KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Endogenous cannabinoid receptor agonist anandamide induces peripheral antinociception by activation of ATP-sensitive K+ channels

AimsThe effects of several potassium (K⁺) channel blockers were studied to determine which K⁺ channels are involved in peripheral antinociception induced by the cannabinoid receptor agonist, anandamide.Main methodsHyperalgesia was induced by subcutaneous injection of 250 μg carrageenan into the plantar surface of the hind paw of rats. The extent of hyperalgesia was measured using a paw pressure test 3 h following carrageenan injection. The weight in grams (g) that elicited a nociceptive response, paw flexion, during the paw pressure test was used as the nociceptive response threshold.Key findingsDoses of 50, 75, and 100 ng of anandamide elicited a dose-dependent antinociceptive effect. Following a 100 ng dose of anandamide no antinociception was observed in the paw that was contralateral to the anandamide injection site, which shows that anandamide has a peripheral site of action. Pretreatment with 20, 40 and 80 μg AM251, a CB₁ receptor antagonist, caused a dose-dependent decrease in anandamide-induced antinociception, suggesting that the CB₁ receptor is directly involved in anandamide effect. Treatment with 40, 80 and 160 μg glibenclamide, an ATP-sensitive K⁺ channel blocker, caused a dose-dependent reversal of anandamide-induced peripheral antinociception. Treatment with other K⁺ channel antagonists, tetraethylammonium (30 μg), paxilline (10 μg) and dequalinium (50 μg), had no effect on the induction of peripheral antinociception by anandamide.SignificanceThis study provides evidence that the peripheral antinociceptive effect of the cannabinoid receptor agonist, anandamide, is primarily caused by activation of ATP-sensitive K⁺ channels and does not involve other potassium channels.     

Functional validation of Ca2 +-binding residues from the crystal structure of the BK ion channel

BK channels are dually regulated by voltage and Ca^2 +, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca^2 + concentration is sensed by a large cytoplasmic region in the channel known as “gating ring”, which is formed by four tandems of regulator of conductance for K⁺ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca^2 + coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca^2 +. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca^2 + in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca^2 + sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca^2 + coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca^2 + binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca^2 + binding sites regulating the Ca^2 + dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.     

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K⁺ (Kv) channels.Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells.Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a K_d value of 1.48 μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM^? 1 s^? 1 and 2.59 ± 0.14 s^? 1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2 Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002.SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.     

Flow-induced activation of TRPV5 and TRPV6 channels stimulates Ca2 +-activated K+ channel causing membrane hyperpolarization

TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca^2 + reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP₂, protons, and divalent ions Ca^2 + and Mg^2 +. Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 min. Fluid flow stimulated TRPV5 and 6-mediated Ca^2 + entry and increased intracellular Ca^2 + concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La^3 +. In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K⁺ secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.     

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway

ObjectivesThis study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC).MethodsIsometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na⁺/K⁺-pump and Na⁺,K⁺,2Cl^-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the ⁸⁶Rb influx, respectively.ResultsNaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K⁺]_o=30 mM). At 10^?4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10^?3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10^?4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na⁺,K⁺,2Cl^-cotransport, whereas the inhibition seen at 10^?3 M NaHS was suppressed in the presence of K⁺ channel blocker TEA. In cultured SMC, 5×10^?5 M NaHS increased Na⁺,K⁺,2Cl^- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, ⁴⁵Ca influx was enhanced in the presence of 10^?4 M NaHS and suppressed under elevation of [NaHS] up to 10^?3 M. ⁴⁵Ca influx triggered by 10^?4 M NaHS was abolished by bumetanide and L-type Ca²⁺ channel blocker nicardipine.ConclusionsOur results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na⁺,K⁺,2Cl^-cotransport and Ca²⁺ influx via L-type channels.     

Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots

Saline environments of terrestrial halophytes are often prone to waterlogging, yet the effects on halophytes of combined salinity and waterlogging have rarely been studied. Either salinity or hypoxia (low O₂) alone can interfere with K⁺ homeostasis, therefore the combination of salinity or hypoxia is expected to impact significantly on K⁺ retention in roots. We studied mechanisms of tolerance to the interaction of salinity with hypoxia in Puccinellia ciliata and Thinopyrum ponticum, halophytic grasses that differ in waterlogging tolerance. Plants were exposed to aerated and stagnant saline (250 mM NaCl) treatments with low (0.25 mM) and high (4 mM) K⁺ levels; growth, net ion fluxes and tissue ion concentrations were determined. P. ciliata was more tolerant than T. ponticum to stagnant-saline treatment, producing twice the biomass of adventitious roots, which accumulated high levels of Na⁺, and had lower shoot Na⁺. After 24 h of saline hypoxic treatment, MIFE measurements revealed a net uptake of K⁺ (∼40 nmol m⁻² s⁻¹) for P. ciliata, but a net loss of K⁺ (∼20 nmol m⁻² s⁻¹) for the more waterlogging sensitive T. ponticum. NaCl alone induced K⁺ efflux from roots of both species, with channel blocker tests implicating GORK-like channels. P. ciliata had constitutively a more negative root cell membrane potential than T. ponticum (−150 versus −115 mV). Tolerance to salinity and hypoxia in P. ciliata is related to increased production of adventitious roots, regulation of shoot K⁺/Na⁺, and a superior ability to maintain negative membrane potential in root cells, resulting in greater retention of K⁺.     

Cerebral vasodilator properties of Danshen and Gegen: A study of their combined efficacy and mechanisms of actions

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED₅₀, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a K_ATP channel inhibitor glibenclamide, a non-selective K⁺ channel inhibitor tetraethylammonium (TEA), and by a combination of K⁺ channel inhibitors (glibenclamide + TEA + iberiotoxin + 4-aminopyridine + barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of K_ATP currents and inhibited Ca²⁺ influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl₂-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of K_ATP channels and inhibition of Ca²⁺ influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.     

Effects of potassium ion supplementation on survival and ion regulation in Gulf killifish Fundulus grandis larvae reared in ion deficient saline waters

Teleost fish often live in an environment in which osmoregulatory mechanisms are critical for survival and largely unknown in larval fish. The effects of a single important marine ion (K⁺) on survival and ion regulation of larval Gulf killifish, an estuarine, euryhaline teleost, were determined. A four-week study was completed in four separate recirculating systems with newly hatched larvae. Salinity in all four systems was maintained between 9.5 and 10‰. Two systems were maintained using crystal salt (99.6% NaCl) with K⁺ supplementation (1.31 ± 0.04 mmol/L and 2.06 ± 0.04 mmol/L K⁺; mean ± SEM), one was maintained with crystal salt and no K⁺ supplementation (0.33 ± 0.05 mmol/L K⁺), the fourth system was maintained using a standard marine mix salt (2.96 ± 0.04 mmol/L K⁺), the salt mix also included standard ranges of other ions such as calcium and magnesium. Larvae were sampled throughout the experiment for dry mass, Na⁺/K⁺-ATPase (NKA) activity, whole body ion composition, relative gene expression (NKA, Na⁺/K⁺/2Cl^? cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR)), and immunocytochemistry staining for NKA, NKCC, and CFTR. Larvae stocked into water with no K⁺ supplementation resulted in 100% mortality within 24 h. Mortality and dry mass were significantly influenced by K⁺ concentration (P ≤ 0.05). No differences were observed among treatment groups for NKA activity. At 1 dph NKA mRNA expression was higher in the 0.3 mmol [K⁺] group than in other treatment groups and at 7 dph differences in intestinal NKA and CFTR staining were observed. These data indicate that the rearing of larval Gulf killifish may be possible in ion deficient water utilizing specific ion supplementation.     

Endothelium-dependent and -independent vasorelaxation induced by CIJ-3-2F,a novel benzyl-furoquinoline with antiarrhythmic action,in rat aorta

AimsThis study was designed to examine the mechanism of relaxation induced by CIJ-3-2F, a benzyl-furoquinoline antiarrhythmic agent, in rat thoracic aorta at the tissue and cellular levels.Main methodsIsometric tension of rat aortic ring was measured in response to drugs. Ionic channel activities in freshly dissociated aortic vascular smooth muscle cells (VSMCs) were investigated using a whole-cell patch-clamp technique.Key findingsCIJ-3-2F relaxed both phenylephrine (PE) and high KCl (60 mM)-induced contractions with respective pEC₅₀ (-log EC₅₀) values of 6.91 ± 0.07 and 6.32 ± 0.06. Removal of endothelium or pretreatment with nitric oxide (NO)-pathway inhibitors N^ω-nitro-l-arginine methyl ester (L-NAME), N^G-monomethyl-l-arginine (L-NMMA), N⁵-(1-iminoethyl)-l-ornithine (L-NIO), hemoglobin, methylene blue or 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ) reduced the relaxant effect of CIJ-3-2F. Relaxation to CIJ-3-2F was also attenuated by K⁺ channel blockers tetraethylammonium (TEA) or 4-aminopyridine (4-AP), but not by charybdotoxin plus apamin, iberiotoxin, glibenclamide, or BaCl₂. CIJ-3-2F non-competitively antagonized the contractions induced by PE, Ca²⁺, and Bay K8644 in endothelium-denuded rings. In addition, CIJ-3-2F inhibited both the phasic and tonic contractions induced by PE but did not affect the transient contraction induced by caffeine. CIJ-3-2F reduced the Ba²⁺ inward current through L-type Ca²⁺ channel (IC₅₀ = 4.1 μM) and enhanced the voltage-dependent K⁺ (K_v) current in aortic VSMCs.SignificanceThese results suggest that CIJ-3-2F induced both endothelium-dependent and -independent vasorelaxation; the former is likely mediated by the NO/cGMP pathway whereas the latter is probably mediated through inhibition of Ca²⁺ influx or inositol 1,4,5-triphosphate (IP₃)-sensitive intracellular Ca²⁺ release, or through activation of K_v channels.     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

Effects of cilnidipine on sympathetic outflow and sympathetic arterial pressure and heart rate regulations in rats

AimsCilnidipine is a unique Ca^2 + channel blocker that inhibits both L-type and N-type Ca^2 + channels. The present study aimed to assess the effects of intravenous cilnidipine on sympathetic outflow and sympathetic arterial pressure (AP) and heart rate (HR) regulations.Main methodsCarotid sinus baroreceptor regions were isolated from the systemic circulation in anesthetized and vagotomized Wistar Kyoto rats. Changes in efferent sympathetic nerve activity (SNA), AP and HR in response to a stepwise input of carotid sinus pressure were examined before and during intravenous cilnidipine administration (30 μg/kg bolus + 100 μg kg^? 1 h^? 1 infusion, n = 6).Key findingsCilnidipine significantly reduced the AP response range (from 68.0 ± 10.2 to 34.6 ± 4.1 mmHg, P = 0.007) but did not affect the SNA response range (from 90.4 ± 10.3 to 84.7 ± 9.5%, P = 0.297) or the HR response range (from 50.4 ± 10.1 to 48.1 ± 6.2 beats/min, P = 0.719).SignificanceCilnidipine, at a depressor dose used in the present study, does not acutely suppress sympathetic outflow from the central nervous system. Also, it spared the sympathetic HR response, suggesting that N-type Ca^2 + channel blocking action at the cardiac sympathetic nerve endings may be a modest one.     

Protection against cardiac injury by small Ca2 +-sensitive K+ channels identified in guinea pig cardiac inner mitochondrial membrane

We tested if small conductance, Ca^2 +‐sensitive K⁺ channels (SK_Ca) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O₂‐derived free radicals are required to initiate protection via SK_Ca channels, and, importantly, if SK_Ca channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O₂^?), and m[Ca^2 +] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK_Ca and IK_Ca channel opener DCEBIO (DCEB) was given for 10 min and ended 20 min before IR. Either TBAP, a dismutator of O₂^?, NS8593, an antagonist of SK_Ca isoforms, or other K_Ca and K_ATP channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O₂^? and m[Ca^2 +], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK_Ca channels to mitochondria and IMM was evidenced by a) identification of purified mSK_Ca protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca^2 +]‐dependence of mSK_Ca channels in planar lipid bilayers, and d) matrix K⁺ influx induced by DCEB and blocked by SK_Ca antagonist UCL1684. This study shows that 1) SK_Ca channels are located and functional in IMM, 2) mSK_Ca channel opening by DCEB leads to protection that is O₂^? dependent, and 3) protection by DCEB is evident beginning during ischemia.     

Docking,characterization and investigation of β-cyclodextrin complexed with farnesol,an acyclic sesquiterpene alcohol,produces orofacial antinociceptive profile in experimental protocols

In this study, an inclusion complex with β-cyclodextrin and farnesol (βCD/FAR) was used to improve the physico-chemical and pharmacological properties of farnesol. The samples were obtained using the physical mixture, paste and slurry complexation methods and characterized by variety of methods. To evaluate the pharmacological effect, an animal model of orofacial pain induced by formalin, glutamate and capsaicin was used, and its possible mechanisms of action were evaluated. The slurry complexation method was produced with a formation energy of 3.45 kcal/mol and exhibited a better complexation profile as it presented smaller, deformed crystals compared to the other methods, with a stable complex formed. The formation energy was 3.45 kcal/mol. In the orofacial pain test induced by formalin, capsaicin and glutamate the results show that farnesol and its complex at doses of 50 and 100 mg/kg significantly decreased (p < 0.001) face-rubbing behavior. In the investigation of the mechanism of action, the administration of glibenclamide and ondansetron modified the antinociceptive effect of the farnesol, suggesting the possible participation of the ATP-sensitive K⁺ channel (K⁺_ATP) and 5-hydroxytryptamine (5-HT₃) channels/receptors. A rota-rod test did not show any significant alterations in motor performance in the groups treated with farnesol and its complex. In conclusion, farnesol and βCD/FAR reduced orofacial pain, possibly mediated by K⁺_ATP channels and 5-HT₃ receptors.     

Quaternary structure of KATP channel SUR2A nucleotide binding domains resolved by synchrotron radiation X-ray scattering

Heterodimeric nucleotide binding domains NBD1/NBD2 distinguish the ATP-binding cassette protein SUR2A, a recognized regulatory subunit of cardiac ATP-sensitive K⁺ (K_ATP) channels. The tandem function of these core domains ensures metabolism-dependent gating of the Kir6.2 channel pore, yet their structural arrangement has not been resolved. Here, purified monodisperse and interference-free recombinant particles were subjected to synchrotron radiation small-angle X-ray scattering (SAXS) in solution. Intensity function analysis of SAXS profiles resolved NBD1 and NBD2 as octamers. Implemented by ab initio simulated annealing, shape determination prioritized an oblong envelope wrapping NBD1 and NBD2 with respective dimensions of 168 × 80 × 37 Å³ and 175 × 81 × 37 Å³ based on symmetry constraints, validated by atomic force microscopy. Docking crystal structure homology models against SAXS data reconstructed the NBD ensemble surrounding an inner cleft suitable for Kir6.2 insertion. Human heart disease-associated mutations introduced in silico verified the criticality of the mapped protein–protein interface. The resolved quaternary structure delineates thereby a macromolecular arrangement of K_ATP channel SUR2A regulatory domains.     

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [³H]-ouabain binding (to analyze K⁺ site of Na⁺, K⁺-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [³H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2 mg/kg) and clozapine (3, 10 and 30 mg/kg). Animals were sacrificed 18 h later, cerebral cortices harvested, membrane fractions prepared and high affinity [³H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10 mg/kg clozapine. Rats were administered with neurotensin (3, 10 y 30 μg, i.c.v.) and 60 min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [³H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30 μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [³H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2 mg/kg, i.p.) or clozapine (10 mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na⁺, K⁺-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na⁺, K⁺-ATPase at central synapses.     

Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2 + to the second metal binding site

BackgroundFluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase–DNA covalent complex as a topoisomerase–fluoroquinolone–DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction.MethodsWe conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg^2 +-, Mn^2 +-, or Ca^2 +-supported DNA cleavage activity of Escherichia coli Topo IV.ResultsIn the absence of any drug, 20–30 mM Mg^2 + was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn^2 + or Ca^2 + was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg^2 + concentrations where Topo IV alone could not efficiently cleave DNA.Conclusions and general significanceAt low Mg^2 + concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg^2 + binding to metal binding site B through the structural distortion in DNA. As Mg^2 + concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg^2 + at site B or inhibition the binding of Mg^2 + to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg^2 + binding.     

The inhibitory effect of curcumin on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of curcumin, the principal active compound of turmeric, on voltage-dependent K⁺ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Curcumin reduced the Kv current in a dose-dependent manner with an apparent K_d value of 1.07 ± 0.03 μM. Although curcumin did not alter the kinetics of Kv current activation, it predominantly accelerated the decay rate of channel inactivation. The association and dissociation rate constants of curcumin were 1.35 ± 0.05 μM^?1 s^?1 and 1.47 ± 0.17 s^?1, respectively. Curcumin did not alter the steady-state activation or inactivation curves. Application of train pulses (1 or 2 Hz) increased curcumin-induced blockade of the Kv current, and the recovery time constant also increased in the presence of curcumin suggesting, that the inhibitory action of Kv currents by curcumin was use-dependent. From these results, we concluded that curcumin inhibited vascular Kv current in a state-, time-, and use-dependent manner.