scholarly journals ML277 regulates KCNQ1 single-channel amplitudes and kinetics, modified by voltage sensor state

2021 ◽  
Vol 153 (12) ◽  
Author(s):  
Jodene Eldstrom ◽  
Donald A. McAfee ◽  
Ying Dou ◽  
Yundi Wang ◽  
David Fedida
Keyword(s):  
Single Channel ◽  
Therapeutic Potential ◽  
Induced Pluripotent Stem Cell ◽  
Voltage Sensor ◽  
K Channel ◽  
Cardiac Repolarization ◽  
Wild Type ◽  
Mutant Channel ◽  
Induced Pluripotent ◽  
Sensor State

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell–derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.

scholarly journals Therapeutics potentiating microglial p21-Nrf2 axis can rescue neurodegeneration caused by neuroinflammation

2020 ◽  
Vol 6 (46) ◽  
pp. eabc1428
Author(s):  
A. Nakano-Kobayashi ◽  
A. Fukumoto ◽  
A. Morizane ◽  
D. T. Nguyen ◽  
T. M. Le ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Neuronal Survival ◽  
Disease Model ◽  
Neuronal Loss ◽  
Induced Pluripotent Stem Cell ◽  
Related Factor ◽  
Induced Pluripotent ◽  
Novel Therapeutic

Neurodegenerative disorders are caused by progressive neuronal loss, and there is no complete treatment available yet. Neuroinflammation is a common feature across neurodegenerative disorders and implicated in the progression of neurodegeneration. Dysregulated activation of microglia causes neuroinflammation and has been highlighted as a treatment target in therapeutic strategies. Here, we identified novel therapeutic candidate ALGERNON2 (altered generation of neurons 2) and demonstrate that ALGERNON2 suppressed the production of proinflammatory cytokines and rescued neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)–induced Parkinson’s disease model. ALGERNON2 stabilized cyclinD1/p21 complex, leading to up-regulation of nuclear factor erythroid 2–related factor 2 (Nrf2), which contributes to antioxidative and anti-inflammatory responses. Notably, ALGERNON2 enhanced neuronal survival in other neuroinflammatory conditions such as the transplantation of induced pluripotent stem cell–derived dopaminergic neurons into murine brains. In conclusion, we present that the microglial potentiation of the p21-Nrf2 pathway can contribute to neuronal survival and provide novel therapeutic potential for neuroinflammation-triggered neurodegeneration.

scholarly journals A Conserved Arginine Residue in the Pore Region of an Inward Rectifier K Channel (IRK1) as an External Barrier for Cationic Blockers

1997 ◽  
Vol 110 (6) ◽  
pp. 665-677 ◽  
Author(s):  
Ravshan Z. Sabirov ◽  
Tomoko Tominaga ◽  
Akiko Miwa ◽  
Yasunobu Okada ◽  
Shigetoshi Oiki
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
K Channel ◽  
Channel Blockers ◽  
Channel Activity ◽  
Wild Type ◽  
K Channels ◽  
Blocking Rate ◽  
External Barrier ◽  
Kir2.1 Channel

The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg148, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2.1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K+ > Rb+ > NH4+ >> Na+) was preserved. The coexpression made the IRK1 channel more sensitive to extracellular block by Mg2+ and Ca2+, and turned this blockade from a voltage-independent to a -dependent process. The sensitivity of the mutant channels to Mg2+ was enhanced at higher pH and by an increased ratio of mutant:WT mRNA, suggesting that the charge on the Arg site controlled the sensitivity. The blocking rate of open channel blockers, such as Cs+ and Ba2+, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg2+ or Ca2+ and that to the barrier peak for block by Cs+ or Ba2+ suggest that Arg148 is located between the external blocking site for Mg2+ or Ca2+ and the deeper blocking site for Cs+ or Ba2+ in the IRK1 channel. It is concluded that Arg148 serves as a barrier to cationic blockers, keeping Mg2+ and Ca2+ out from the electric field of the membrane.

scholarly journals Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

2021 ◽  
Vol 22 (24) ◽  
pp. 13500
Author(s):  
Beatrice Badone ◽  
Carlotta Ronchi ◽  
Francesco Lodola ◽  
Anika E. Knaust ◽  
Arne Hansen ◽  
...  
Keyword(s):  
Functional Data ◽  
Pluripotent Stem Cell ◽  
Protein Localization ◽  
Induced Pluripotent Stem Cell ◽  
Wild Type ◽  
Time To Peak ◽  
Activation Mechanisms ◽  
Natural Inhibitor ◽  
Induced Pluripotent

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a “superinhibition”. Aim: To test in cardiomyocytes (hiPSC-CMs) derived from a PLN p.Arg14del carrier whether (1) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093). Methods: Ca2+ transients (CaT) were recorded at 36 °C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN where immunolabeled in single hiPSC-CMs. Mutant preparations (MUT) were compared to isogenic wild-type ones (WT), obtained by mutation reversal. Results: WT and MUT differed for the following properties: (1) CaT time to peak (tpeak) and half-time of CaT decay were shorter in MUT; (2) several CaT profiles were identified in WT, “hyperdynamic” ones largely prevailed in MUT; (3) whereas tpeak rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca2+ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 turned all the above properties to resemble those of MUT; when applied to MUT, PST3093 had a smaller or negligible effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs. Conclusions: Functional data converge to argue for PLN p.Arg14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, possibly including dysregulation of Ca2+-dependent transcription.

scholarly journals Pluripotency and Immunomodulatory Signatures of Canine Induced Pluripotent Stem Cell-derived Mesenchymal Stromal Cells Are Similar to Harvested Mesenchymal Stromal Cells

2020 ◽  
Author(s):  
Arash Shahsavari ◽  
Prasanna Weeratunga ◽  
Dmitry A. Ovchinnikov ◽  
Deanne Whitworth
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Epithelial To Mesenchymal Transition ◽  
Therapeutic Potential ◽  
Induced Pluripotent Stem Cell ◽  
Interferon Γ ◽  
Mesenchymal Transition ◽  
Cox 2 ◽  
Induced Pluripotent ◽  
Tgf Β1

Abstract Background: With a view towards harnessing the therapeutic potential of canine mesenchymal stromal cells (cMSCs) as modulators of inflammation and the immune response, and to avoid the issues of the variable quality and quantity of harvested cMSCs, we examined the immunomodulatory properties of cMSCs derived from canine induced pluripotent stem cells (ciMSCs), and compared them to cMSCsharvested from adipose tissue (cAT-MSC) and bone marrow (cBM-MSC).Methods and results: Deep sequencing of the ciMSC transcriptome confirmed that ciMSCsexpress more genes in common with cBM-MSCsthan with the ciPSCs from which they were derived. Both ciMSCs and cBM-MSCsexpress a range of pluripotency factors in common withthe ciPSCsincluding NANOG, POU5F1 (OCT-4), SOX-2, KLF-4, LIN-28A, MYC, LIF, LIFR, and TERT. However, ESRRB and PRDM-14, both factors associated with naïve, rather than primed, pluripotency were expressed only in the ciPSCs. LOXL-2, which is involved in epithelial to mesenchymal transition (EMT), is also expressed in ciMSCs and cBM-MSCs but notciPSCs. ciMSCsconstitutively express the immunomodulatory factors iNOS, GAL-9, TGF-β1, PTGER-2αand VEGF, and the pro-inflammatory mediators COX-2,IL-1βand IL-8.When stimulated with the canine pro-inflammatory cytokines tumor necrosis factor-α (cTNF-α), interferon-γ (cIFN-γ), or a combination of both, ciMSCsupregulated their expression ofIDO,iNOS, GAL-9,HGF, TGF-β1, PTGER-2α, VEGF, COX-2, IL-1β andIL-8.When co-cultured with mitogen-stimulated lymphocytes, ciMSCsdownregulated their expression of iNOS, HGF, TGF-β1andPTGER-2α, while increasing their expression of COX-2, IDO and IL-1β. Conclusions: Taken together, these findings suggest that ciMSCs possess similar immunomodulatory capabilities as harvested cMSCs and support further investigation into the potential use ofciMSCsfor the management of canine immune-mediated and inflammatory disorders.

Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2)

1998 ◽  
Vol 275 (3) ◽  
pp. F415-F422 ◽  
Author(s):  
Gordon G. MacGregor ◽  
Jason Z. Xu ◽  
Carmel M. McNicholas ◽  
Gerhard Giebisch ◽  
Steven C. Hebert
Keyword(s):  
Protein Kinase ◽  
Open Channel ◽  
Single Channel ◽  
K Channel ◽  
Wild Type ◽  
Site Mutation ◽  
Patch Clamp Technique ◽  
Active Channels ◽  
Channel Configuration ◽  
Kinase A

The activity of the cloned renal K+ channel (ROMK2) is dependent on a balance between phosphorylation and dephosphorylation. There are only three protein kinase A (PKA) sites on ROMK2, with the phosphorylated residues being serine-25 (S25), serine-200 (S200), and serine-294 (S294) (Z.-C. Xu, Y. Yang, and S. C. Hebert. J. Biol. Chem. 271: 9313–9319, 1996). We previously mutated these sites from serine to alanine to study the contribution of each site to overall channel function. Here we have studied each of these single PKA site mutants using the single-channel configuration of the patch-clamp technique. Both COOH-terminal mutations at sites S200A and S294A showed a decreased open channel probability ( P o), whereas the NH2-terminal mutation at site S25A showed no change in P o compared with wild-type ROMK2. The decrease in P o for the S200A and S294A mutants was caused by the additional presence of a long closed state. In contrast, the occurrence of the S25A channel was ∼66% less, suggesting fewer active channels at the membrane. The S200A and S294A channels had different kinetics compared with wild-type ROMK2 channels, showing an increased occurrence of sublevels. Similar kinetics were observed when wild-type ROMK2 was excised and exposed to dephosphorylating conditions, indicating that these effects are specifically a property of the partially phosphorylated channel and not due to an unrelated effect of the mutation.

scholarly journals Transplantation of Mouse Induced Pluripotent Stem Cell-Derived Podocytes in a Mouse Model of Membranous Nephropathy Attenuates Proteinuria

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Amin Ahmadi ◽  
Reza Moghadasali ◽  
Vahid Ezzatizadeh ◽  
Zeinab Taghizadeh ◽  
Seyed Mahdi Nassiri ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Membranous Nephropathy ◽  
Therapeutic Strategy ◽  
Therapeutic Potential ◽  
Induced Pluripotent Stem Cell ◽  
Glomerular Diseases ◽  
Heavy Proteinuria ◽  
Immune Mediated ◽  
Induced Pluripotent ◽  
Shed Light

Abstract Injury to podocytes is a principle cause of initiation and progression of both immune and non-immune mediated glomerular diseases that result in proteinuria and decreased function of the kidney. Current advances in regenerative medicine shed light on the therapeutic potential of cell-based strategies for treatment of such disorders. Thus, there is hope that generation and transplantation of podocytes from induced pluripotent stem cells (iPSCs), could potentially be used as a curative treatment for glomerulonephritis caused by podocytes injury and loss. Despite several reports on the generation of iPSC-derived podocytes, there are rare reports about successful use of these cells in animal models. In this study, we first generated a model of anti-podocyte antibody-induced heavy proteinuria that resembled human membranous nephropathy and was characterized by the presence of sub-epithelial immune deposits and podocytes loss. Thereafter, we showed that transplantation of functional iPSC-derived podocytes following podocytes depletion results in recruitment of iPSC-derived podocytes within the damaged glomerulus, and leads to attenuation of proteinuria and histological alterations. These results provided evidence that application of iPSCs-derived renal cells could be a possible therapeutic strategy to favorably influence glomerular diseases outcomes.

scholarly journals Use of human iPSCs and kidney organoids to develop a cysteamine/mTOR inhibition combination therapy to treat cystinosis

2019 ◽  
Author(s):  
Jennifer A. Hollywood ◽  
Aneta Przepiorski ◽  
Patrick T. Harrison ◽  
Ernst J. Wolvetang ◽  
Alan J. Davidson ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Therapeutic Potential ◽  
Induced Pluripotent Stem Cell ◽  
Lysosomal Storage ◽  
Autophagy Flux ◽  
Mtor Inhibition ◽  
Cystine Transporter ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Kidney Organoids

AbstractCystinosis is a lysosomal storage disease caused by mutations inCTNS, encoding a cystine transporter, and in its severest form is characterized by cystine accumulation, renal proximal tubule dysfunction and kidney failure. Cystinosis is treated with the cystine-depleting drug cysteamine, however this only slows progression of the disease and there is an urgent need for better treatments. Here, we have generated and characterized the first human induced pluripotent stem cell (iPSC) and kidney organoid models of cystinosis. These models exhibit elevated cystine and cysteine levels, enlarged lysosomes and a block in basal autophagy flux. Cysteamine treatment ameliorates this phenotype except for the basal autophagy flux defect. We found that treatment with Everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes and activates autophagy but does not rescue the cystine/cysteine loading defect. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and Everolimus corrects all of the observed phenotypes indicating that a combination therapy has therapeutic potential to improve the treatment of cystinosis.

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