Tannic acid activates the Kv7.4 and Kv7.3/7.5 K+ channels expressed in HEK293 cells and reduces tension in the rat mesenteric arteries

2016 ◽  
Vol 68 (4) ◽  
pp. 494-502 ◽  
Author(s):  
Yuanyuan Zhang ◽  
Xi Chu ◽  
Ling Liu ◽  
Nan Zhang ◽  
Hui Guo ◽  
...  
Keyword(s):  
Tannic Acid ◽  
Mesenteric Arteries ◽  
Hek293 Cells ◽  
K Channels

Abstract P501: Editing of Myosin Phosphatase Gene as a Novel Approach for Vasodilator Sensitization and Lowering of Blood Pressure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Mariam Meddeb ◽  
Jeanine Ursitti ◽  
John Reho ◽  
Steven A Fisher
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Leucine Zipper ◽  
Splice Variants ◽  
Species Conservation ◽  
Mesenteric Arteries ◽  
Hek293 Cells ◽  
Myosin Phosphatase ◽  
Novel Approach

Myosin Phosphatase (MP) is the primary effector of vascular smooth muscle (VSM) relaxation and a key end target of signaling pathways that regulate vessel tone. Regulated splicing of alternative Exon24 (E24) of Myosin Phosphatase Regulatory/ Targeting subunit (MYPT1) sets vasodilator sensitivity. Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGK1α binding and NO/cGMP activation of MP resulting in vasodilation. Inclusion of 31 nt E24 shifts the reading frame coding for a Mypt1 isoform with a distinct C-terminus (LZ-) that is unresponsive to NO/cGMP. We are using two editing approaches to test the function of Mypt1 E24 splice variants in the control of BP in vivo. First, LoxP sites were inserted in introns flanking E24, crossed with smMHCCre ER , and treated with Tamoxifen to achieve smooth muscle-specific cKO of E24 (SMcKO E24), thereby converting Mypt1 to the LZ+ isoform. E24 cKO mice had mean BP that was 15 + 3 mmHg lower than control (n=3-5; p<0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC 50 : 2.1+0.5 nM vs 18.2+5.6 μM; n=5-6, p<0.05). We now are developing CRISPR/CAS9 editing of Mypt1 for translation into humans with hypertension. Guide(g)RNAs targeting E24 were designed using Benchling.com and selected for further study based on predicted efficacy, specificity (>10%,>60%) and cross-species conservation. Plasmids were generated by sub-cloning of oligonucleotides into the parent pX601 plasmid for the purpose of co-expression of gRNA and saCas9. These plasmids were transfected into HEK293 cells singly and in combinations and Mypt1 gene editing assayed by PCR, Surveyor nuclease assays and sequencing of genomic DNA. Single gRNAs yielded deletions of 1-3 nt. Combinations yielded deletions of 104-334 nt that removed >80% of E24 with an efficiency of editing that varied from 10% (gRNAs 6+9 and 5+9) to 40% (gRNAs 6+11 and 5+11). We have now generated AAVgE24 and are testing their efficiency of editing of VSM in vivo. These studies support that AAV mediated CRISPR/Cas9 editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans.

scholarly journals Validation of a [3H]Astemizole Binding Assay in HEK293 Cells Expressing HERG K+ Channels

2004 ◽  
Vol 95 (3) ◽  
pp. 311-319 ◽  
Author(s):  
Peter J.S. Chiu ◽  
Karen F. Marcoe ◽  
Sidney E. Bounds ◽  
Chun-Hsiung Lin ◽  
Jin-Jye Feng ◽  
...  
Keyword(s):  
Binding Assay ◽  
Hek293 Cells ◽  
K Channels

scholarly journals Counteracting suppression of CFTR and voltage-gated K+ channels by a bacterial pathogenic factor with the natural product tannic acid

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yajamana Ramu ◽  
Yanping Xu ◽  
Hyeon-Gyu Shin ◽  
Zhe Lu
Keyword(s):  
Lung Disease ◽  
Natural Product ◽  
Tannic Acid ◽  
Host Immunity ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Chemical Library ◽  
K Channels ◽  
Voltage Gated ◽  
Cystic Fibrosis Transmembrane

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause recurring bacterial infection in CF patients' lungs. However, the severity of CF lung disease correlates poorly with genotype. Antibiotic treatment helps dramatically prolong patients' life. The lung disease generally determines prognosis and causes most morbidity and mortality; early control of infections is thus critical. Staphylococcus aureus is a main cause of early infection in CF lungs. It secretes sphingomyelinase (SMase) C that can suppress CFTR activity. SMase C also inhibits voltage-gated K+ channels in lymphocytes; inhibition of these channels causes immunosuppression. SMase C's pathogenicity is further illustrated by the demonstration that once Bacillus anthracis is engineered to express high levels of SMase C, the resulting mutant can evade the host immunity elicited by a live vaccine because additional pathogenic mechanisms are created. By screening a chemical library, we find that the natural product tannic acid is an SMase C antidote.

scholarly journals ACTH Inhibits bTREK-1 K+ Channels through Multiple cAMP-dependent Signaling Pathways

2008 ◽  
Vol 132 (2) ◽  
pp. 279-294 ◽  
Author(s):  
Haiyan Liu ◽  
Judith A. Enyeart ◽  
John J. Enyeart
Keyword(s):  
Hek293 Cells ◽  
Resting Membrane Potential ◽  
Zona Fasciculata ◽  
Nucleotide Exchange ◽  
Pipette Solution ◽  
K Channels ◽  
Whole Cell Patch Clamp ◽  
Dependent Protein ◽  
Secretion Inhibition ◽  
And Function

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels that set the resting membrane potential and function pivotally in the physiology of cortisol secretion. Inhibition of these K+ channels by adrenocorticotropic hormone (ACTH) or cAMP is coupled to depolarization and Ca2+ entry. The mechanism of ACTH and cAMP-mediated inhibition of bTREK-1 was explored in whole cell patch clamp recordings from AZF cells. Inhibition of bTREK-1 by ACTH and forskolin was not affected by the addition of both H-89 and PKI(6–22) amide to the pipette solution at concentrations that completely blocked activation of cAMP-dependent protein kinase (PKA) in these cells. The ACTH derivative, O-nitrophenyl, sulfenyl-adrenocorticotropin (NPS-ACTH), at concentrations that produced little or no activation of PKA, inhibited bTREK-1 by a Ca2+-independent mechanism. Northern blot analysis showed that bovine AZF cells robustly express mRNA for Epac2, a guanine nucleotide exchange protein activated by cAMP. The selective Epac activator, 8-pCPT-2′-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC50 = 0.63 μM) at concentrations that did not activate PKA. Inhibition by this agent was unaffected by PKA inhibitors, including RpcAMPS, but was eliminated in the absence of hydrolyzable ATP. Culturing AZF cells in the presence of ACTH markedly reduced the expression of Epac2 mRNA. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 current in AZF cells that had been treated with ACTH for 3–4 d while inhibition by 8-br-cAMP was not affected. 8-pCPT-2′-O-Me-cAMP failed to inhibit bTREK-1 expressed in HEK293 cells, which express little or no Epac2. These findings demonstrate that, in addition to the well-described PKA-dependent TREK-1 inhibition, ACTH, NPS-ACTH, forskolin, and 8-pCPT-2′-O-Me-cAMP also inhibit these K+ channels by a PKA-independent signaling pathway. The convergent inhibition of bTREK-1 through parallel PKA- and Epac-dependent mechanisms may provide for failsafe membrane depolarization by ACTH.

Normal function of HERG K+ channels expressed in HEK293 cells requires basal protein kinase B activity

FEBS Letters ◽  
2002 ◽  
Vol 534 (1-3) ◽  
pp. 125-132 ◽  
Author(s):  
Yiqiang Zhang ◽  
Huizhen Wang ◽  
Jingxiong Wang ◽  
Hong Han ◽  
Stanley Nattel ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Normal Function ◽  
Hek293 Cells ◽  
K Channels

scholarly journals Dihydropyridine Ca2+ channel antagonists and agonists block Kv4.2, Kv4.3 and Kv1.4 K+ channels expressed in HEK293 cells

2003 ◽  
Vol 139 (3) ◽  
pp. 533-544 ◽  
Author(s):  
Noriyuki Hatano ◽  
Susumu Ohya ◽  
Katsuhiko Muraki ◽  
Wayne Giles ◽  
Yuji Imaizumi
Keyword(s):  
Hek293 Cells ◽  
K Channels
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