IL-1β, while it only partly diminished the long-term effect of IL-1β. These results may provide important molecular mechanisms for therapeutic strategies targeting BK channel in inflammation-related diseases.     

PACAP stimulates insulin secretion by PAC1 receptor and ion channels in β-cells

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays a crucial role in the endocrine system. The present study aimed to investigate the effect of PACAP38 on insulin secretion and the underlying mechanism in rat pancreatic β-cells. The insulin secretion results showed that PACAP38 stimulated insulin secretion in a glucose- and dose-dependent manner. The insulinotropic effect was mediated by PAC₁ receptor, but not by VPAC₁ and VPAC₂ receptors. Inhibition of adenylyl cyclase and protein kinase A suppressed PACAP38-augmented insulin secretion. Glucose-regulated insulin secretion is dependent on a series of electrophysiological activities. Current-clamp technology suggested that PACAP38 prolonged action potential duration. Voltage-clamp recordings revealed that PACAP38 blocked voltage-dependent potassium currents, and this effect was reversed by inhibition of PAC₁ receptor, adenylyl cyclase, or protein kinase A. Activation of Ca²⁺ channels by PACAP38 was also observed, which could be antagonized by the PAC₁ receptor antagonist. In addition, calcium-imaging analysis indicated that PACAP38 increased intracellular Ca²⁺ concentration, which was decreased by PAC₁ receptor antagonist. These findings demonstrate that PACAP38 stimulates glucose-induced insulin secretion mainly by acting on PAC₁ receptor, inhibiting voltage-dependent potassium channels, activating Ca²⁺ channels and increasing intracellular Ca²⁺ concentration. Further, PACAP blocks voltage-dependent potassium currents via the adenylyl cyclase/protein kinase A signaling pathway.     

Chronic hypoxia in cultured human podocytes inhibits BKCa channels by upregulating its β4-subunit

Acyl-CoA synthetase (ACS4) is an arachidonate preferring acyl-CoA synthetase and has been shown to play an important role in lipid metabolism. Although arachidonate and decosahexanoate play a key role in lipid metabolism and in the brain, the mechanisms of its action are still not clearly understood. In this study, we identified brain-type ACS4 mRNA by 5'-RACE. We also confirmed that the mRNA in TT2 ES cells encoded a novel ACS4 protein, which contains 41 amino acids at its N-terminus. Furthermore, we found that ACS4 proteins were up-regulated in PC-12 cell by stimulation with nerve growth factor (NGF). Importantly, knockout of ACS4 in ES cells markedly attenuated neuronal differentiation induced by NGF and all-trans retinoic acids (RA). These results indicate that the ACS4 protein specifically expressed in brain plays an important role in arachidonate metabolism and neuronal differentiation in the brain.     

The p101 subunit of PI3Kγ restores activation by Gβ mutants deficient in stimulating p110γ

G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gβγ complexes. We have demonstrated previously that Gβγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gβ residues responsible for the Gβγ-PI3Kγ interaction, we examined Gβ1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gβ residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gβ1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.     

The antimicrobial peptide,human β-defensin-1, potentiates in vitro osteoclastogenesis via activation of the p44/42 mitogen-activated protein kinases

Previous studies have demonstrated increased expression and raised levels of human β-defensin (hBD)-1 in gingival tissue and crevicular fluid of patients with chronic periodontitis and peri-implantitis, oral bone-resorbing diseases caused by enhanced osteoclastogenesis. Therefore, we aimed to investigate the effect of hBD-1 on osteoclast formation and function and to elucidate the involved signaling pathway in vitro. Human peripheral blood mononuclear cells (PBMCs) were first incubated with various doses of hBD-1 and cell viability was assayed by MTT. PBMCs were treated with macrophage-colony stimulating factor and receptor activator of nuclear factor kappa-B ligand (RANKL) in the presence or absence of non-toxic doses of hBD-1. In vitro osteoclastogenesis was analyzed by tartrate-resistant acid phosphatase (TRAP) staining, osteoclast-specific gene expression, and a resorption pit assay. Involvement of mitogen-activated protein kinases (MAPKs) was studied by immunoblotting and specific MAPK inhibitors. HBD-1 potentiated induction of in vitro osteoclastogenesis by RANKL, as shown by significantly increased number of TRAP-positive multinuclear cells and resorption areas on the dentin slices, and further up-regulated expressions of osteoclast-specific genes compared to those by RANKL treatment (p < 0.05). However, hBD-1 treatment without RANKL failed to induce formation of osteoclast-like cells. A significant and further increase in transient phosphorylation of the p44/42 MAPKs was demonstrated by hBD-1 co-treatment (p < 0.05), consistent with the inhibitory effect by pretreatment with U0126 or PD98059 on hBD-1-enhanced osteoclastogenesis. Collectively, hBD-1 potentiates the induction of in vitro osteoclastogenesis by RANKL via enhanced phosphorylation of the p44/42 MAPKs.     

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing.     

The activation of CD99 inhibits cell-extracellular matrix adhesion by suppressing β1 integrin affinity

CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with Mn(2+) or by β(1) integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated β(1) integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the β(1) stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matrix adhesion by suppressing β(1) integrin affinity. [BMB reports 2012; 45(3): 159-164].     

Hypoxia potentiates LPS-induced inflammatory response and increases cell death by promoting NLRP3 inflammasome activation in pancreatic β cells

Hypoxia and islet inflammation are involved in β-cell failure in type 2 diabetes (T2D). Elevated plasma LPS levels have been verified in patients with T2D, and hypoxia occurs in islets of diabetic mice. Activation of inflammasomes in ischemic or hypoxic conditions was identified in various tissues. Here, we investigated whether hypoxia activates the inflammasome in β cells and the possible mechanisms involved. In mouse insulinoma cell line 6 (MIN6), hypoxia (1% O₂) primes the NLRP3 inflammasome along with NF-κB signaling activation. Our results demonstrate that hypoxia can activate the NLRP3 inflammasome in LPS-primed MIN6 to result in initiating the β cell inflammatory response and cell death in vitro. Reactive oxygen species (ROS) and the thioredoxin-interacting protein (TXNIP) are up-regulated in response to hypoxia. Finally, the role of the ROS-TXNIP axis in mediating the activation of the NLRP3 inflammasome and cell death was characterized by pretreating with the ROS scavenger N-acetylcysteine (NAC) and performing TXNIP knockdown experiments in MIN6. Our data indicate for the first time that the inflammasome is involved in the inflammatory response and cell death in hypoxia-induced β cells through the ROS-TXNIP-NLRP3 axis in vitro. This provides new insight into the relationship between hypoxia and inflammation in T2D.     

A novel role of proteasomal β1 subunit in tumorigenesis

p27^Kip1 is a key cell-cycle regulator whose level is primarily regulated by the ubiquitin–proteasome degradation pathway. Its β1 subunit is one of seven β subunits that form the β-ring of the 20S proteasome, which is responsible for degradation of ubiquitinated proteins. We report here that the β1 subunit is up-regulated in oesophageal cancer tissues and some ovarian cancer cell lines. It promotes cell growth and migration, as well as colony formation. β1 binds and degrades p27^Kip1directly. Interestingly, the lack of phosphorylation at Ser¹⁵⁸ of the β1 subunit promotes degradation of p27^Kip1. We therefore propose that the β1 subunit plays a novel role in tumorigenesis by degrading p27^Kip1.     

β-estradiol activates BK(Ca) in mesenteric artery smooth muscle cells of post-menopause women

The aim of the present study was to study the effect of β-estradiol (β-E(2)) on the large-conductance Ca(2+)-activated potassium (BK(Ca)) channel in mesenteric artery smooth muscle cells (SMCs). The mesenteric arteries were obtained from post-menopause female patients with abdominal surgery, and the SMCs were isolated from the arteries using an enzymatic disassociation. According to the sources, the SMCs were divided into non-hypertension (NH) and essential hypertension (EH) groups. Single channel patch clamp technique was used to investigate the effect of β-E(2) and ICI 182780 (a specific blocker of estrogen receptor) on BK(Ca) in the SMCs. The results showed the opening of BK(Ca) in the SMCs was voltage and calcium dependent, and could be blocked by IbTX. β-E(2) (100 μmol/L) significantly increased open probability (Po) of BK(Ca) in both NH and EH groups. After β-E(2) treatment, NH group showed higher Po of BK(Ca) compared with EH group. ICI 182780 could inhibit the activating effect of β-E(2) on BK(Ca) in no matter NH or EH groups. These results suggest β-E(2) activates BK(Ca) in mesenteric artery SMCs from post-menopause women via estrogen receptor, but hypertension may decline the activating effect of β-E(2) on BK(Ca).     

Regulation of voltage-gated K channels by glucose metabolism in pancreatic β-cells

Regulation of delayed rectifier-type K⁺ channels (Kv-channels) by glucose was studied in rat pancreatic β-cells. The Kv-channel current was increased in amplitudes by increasing glucose concentration from 2.8 to 16.6 mM, while it was decreased by 2.8 mM glucose in a reversible manner (down-regulation) in both perforated and conventional whole-cell modes. The current was decreased by FCCP, intrapipette 0 mM ATP or AMPPNP. Glyceraldehyde, pyruvic acid, 2-ketoisocaproic acid, and 10 mM MgATP prevented the down-regulation induced by 2.8 mM or less glucose. The residual current after treatment with Kv2.1-specific blocker, guangxitoxin-1E, was unchanged by lowering or increasing glucose concentration. We conclude that glucose metabolism regulates Kv2.1 channels in rats β-cells via altering MgATP levels.     

Poly(ADP-ribose) polymerase 1 is indispensable for transforming growth factor-β Induced Smad3 activation in vascular smooth muscle cell

Background

Transforming growth factor type-β (TGF-β)/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS) generation also mediates TGF-β signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose) polymerase 1 (PARP1), a downstream effector of ROS, on TGF-β signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs).

Methods and Results

TGF-β1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-β1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB) or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetami (PJ34), or PARP1 siRNA. TGF-β1 treatment promoted poly(ADP-ribosy)lation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosy)lation enhanced Smad-Smad binding element (SBE) complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-β1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs.

Conclusions

PARP1 is indispensable for TGF-β1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3 pathway.