Development of charybdotoxin Q18F variant as a selective peptide blocker of neuronal BK(α + ß4) channel for the treatment of epileptic seizures.

Epilepsy is the results from the imbalance between inhibition and excitation in neural circuits, which is mainly treated by some chemical drugs with side effects. Gain-of-function of BK channels or knockout of its ß4 subunit associates with spontaneous epilepsy. Currently, few reports were published about the efficacy of BK(α + ß4) channel modulators in epilepsy prevention. Charybdotoxin is a non-specific inhibitor of BK and other K+ channels. Here, by nuclear magnetic resonance (NMR) and other biochemical techniques, we found that charybdotoxin might interact with the extracellular loop of human ß4 subunit (i.e., hß4-loop) of BK(α + ß4) channel at a molar ratio 4:1 (hß4-loop vs. charybdotoxin). Charybdotoxin enhanced its ability to prevent K+ current of BK(α + ß4 H101Y) channel. The charybdotoxin Q18F variant selectively reduced the neuronal spiking frequency and increased interspike intervals of BK(α + ß4) channel by π-π stacking interactions between its residue Phe18 and residue His101 of hß4-loop. Moreover, intrahippocampal infusion of charybdotoxin Q18F variant significantly increased latency time of seizure, reduced seizure duration and seizure numbers on pentylenetetrazole-induced pre-sensitized rats, inhibited hippocampal hyperexcitability and c-Fos expression, and displayed neuroprotective effects on hippocampal neurons. These results implied that charybdotoxin Q18F variant could be potentially used for intractable epilepsy treatment by therapeutically targeting BK(α + ß4) channel.

Calcitonin Gene Related Peptide, Adrenomedullin, and Adrenomedullin 2 Function in Uterine Artery During Human Pregnancy.

RATIONALE: Calcitonin gene-related peptide (CGRP) and its family members adrenomedullin (ADM) and adrenomedullin 2 (ADM2; also known as intermedin) support vascular adaptions in rat pregnancy. OBJECTIVE: This study aimed to assess the relaxation response of uterine artery (UA) for CGRP, ADM, and ADM2 in nonpregnant and pregnant women and identify the involved mechanisms. FINDINGS: (1) Segments of UA from nonpregnant women that were precontracted with U46619 (1µM) in vitro are insensitive to the hypotensive effects of CGRP, ADM, and ADM2; (2) CGRP, ADM, and ADM2 (0.1-100nM) dose dependently relax UA segments from pregnant women with efficacy for CGRP > ADM = ADM2; (3) the relaxation responses to CGRP, ADM, and ADM2 are differentially affected by the inhibitors of nitric oxide (NO) synthase (L-NAME), adenylyl cyclase (SQ22536), apamin, and charybdotoxin; (4) UA smooth muscle cells (UASMC) express mRNA for calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP)1 and RAMP2 but not RAMP3; (5) receptor heterodimer comprising CRLR/RAMP1 and CRLR/RAMP2 but not CRLR/RAMP3 is present in UA; (6) soluble fms-like tyrosine kinase (sFLT-1) and TNF-α treatment decrease the expression of RAMP1 mRNA (P < 0.05) in UASMC; and (7) sFLT-1 treatment impairs the association of CRLR with all 3 peptides while TNF-α inhibits the interaction of CGRP but not ADM or ADM2 with CRLR in UASMC (P < 0.05). CONCLUSIONS: Relaxation sensitivity of UA for CGRP, ADM, and ADM2 is increased during pregnancy via peptide-specific involvement of NO system and endothelium-derived hyperpolarizing factors; vascular disruptors such as sFLT-1 and TNFα adversely impact their receptor system in UASMC.

Which proteinase-activated receptor-1 antagonist is better?: Evaluation of vorapaxar and parmodulin-2 effects on human left internal mammary artery endothelial function.

OBJECTIVE: Endothelial dysfunction occurs as an early event in cardiovascular disease. Previously, vorapaxar, a proteinase-activated receptor-1 antagonist, was shown to cause endothelial damage in a cell culture study. Therefore, our study aimed to compare the effects of vorapaxar and parmodulin-2, proteinase-activated receptor-1 biased agonist, on human left internal mammary artery endothelial function in vitro. METHOD: Isolated arteries were hung in the organ baths. Acetylcholine responses (10-11-10-6 M) were obtained in endothelium-intact tissues the following incubation with vorapaxar/parmodulin-2 (10-6 M) to determine the effects of these molecules on the endothelium-dependent relaxation. Subsequently, endothelium-dependent relaxation responses of tissues were investigated in the presence of L-NAME (10-4 M), L-arginine (10-5 M), indomethacin (10-5 M), and charybdotoxin-apamin (10-7 M) in addition to vorapaxar/parmodulin-2 incubation. Besides, the effect of these molecules on endothelium-independent relaxation response was evaluated with sodium nitroprusside (10-11-10-6 M). Finally, the sections of human arteries were imaged using a transmission electron microscope, and the integrity of the endothelial layer was evaluated. RESULTS: We found that vorapaxar caused significant endothelial dysfunction by disrupting nitric oxide and endothelium-derived hyperpolarizing factor-dependent relaxation mechanisms. Parmodulin-2 did not cause endothelial damage. Neither vorapaxar nor parmodulin-2 disrupted endothelium-independent relaxation responses. The effect of vorapaxar on the endothelial layer was supported by the transmission electron microscope images. CONCLUSION: Parmodulin-2 may be a better option than vorapaxar in treating cardiovascular diseases since it can inhibit PAR-1 without caused endothelial dysfunction.

Molecular mechanism of apelin-13 regulation of colonic motility in rats.

Apelin is a novel neuropeptide identified as the endogenous ligand for the apelin receptor. Apelin and its receptor are widely distributed in the gastrointestinal tract. Studies have reported that apelin-13 is involved in modulating gastrointestinal motility; however, the evidence is insufficient and the relevant mechanism is still not fully clear. Consequently, our study designed to explore the effect induced by exogenous apelin-13, to analyze the mechanism of action in isolated rat colons and colonic smooth muscle cells. The spontaneous contractions of colonic smooth muscle strips from rat were measured in an organ bath system. L-type calcium currents and large conductance Ca2+-activated K+ (BKCa) currents in rat colonic smooth muscle cells were investigated using the electrophysiological patch-clamp technique. Apelin-13 decreased the spontaneous contractile activity of colonic smooth muscle strips in a dose-dependent manner, and the inhibitory effect was not abolished by tetrodotoxin. The electrophysiological recordings revealed that apelin-13 reduced the crest currents of L-type calcium in a concentration-dependent manner in colonic smooth muscle cells at the test potential of 0 mV. Moreover, apelin-13 moved the current-voltage (I-V) curves of L-type calcium channels upward, but did not change their contour. Furthermore, the characteristics of L-type calcium channels with steady-state activation and steady-state inactivation were not significantly changed. Similarly, application of apelin-13 also significantly decreased BKCa currents in a concentration-dependent manner. In conclusion, apelin-13 inhibited the spontaneous contractile activity of isolated rat colons via the suppression of L-type calcium channels and BKCa channels in colonic smooth muscle cells.

Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells.

Ion channels play pivotal role in the physiological and pathological function of immune cells. As immune cells represent a functionally diverse population, subtype-specific functional studies, such as single-cell electrophysiology require proper subset identification and separation. Magnetic-activated cell sorting (MACS) techniques provide an alternative to fluorescence-activated cell sorting (FACS), however, the potential impact of MACS-related beads on the biophysical and pharmacological properties of the ion channels were not studied yet. We studied the aforementioned properties of the voltage-gated Kv1.3 K+ channel in activated CD4+ T-cells as well as the membrane capacitance using whole-cell patch-clamp following immunomagnetic positive separation, using the REAlease® kit. This kit allows three experimental configurations: bead-bound configuration, bead-free configuration following the removal of magnetic beads, and the label-free configuration following removal of CD4 recognizing antibody fragments. As controls, we used FACS separation as well as immunomagnetic negative selection. The membrane capacitance and of the biophysical parameters of Kv1.3 gating, voltage-dependence of steady-state activation and inactivation kinetics of the current were not affected by the presence of MACS-related compounds on the cell surface. We found subtle differences in the activation kinetics of the Kv1.3 current that could not be explained by the presence of MACS-related compounds. Neither the equilibrium block of Kv1.3 by TEA+ or charybdotoxin (ChTx) nor the kinetics of ChTx block are affected by the presence of the magnetics beads on the cell surface. Based on our results MACS is a suitable method to separate cells for studying ion channels in non-excitable cells, such as T-lymphocytes.

Vasodilatory Effect of Phellinus linteus Extract in Rat Mesenteric Arteries.

Phellinus linteus is a well-known medicinal mushroom that is widely used in Asian countries. In several experimental models, Phellinus linteus extracts were reported to have various biological effects, including anti-inflammatory, anti-cancer, hepatoprotective, anti-diabetic, neuroprotective, and anti-angiogenic activity. In the present study, several bioactive compounds, including palmitic acid ethyl ester and linoleic acid, were identified in Phellinus linteus. The intermediate-conductance calcium-activated potassium channel (IKCa) plays an important role in the regulation of the vascular smooth muscle cells' (VSMCs) contraction and relaxation. The activation of the IKCa channel causes the hyperpolarization and relaxation of VSMCs. To examine whether Phellinus linteus extract causes vasodilation in the mesenteric arteries of rats, we measured the isometric tension using a wire myograph. After the arteries were pre-contracted with U46619 (a thromboxane analogue, 1 µM), Phellinus linteus extract was administered. The Phellinus linteus extract induced vasodilation in a dose-dependent manner, which was independent of the endothelium. To further investigate the mechanism, we used the non-selective K+ channel blocker tetraethylammonium (TEA). TEA significantly abolished Phellinus linteus extract-induced vasodilation. Thus, we tested three different types of K+ channel blockers: iberiotoxin (BKca channel blocker), apamin (SKca channel blocker), and charybdotoxin (IKca channel blocker). Charybdotoxin significantly inhibited Phellinus linteus extract-induced relaxation, while there was no effect from apamin and iberiotoxin. Membrane potential was measured using the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) in the primary isolated vascular smooth muscle cells (VSMCs). We found that the Phellinus linteus extract induced hyperpolarization of VSMCs, which is associated with a reduced phosphorylation level of 20 KDa myosin light chain (MLC20).

Intercalated cell BKα subunit is required for flow-induced K+ secretion.

BK channels are expressed in intercalated cells (ICs) and principal cells (PCs) in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK α subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin-sensitive (ChTX-sensitive) K+ currents were readily detected in control ICs but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice versus controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated epithelial Na+ channel-mediated (ENaC-mediated) Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice.

Altered cyclooxygenase-1 and enhanced thromboxane receptor activities underlie attenuated endothelial dilatory capacity of omental arteries in obesity.

AIMS: Obesity is a risk factor for endothelial dysfunction, the severity of which is likely to vary depending on extent and impact of adiposity on the vasculature. This study investigates the roles of cyclooxygenase isoforms and thromboxane receptor activities in the differential endothelial dilatory capacities of arteries derived from omental and subcutaneous adipose tissues in obesity. MAIN METHODS: Small arteries were isolated from omental and subcutaneous adipose tissues obtained from consented morbidly obese patients (n = 65, BMI 45 ±â€¯6 kg m-2 [Mean ±â€¯SD]) undergoing bariatric surgery. Relaxation to acetylcholine was studied by wire myography in the absence or presence of indomethacin (10 µM, cyclooxygenase inhibitor), FR122047 (1 µM, cyclooxygenase-1 inhibitor), Celecoxib (4 µM, cyclooxygenase-2 inhibitor), Nω-Nitro-L-arginine methyl ester (L-NAME, 100 µM, nitric oxide synthase inhibitor) or combination of apamin (0.5 µM) and charybdotoxin (0.1 µM) that together inhibit endothelium-derived hyperpolarizing factor (EDHF). Contractions to U46619 (thromboxane A2 mimetic) were also studied. KEY FINDINGS: Acetylcholine relaxation was significantly attenuated in omental compared with subcutaneous arteries from same patients (p < 0.01). Indomethacin (p < 0.01) and FR122047 (p < 0.001) but not Celecoxib significantly improved the omental arteriolar relaxation. Cyclooxygenase-1 mRNA and U46619 contractions were both increased in omental compared with subcutaneous arteries (p < 0.05). L-NAME comparably inhibited acetylcholine relaxation in both arteries, while apamin+charybdotoxin were less effective in omental compared with subcutaneous arteries. SIGNIFICANCE: The results show that the depot-specific reduction in endothelial dilatory capacity of omental compared with subcutaneous arteries in obesity is in large part due to altered cyclooxygenase-1 and enhanced thromboxane receptor activities, which cause EDHF deficiency.

L-type Ca2+ channels and charybdotoxin-sensitive Ca2+-activated K+ channels are required for reduction of GABAergic activity induced by ß2-adrenoceptor in the prefrontal cortex.

Whereas ß2-adrenoceptor (ß2-AR) has been reported to reduce GABAergic activity in the prefrontal cortex (PFC), the underlying cellular and molecular mechanisms have not been completely determined. Here, we showed that ß2-AR agonist Clenbuterol (Clen) decreased GABAergic transmission onto PFC layer V/VI pyramidal neurons via a presynaptic mechanism without altering postsynaptic GABA receptors. Clen decreased the action potential firing rate but increased the burst afterhyperpolarization (AHP) amplitude in PFC interneurons. Application of L-type Ca2+ channel or charybdotoxin-sensitive Ca2+-activated K+ channel inhibitors blocked Clen-induced decreases in action potential firing rate, spontaneous inhibitory postsynaptic current (sIPSC) frequency and Clen-induced enhancement of AHP amplitude, suggesting that the effects of Clen involves L-type Ca2+ Channels and charybdotoxin-sensitive Ca2+-activated K+ channels. Our results provide a potential cellular mechanism by which Clen controls GABAergic neuronal activity in PFC.

Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states.

In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Chlorotoxin targets ERα/VASP signaling pathway to combat breast cancer.

Breast cancer is one of the most common malignant tumors among women worldwide. About 70-75% of primary breast cancers belong to estrogen receptor (ER)-positive breast cancer. In the development of ER-positive breast cancer, abnormal activation of the ERα pathway plays an important role and is also a key point leading to the failure of clinical endocrine therapy. In this study, we found that the small molecule peptide chlorotoxin (CTX) can significantly inhibit the proliferation, migration and invasion of breast cancer cells. In in vitro study, CTX inhibits the expression of ERα in breast cancer cells. Further studies showed that CTX can directly bind to ERα and change the protein secondary structure of its LBD domain, thereby inhibiting the ERα signaling pathway. In addition, we also found that vasodilator stimulated phosphoprotein (VASP) is a target gene of ERα signaling pathway, and CTX can inhibit breast cancer cell proliferation, migration, and invasion through ERα/VASP signaling pathway. In in vivo study, CTX significantly inhibits growth of ER overexpressing breast tumor and, more importantly, based on the mechanism of CTX interacting with ERα, we found that CTX can target ER overexpressing breast tumors in vivo. Our study reveals a new mechanism of CTX anti-ER-positive breast cancer, which also provides an important reference for the study of CTX anti-ER-related tumors.

Human Menstrual Blood-Derived Stromal Cells Promote Recovery of Premature Ovarian Insufficiency Via Regulating the ECM-Dependent FAK/AKT Signaling.

POI is characterized by "absent not abnormal" menstruation with hormonal disorders in woman younger than 40 years of age, and etiological and pathophysiological mechanisms underlying the POI development have not been clearly defined. Recently, due to advantages such as abundant sources and non-invasive methods of harvest, MenSCs have been emerging as a promising treatment strategy for the recovery of female reproductive damage. Here, we demonstrated that MenSCs graft in POI mice after CTX treatment could restore ovarian function by regulating normal follicle development and estrous cycle, reducing apoptosis in ovaries to maintain homeostasis of microenvironment and modulating serum sex hormones to a relatively normal status. Moreover, MenSCs participated in the activation of ovarian transcriptional expression in ECM-dependent FAK/AKT signaling pathway and thus restored ovarian function to a certain extent. MenSCs transplantation was proved to be an effective way to repair ovarian function with low immunogenicity, suggesting its great potential for POI treatment.

Attenuation of autophagy impacts on muscle fibre development, starvation induced stress and fibre regeneration following acute injury.

Autophagy has been implicated as a major factor in the development of a number of diseases of skeletal muscle. However, its role in skeletal muscle homeostasis is still evolving. We examined skeletal muscle architecture in a mouse model, Atg16L1, where autophagy is attenuated but importantly still present. We show that muscle fibres from Atg16L1 mice were smaller than wild-type counterparts, proving a role for this process in the growth of these cells. We show that mild attenuation of autophagy results in accelerated muscle loss during the initial phase of acute starvation. Furthermore, we show that regeneration of skeletal muscle following cardiotoxin (CTX) mediated injury is slower in the Atg16L1 mouse compared to wild-type. Lastly, we show that autophagy controls the integrity of the sarcolemma. Attenuated autophagy makes muscle fibres more susceptible to infiltration by circulating immunoglobulins following muscle injury with CTX. These fibres internalise dystrophin and nNOS. Importantly these fibres are able to restore dystrophin and nNOS localisation and do not die. In conclusion, these studies shed new light into the ability of skeletal muscle fibres to cope with injury and establish a link between the fine-tuning of autophagy and skeletal muscle regeneration.

Characterization of the σ-Pore in Mutant hKv1.3 Potassium Channels.

BACKGROUND/AIMS: The replacement of the amino acid valine at position 388 (Shaker position 438) in hKv1.3 channels or at the homologue position 370 in hKv1.2 channels resulted in a channel with two different ion conducting pathways: One pathway was the central, potassium-selective α-pore, that was sensitive to block by peptide toxins (CTX or KTX in the hKv1.3_V388C channel and CTX or MTX in the hKv1.2_V370C channel). The other pathway (σ-pore) was behind the central α-pore creating an inward current at potentials more negative than -100 mV, a potential range where the central α-pore was closed. In addition, current through the σ-pore could not be reduced by CTX, KTX or MTX in the hKv1.3_V388C or the hKv1.2_V370C channel, respectively. METHODS: For a more detailed characterization of the σ-pore, we created a trimer consisting of three hKv1.3_V388C α-subunits linked together and characterized current through this trimeric hKv1.3_V388C channel. Additionally, we determined which amino acids line the σ-pore in the tetrameric hKv1.3_V388C channel by replacing single amino acids in the tetrameric hKv1.3_V388C mutant channel that could be involved in σ-pore formation. RESULTS: Overexpression of the trimeric hKv1.3_V388C channel in COS-7 cells yielded typical σ-pore currents at potentials more negative than -100 mV similar to what was observed for the tetrameric hKv1.3_V388C channel. Electrophysiological properties of the trimeric and tetrameric channel were similar: currents could be observed at potentials more negative than -100 mV, were not carried by protons or chloride ions, and could not be reduced by peptide toxins (CTX, MTX) or TEA. The σ-pore was mostly permeable to Na+ and Li+. In addition, in our site-directed mutagenesis experiments, we created a number of new double mutant channels in the tetrameric hKv1.3_V388C background channel. Two of these tetrameric double mutant channels (hKv1.3_V388C_T392Y and hKv1.3_V388C_Y395W) did not show currents through the σ-pore. CONCLUSIONS: From our experiments with the trimeric hKv1.3_V388C channel we conclude that the σ-pore exists in hKv1.3_V388C channels independently of the α-pore. From our site-directed mutagenesis experiments in the tetrameric hKv1.3_V388C channel we conclude that amino acid position 392 and 395 (Shaker position 442 and 445) line the σ-pore.

Solution structure of extracellular loop of human ß4 subunit of BK channel and its biological implication on ChTX sensitivity.

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary ß (ß1-ß4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched ß4 subunit (hß4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (hß4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of ß4 subunit's extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by ß4 are still unclear. To address them, here, we first identified four disulfide bonds in hß4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of ß4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BKα/ß4 and thermodynamic double-mutant cycle analysis, we proposed that BKα subunit might interact with ß4 subunit through the conserved residue Glu264(BKα) coupling with residue Asn123(ß4).

Hyperpolarization by N-(3-oxododecanoyl)-l-homoserine-lactone, a quorum sensing molecule, in rat thymic lymphocytes.

To study the adverse effects of N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule, on mammalian host cells, its effect on membrane potential was examined in rat thymic lymphocytes using flow cytometric techniques with a voltage-sensitive fluorescent probe. As 3-300 µM ODHL elicited hyperpolarization, it is likely that it increases membrane K+ permeability because hyperpolarization is directly linked to changing K+ gradient across membranes, but not Na+ and Cl- gradients. ODHL did not increase intracellular Ca2+ concentration. ODHL also produced a response in the presence of an intracellular Zn2+ chelator. Thus, it is unlikely that intracellular Ca2+ and Zn2+ are attributed to the response. Quinine, a non-specific K+ channel blocker, greatly reduced hyperpolarization. However, because charybdotoxin, tetraethylammonium chloride, 4-aminopyridine, and glibenclamide did not affect it, it is pharmacologically hypothesized that Ca2+-activated K+ channels, voltage-gated K+ channels, and ATP-sensitive K+ channels are not involved in ODHL-induced hyperpolarization. Although the K+ channels responsible for ODHL-induced hyperpolarization have not been identified, it is suggested that ODHL can elicit hyperpolarization in mammalian host cells, disturbing cellular functions.

Inhibitory Effect of Interferons on Contractive Activity of Bovine Mesenteric Lymphatic Vessels and Nodes.

We studied the effect of IFNα-2b and IFNß-1a on phasic and tonic contractions of isolated bovine mesenteric lymphatic vessels and nodes. IFNα-2b and IFNß-1a in concentrations of 250-1000 U/ml produced dose-dependent negative chronotropic and inotropic effects on spontaneous phasic contractions and tonus of lymphatic vessels and nodes. In de-endothelialized lymphatic vessels and nodes, IFNα-2b and IFNß-1a in the same concentrations had less pronounced inhibitory effect on spontaneous contraction and tonus. L-NAME (100 µM) and charybdotoxin (0.1 µM with 0.5 µM apamine) significantly attenuated the inhibitory effect of IFNα-2b on phasic and tonic contractions of lymph nodes. L-NAME (100 µM) and indomethacin (10 µM) significantly reduced the IFNα-2b-induced inhibitory effect on phasic and tonic contractions of lymph node. These results indicate that IFNα-2b and IFNß-1a have a pronounced inhibitory effect on the phasic and tonic contractions of bovine mesenteric lymphatic vessels and nodes. The responses are endothelium-dependent and are determined by production of NO and endothelium-dependent hyperpolarizing factor by endotheliocytes in lymphatic vessels and by production of NO and prostacyclin by endotheliocytes in the lymphatic nodes.

The glycosylation of the extracellular loop of ß2 subunits diversifies functional phenotypes of BK Channels.

Large-conductance Ca2+- and voltage-activated potassium (MaxiK or BK) channels are composed of a pore-forming α subunit (Slo) and 4 types of auxiliary ß subunits or just a pore-forming α subunit. Although multiple N-linked glycosylation sites in the extracellular loop of ß subunits have been identified, very little is known about how glycosylation influences the structure and function of BK channels. Using a combination of site-directed mutagenesis, western blot and patch-clamp recordings, we demonstrated that 3 sites in the extracellular loop of ß2 subunit are N-glycosylated (N-X-T/S at N88, N96 and N119). Glycosylation of these sites strongly and differentially regulate gating kinetics, outward rectification, toxin sensitivity and physical association between the α and ß2 subunits. We constructed a model and used molecular dynamics (MD) to simulate how the glycosylation facilitates the association of α/ß2 subunits and modulates the dimension of the extracellular cavum above the pore of the channel, ultimately to modify biophysical and pharmacological properties of BK channels. Our results suggest that N-glycosylation of ß2 subunits plays crucial roles in imparting functional heterogeneity of BK channels, and is potentially involved in the pathological phenotypes of carbohydrate metabolic diseases.

Role of BKCa in Stretch-Induced Relaxation of Colonic Smooth Muscle.

Stretch-induced relaxation has not been clearly identified in gastrointestinal tract. The present study is to explore the role of large conductance calcium-activated potassium channels (BKCa) in stretch-induced relaxation of colon. The expression and currents of BKCa were detected and the basal muscle tone and contraction amplitude of colonic smooth muscle strips were measured. The expression of BKCa in colon is higher than other GI segments (P < 0.05). The density of BKCa currents was very high in colonic smooth muscle cells (SMCs). BKCa in rat colonic SMCs were sensitive to stretch. The relaxation response of colonic SM strips to stretch was attenuated by charybdotoxin (ChTX), a nonspecific BKCa blocker (P < 0.05). After blocking enteric nervous activities by tetrodotoxin (TTX), the stretch-induced relaxation did not change (P > 0.05). Still, ChTX and iberiotoxin (IbTX, a specific BKCa blocker) attenuated the relaxation of the colonic muscle strips enduring stretch (P < 0.05). These results suggest stretch-activation of BKCa in SMCs was involved in the stretch-induced relaxation of colon. Our study highlights the role of mechanosensitive ion channels in SMCs in colon motility regulation and their physiological and pathophysiological significance is worth further study.

Overexcited MaxiK and KATP channels underlie obstructive jaundice-induced vasoconstrictor hyporeactivity of arterial smooth muscle.

Substantial evidence has shown that obstructive jaundice can induce vascular hyporesponsiveness. The present study was designed to investigate mechanisms of MaxiK channel and KATP underlying cholestasis-induced vascular dysfunction. The isolated thoracic aorta was used to explore norepinephrine (NE)-induced contraction. The function of MaxiK and KATP channels were investigated using whole-cell patch clamp recording. Compared with Sham group, NE-induced vascular contraction was blunted after bile duct ligation (BDL), which could not be ameliorated significantly after endothelial denudation. Charybdotoxin and glibenclamide induced a more pronounced recovery from vascular hyporesponsiveness to NE in BDL group compared with Sham group. BDL significantly promoted the charybdotoxin sensitive MaxiK current and KATP current in isolated aortic smooth muscle cells. In addition, the expression of auxiliary subunits (MaxiK-ß1 and SUR2B) rather pore-forming subunits (MaxiK-α and Kir6.1) was significantly up-regulated after BDL. These findings suggest that MaxiK and KATP channels play an important role in regulating vascular hyporesponsiveness in BDL rats.