Properties and molecular determinants of the natural flavone acacetin for blocking hKv4.3 channels.
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ABSTRACT:
SUBMITTER: Wu HJ
PROVIDER: S-EPMC3603988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cardiac senescence is associated with cardiomyopathy which is a degenerative disease in the aging process of the elderly. The present study investigates using multiple experimental approaches whether the natural flavone acacetin could attenuate myocardial senescence in C57/BL6 mice and H9C2 rat cardiac cells induced by D-galactose. We found that the impaired heart function in D-galactose-induced accelerated aging mice was improved by oral acacetin treatment in a dose-dependent manner. Acacetin significantly countered the increased serum advanced glycation end products, the myocardial telomere length shortening, the increased cellular senescence marker proteins p21 and p53, and the reduced mitophagy signaling proteins PINK1/Parkin and Sirt6 expression in aging mice. In H9C2 rat cardiac cells, acacetin alleviated cell senescence induced by D-galactose in a concentration-dependent manner. Acacetin decreased p21 and p53 expression, up-regulated PINK1/Parkin, LC3II/LC3I ratio, pLKB1, pAMPK and Sirt6, and reversed the depolarized mitochondrial membrane potential in aging cardiac cells. Mitophagy inhibition with 3-methyladenine or silencing Sirt6 abolished the protective effects of acacetin against cardiac senescence. Further analysis revealed that acacetin effect on Sirt6 was mediated by Sirt1 activation and increase of NAD+/NADH ratio. These results demonstrate that acacetin significantly inhibits in vivo and in vitro cardiac senescence induced by D-galactose via Sirt1-mediated activation of Sirt6/AMPK signaling pathway, thereby enhancing mitophagy and preserving mitochondrial function, which suggests that acacetin may be a drug candidate for treating cardiovascular disorders related to aging.
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Project description:The present study investigates the potential signal pathway of acacetin in cardioprotection against ischemia/reperfusion injury using an in vitro hypoxia/reoxygenation model in primary cultured neonatal rat cardiomyocytes and H9C2 cardiomyoblasts. It was found that acacetin (0.3-3 ?M) significantly decreased the apoptosis and reactive oxygen species production induced by hypoxia/reoxygenation injury in cardiomyocytes and H9C2 cardiomyoblasts via reducing the pro-apoptotic proteins Bax and cleaved-caspase-3 and increasing the anti-apoptotic protein Bcl-2. In addition, acacetin not only suppressed the release of pro-inflammatory cytokines TLR-4 and IL-6 induced by hypoxia/reoxygenation injury, but also increased the secretion of anti-inflammatory cytokine IL-10. Moreover, acacetin increased Nrf2 and HO-1 in a concentration-dependent manner, and rescued SOD1 and SOD2 reduction induced by hypoxia/reoxygenation insult. These beneficial effects of acacetin disappeared in cells with silenced Nrf2, suggesting that Nrf2 activation participates in the cardioprotective effect of acacetin against hypoxia/reoxygenation insult. However, acacetin-induced Nrf2 activation was not observed in cells with silenced AMPK and in ventricular tissues of rat hearts treated with the AMPK inhibitor Compound C and subjected to ischemia/reperfusion injury. Our results demonstrate for the first time that AMPK-mediated Nrf2 activation is involved in the cardiomyocytes protection of acacetin against hypoxia/reoxygenation injury by activating a series of intracellular signals involved in anti-oxidation, anti-inflammation, and anti-apoptosis.
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Project description:The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) >> Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.
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Project description:Kv2.1 channels exhibit a U-shaped voltage-dependence of inactivation that is thought to represent preferential inactivation from preopen closed states. However, the molecular mechanisms underlying so-called U-type inactivation are unknown. We have performed a cysteine scan of the S3-S4 and S5-P-loop linkers and found sites that are important for U-type inactivation. In the S5-P-loop linker, U-type inactivation was preserved in all mutant channels except E352C. This mutation, but not E352Q, abolished closed-state inactivation while preserving open-state inactivation, resulting in a loss of the U-shaped voltage profile. The reducing agent DTT, as well as the C232V mutation in S2, restored U-type inactivation to the E352C mutant, which suggests that residues 352C and C232 may interact to prevent U-type inactivation. The R289C mutation, in the S3-S4 linker, also reduced U-type inactivation. In this case, DTT had little effect but application of MTSET restored wild-type-like U-type inactivation behavior, suggestive of the importance of charge at this site. Kinetic modeling suggests that the E352C and R289C inactivation phenotypes largely resulted from reductions in the rate constants for transitions from closed to inactivated states. The data indicate that specific residues within the S3-S4 and S5-P-loop linkers may play important roles in Kv2.1 U-type inactivation.
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Project description:Natural killer (NK) cells are large granular lymphocytes that participate in both innate and adaptive immune responses against tumors and pathogens. They are also involved in other conditions, including organ rejection, graft-versus-host disease, recurrent spontaneous abortions, and autoimmune diseases such as multiple sclerosis. We demonstrate that human NK cells express the potassium channels Kv1.3 and KCa3.1. Expression of these channels does not vary with expression levels of maturation markers but varies between adherent and non-adherent NK cell subpopulations. Upon activation by mitogens or tumor cells, adherent NK (A-NK) cells preferentially up-regulate KCa3.1 and non-adherent (NA-NK) cells preferentially up-regulate Kv1.3. Consistent with this different phenotype, A-NK and NA-NK do not display the same sensitivity to the selective KCa3.1 blockers TRAM-34 and NS6180 and to the selective Kv1.3 blockers ShK-186 and PAP-1 in functional assays. Kv1.3 block inhibits the proliferation and degranulation of NA-NK cells with minimal effects on A-NK cells. In contrast, blocking KCa3.1 increases the degranulation and cytotoxicity of A-NK cells, but not of NA-NK cells. TRAM-34, however, does not affect their ability to form conjugates with target tumor cells, to migrate, or to express chemokine receptors. TRAM-34 and NS6180 also increase the proliferation of both A-NK and NA-NK cells. This results in a TRAM-34-induced increased ability of A-NK cells to reduce in vivo tumor growth. Taken together, our results suggest that targeting KCa3.1 on NK cells with selective blockers may be beneficial in cancer immunotherapy.
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Project description:Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (IKr) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K+channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R-roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 ?M R-roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R-roscovitine's relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.
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Project description:The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.
| S-EPMC3932411 | biostudies-literature
Project description:The conductance, g(j), of many gap junctions depends on voltage between the coupled cells (transjunctional voltage, V(j)) with little effect of the absolute membrane potential (V(m)) in the two cells; others show combined V(j) and V(m) dependence. We examined the molecular determinants of V(m) dependence by using rat connexin 43 expressed in paired Xenopus oocytes. These junctions have, in addition to V(j) dependence, V(m) dependence such that equal depolarization of both cells decreases g(j). The dependence of g(j) on V(m) was abolished by truncation of the C-terminal domain (CT) at residue 242 but not at 257. There are two charged residues between 242 and 257. In full-length Cx43, mutations neutralizing either one of these charges, Arg243Gln and Asp245Gln, decreased and increased V(m) dependence, respectively, suggesting that these residues are part of the V(m) sensor. Mutating both residues together abolished V(m) dependence, although there is no net change in charge. The neutralizing mutations, together or separately, had no effect on V(j) dependence. Thus, the voltage sensors must differ. However, V(j) gating was somewhat modulated by V(m), and V(m) gating was reduced when the V(j) gate was closed. These data suggest that the two forms of voltage dependence are mediated by separate but interacting domains.
| S-EPMC18992 | biostudies-literature
Project description:To explore the molecular basis of the distinct intrinsic membrane properties and other dstinguishing features of functionally defined DRG neuron subtypes, we bulk-sequenced RNA at high depth of genetically-labeled DRG neurons to generate transcriptome profiles of eight major DRG neuron subtypes. The trancriptome profiles revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by the transcriptome pofiles, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns of the DRG neuron subtypes. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties.
2019-05-16 | GSE131230 | GEO