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1.
Tissue Eng Part A ; 29(13-14): 372-383, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37130035

RESUMO

Urine-derived stem cells (USCs) are adult kidney cells that have been isolated from a urine sample and propagated in tissue culture on gelatin-coated plates. Urine is a practical and completely painless source of cells for gene and cell therapy applications. We have isolated, expanded, and optimized transfection of USCs to develop regenerative therapies based on piggyBac transposon modification. USCs from a healthy donor sample were isolated according to established protocols. Within 2 months, 10 clones had been expanded, analyzed, and frozen. Fluorescence-activated cell sorting analysis of individual clones revealed that all 10 clones expressed characteristic USC markers (97-99% positive for CD44, CD73, CD90, and CD146; negative for CD31, CD34, and CD45). The isolated USCs were successfully differentiated along the osteogenic, adipogenic, and chondrogenic lineages, suggesting multipotent differentiation capacity. Additionally, the USCs were differentiated into podocytes positive for NEPHRIN (NPHS1), podocalyxin, and Wilms tumor 1 (WT1). Transfection of USCs with a strongly expressing Green fluorescent protein plasmid was optimized to achieve 61% efficiency in live cells using several commercially available lipophilic reagents. Transgene promoters were compared in five luciferase-expressing piggyBac transposons by live animal imaging. The CMV promoter produced the highest luciferase signal, followed by EF1-α. Finally, HEK-293 and USCs were transfected with piggyBac transposons expressing lactoferrin and DNase1 for treatment of acute kidney injury associated with rhabdomyolysis. We found that both proteins were expressed in USCs and that lactoferrin was successfully secreted into the cell culture media. In conclusion, USCs represent a clinically relevant cell type that can express nonviral transgenes. Impact statement Acute kidney injury (AKI) affects over 13 million people worldwide each year, with hospitalization rates on the rise. There are no therapies that directly regenerate the kidney after AKI. Each human kidney contains approximately one million nephrons that process ∼100 L of urinary filtrate each day. Thousands of kidney cells become detached and are excreted in the urine. A small percentage of these cells can be clonally derived into urine-derived stem cells. We have optimized methods for genome engineering of adult human urine-derived stem cells for future applications in regenerative approaches to treat kidney injury.


Assuntos
Injúria Renal Aguda , Lactoferrina , Adulto , Animais , Humanos , Lactoferrina/genética , Células HEK293 , Células-Tronco , Diferenciação Celular , Desoxirribonucleases/metabolismo
2.
Stem Cell Res Ther ; 13(1): 355, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35883199

RESUMO

BACKGROUND: In diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not been compared. METHODS: We characterized our accelerated protocol by guiding the cells through differentiation with four different medias into MIXL1+ primitive streak cells with Activin A and CHIR for Wnt activation, intermediate mesoderm PAX8+ cells via increasing the CHIR concentration, nephron progenitors with FGF9 and Heparin for stabilization, and finally into differentiated podocytes with Activin A, BMP-7, VEGF, reduced CHIR, and retinoic acid. The podocyte morphology was characterized by scanning and transmission electron microscopy and by flow cytometry analysis for podocyte markers. To confirm cellular identity and niche localization, we performed cell recombination assays combining iPSC-podocytes with dissociated mouse embryonic kidney cells. Finally, to test iPSC-derived podocytes for the modeling of diabetic kidney disease, human podocytes were exposed to high glucose. RESULTS: Podocyte markers were expressed at similar or higher levels for our accelerated protocol as compared to previously published protocols that require longer periods of tissue culture. We confirmed that the human podocytes derived from induced pluripotent stem cells in twelve days integrated into murine glomerular structures formed following seven days of culture of cellular recombinations. We found that the high glucose-treated human podocytes displayed actin rearrangement, increased cytotoxicity, and decreased viability. CONCLUSIONS: We found that our accelerated 12-day method for the differentiation of podocytes from human-induced pluripotent stem cells yields podocytes with comparable marker expression to longer podocytes. We also demonstrated that podocytes created with this protocol have typical morphology by electron microscopy. The podocytes have utility for diabetes modeling as evidenced by lower viability and increased cytotoxicity when treated with high glucose. We found that multiple, diverse methods may be utilized to create iPSC-podocytes, but closely mimicking developmental cues shortened the time frame required for differentiation.


Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Células-Tronco Pluripotentes Induzidas , Podócitos , Animais , Diabetes Mellitus/metabolismo , Nefropatias Diabéticas/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glomérulos Renais/metabolismo , Camundongos , Podócitos/metabolismo
3.
Cell Mol Bioeng ; 13(1): 17-26, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32030105

RESUMO

INTRODUCTION: Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. METHODS: We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. RESULTS: piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. CONCLUSIONS: Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

4.
Genesis ; 58(5): e23357, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32078250

RESUMO

Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co-injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off-target editing. No off-target indels were observed for the top 10 predicted off-targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Cistinúria/genética , Mutação , Animais , Sistemas CRISPR-Cas , Cisteína/urina , Cistinúria/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL
5.
BMC Nephrol ; 20(1): 227, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221135

RESUMO

BACKGROUND: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1. METHODS: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay. RESULTS: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1-/- mice. Male Slc3a1-/- mice exhibited lower weights compared to Slc3a1+/+. Slc3a1-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals. CONCLUSIONS: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.


Assuntos
Nitrogênio da Ureia Sanguínea , Cistinúria/diagnóstico por imagem , Cistinúria/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/deficiência , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Cistinúria/genética , Feminino , Masculino , Camundongos , Camundongos Knockout
6.
Forensic Sci Med Pathol ; 14(3): 367-371, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29881912

RESUMO

We report a case of a woman who experienced intrauterine fetal death at full term pregnancy, and then died suddenly soon after learning about the death of her fetus. At autopsy, previously undiagnosed neurofibromatosis and an adrenal gland pheochromocytoma were discovered in the mother. Genetic screening also revealed a novel KCNH2mutation in both fetus and mother indicating type 2 congenital long-QT syndrome (LQTS). A catecholamine surge was suspected as the precipitating event of fetal cardiac arrhythmia and sudden fetal death, while the addition of emotional stress provoked a lethal cardiac event in the mother. This case illustrates the potential for lethal interactions between two occult diseases (pheochromocytoma, LQTS).


Assuntos
Canal de Potássio ERG1/genética , Morte Fetal , Síndrome do QT Longo/genética , Mutação , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Morte Súbita Cardíaca , Feminino , Parada Cardíaca/etiologia , Humanos , Neurofibromatoses/diagnóstico , Feocromocitoma/patologia , Gravidez
7.
Nat Commun ; 9(1): 1325, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29636469

RESUMO

A cell therapy platform permitting long-term delivery of peptide hormones in vivo would be a significant advance for patients with hormonal deficiencies. Here we report the utility of antigen-specific T lymphocytes as a regulatable peptide delivery platform for in vivo therapy. piggyBac transposon modification of murine cells with luciferase allows us to visualize T cells after adoptive transfer. Vaccination stimulates long-term T-cell engraftment, persistence, and transgene expression enabling detection of modified cells up to 300 days after adoptive transfer. We demonstrate adoptive transfer of antigen-specific T cells expressing erythropoietin (EPO) elevating the hematocrit in mice for more than 20 weeks. We extend our observations to human T cells demonstrating inducible EPO production from Epstein-Barr virus (EBV) antigen-specific T lymphocytes. Our results reveal antigen-specific T lymphocytes to be an effective delivery platform for therapeutic molecules such as EPO in vivo, with important implications for other diseases that require peptide therapy.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Elementos de DNA Transponíveis/imunologia , Eritropoetina/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Transferência Adotiva , Animais , Engenharia Celular/métodos , Eritropoetina/imunologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hematopoese/efeitos dos fármacos , Hematopoese/imunologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Camundongos , Ovalbumina/administração & dosagem , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/transplante , Transgenes , Vacinação
8.
J Vis Exp ; (131)2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29364221

RESUMO

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.


Assuntos
DNA/administração & dosagem , Pelve Renal/fisiologia , Rim/fisiologia , Plasmídeos/administração & dosagem , Biossíntese de Proteínas/genética , Transfecção/métodos , Animais , DNA/genética , Hidrodinâmica , Injeções , Rim/metabolismo , Masculino , Camundongos , Plasmídeos/genética , Transgenes
9.
Sci Rep ; 7: 44904, 2017 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-28317878

RESUMO

Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice.


Assuntos
Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Rim/metabolismo , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Animais , Eritropoetina/genética , Eritropoetina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Hidrodinâmica , Imunossupressores/farmacologia , Rim/patologia , Masculino , Camundongos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Transfecção/métodos
10.
Sci Adv ; 2(9): e1501228, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27626070

RESUMO

The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K(+)) channel to enable K(+) recycling coupled to transepithelial chloride ion (Cl(-)) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K(+) recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated "up" state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the "CF gender gap."


Assuntos
Fibrose Cística/metabolismo , Canal de Potássio KCNQ1/química , Complexos Multiproteicos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Animais , Canais de Cloreto/química , Biologia Computacional/métodos , Fibrose Cística/patologia , Fenômenos Eletrofisiológicos , Células Epiteliais/química , Células Epiteliais/metabolismo , Humanos , Canal de Potássio KCNQ1/metabolismo , Complexos Multiproteicos/metabolismo , Potássio/química , Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Domínios Proteicos
11.
Nucleic Acids Res ; 43(3): 1770-82, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25605795

RESUMO

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5'TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.


Assuntos
Cromossomos Artificiais Bacterianos , Elementos de DNA Transponíveis , Genoma Humano , Regiões 5' não Traduzidas , Dano ao DNA , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Plasmídeos , Reação em Cadeia da Polimerase , Transgenes , Transposases/metabolismo
12.
Biochemistry ; 50(50): 10851-9, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22085289

RESUMO

KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.


Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Lipossomos/química , Lipossomos/metabolismo , Micelas , Microinjeções , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oócitos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Xenopus laevis
13.
J Biol Chem ; 286(6): 4141-9, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21118809

RESUMO

Voltage-gated potassium (K(V)) channels, such as KCNQ1 (K(V)7.1), are modulated by accessory subunits and regulated by intracellular second messengers. Accessory subunits belonging to the KCNE family exert diverse functional effects on KCNQ1, have been implicated in the pathogenesis of various genetic disorders of heart rhythm, and contribute to transducing intracellular signaling events into changes in K(V) channel activity. We investigated the interactions between calmodulin (CaM), the ubiquitous Ca(2+)-transducing protein that binds and confers Ca(2+) sensitivity to the biophysical properties of KCNQ1, and KCNE4. These studies were motivated by the observed similarities between the suppression of KCNQ1 function by pharmacological disruption of KCNQ1-CaM interactions and the effects of KCNE4 co-expression on the channel. We determined that KCNE4, but not KCNE1, can biochemically interact with CaM and that this interaction is Ca(2+)-dependent and requires a tetraleucine motif in the juxtamembrane region of the KCNE4 C terminus. Furthermore, disruption of the KCNE4-CaM interaction either by mutagenesis of the tetraleucine motif or by acute Ca(2+) chelation impairs the ability of KCNE4 to inhibit KCNQ1. Our findings have potential relevance to KCNQ1 regulation both by KCNE accessory subunits and by an important intracellular signaling molecule.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Regulação da Expressão Gênica/fisiologia , Canal de Potássio KCNQ1/biossíntese , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Calmodulina/genética , Cricetinae , Cricetulus , Humanos , Canal de Potássio KCNQ1/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética
14.
Channels (Austin) ; 4(2): 108-14, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20139709

RESUMO

Voltage-gated potassium channels are often assembled with accessory proteins that increase their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (K(V)) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some K(V) channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (I(Ks)). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of I(Ks). Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent I(Ks) expression following prKCNE1 injection. By contrast, incubation with brefeldin A prevented KCNQ1 modulation by prKCNE1. Moreover, injection of the trafficking-deficient KCNE1-L51H reduced KCNQ1 currents. Together, these observations indicate that while assembly of KCNE1 with KCNQ1 does not require co-translation, functional KCNQ1-prKCNE1 channels assemble early in the secretory pathway and reach the plasma membrane via vesicular trafficking.


Assuntos
Ativação do Canal Iônico , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potássio/metabolismo , Animais , Brefeldina A/farmacologia , Membrana Celular/metabolismo , Cicloeximida/farmacologia , Retículo Endoplasmático , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio KCNQ1/biossíntese , Canal de Potássio KCNQ1/efeitos dos fármacos , Canal de Potássio KCNQ1/genética , Cinética , Potenciais da Membrana , Canais de Potássio de Abertura Dependente da Tensão da Membrana/biossíntese , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Proteínas Recombinantes/metabolismo , Xenopus laevis
15.
Biochemistry ; 49(4): 653-5, 2010 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-20044833

RESUMO

Voltage-gated potassium channel modulatory membrane protein KCNE3 was overexpressed and purified into both micelles and bicelles. Remarkably, microinjection of KCNE3 in bicelles into Xenopus oocytes resulted in functional co-assembly with the human KCNQ1 channel expressed therein. Microinjection of LMPC micelles containing KCNE3 did not result in channel modulation, indicating that bicelles sometimes succeed at delivering a membrane protein into a cellular membrane when classical micelles fail. Backbone NMR resonance assignments were completed for KCNE3 in both bicelles and LMPC, indicating that the secondary structure distribution in KCNE3's N-terminus and transmembrane domains exhibits only modest differences from that of KCNE1, even though these KCNE family members have very different effects on KCNQ1 channel function.


Assuntos
Proteínas de Membrana/metabolismo , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Potenciais da Membrana , Proteínas de Membrana/química , Micelas , Microinjeções , Modelos Biológicos , Dados de Sequência Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Xenopus/metabolismo
16.
J Gen Physiol ; 134(3): 207-17, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19687231

RESUMO

Modulation of voltage-gated potassium (KV) channels by the KCNE family of single transmembrane proteins has physiological and pathophysiological importance. All five KCNE proteins (KCNE1-KCNE5) have been demonstrated to modulate heterologously expressed KCNQ1 (KV7.1) with diverse effects, making this channel a valuable experimental platform for elucidating structure-function relationships and mechanistic differences among members of this intriguing group of accessory subunits. Here, we specifically investigated the determinants of KCNQ1 inhibition by KCNE4, the least well-studied KCNE protein. In CHO-K1 cells, KCNQ1, but not KCNQ4, is strongly inhibited by coexpression with KCNE4. By studying KCNQ1-KCNQ4 chimeras, we identified two adjacent residues (K326 and T327) within the extracellular end of the KCNQ1 S6 segment that determine inhibition of KCNQ1 by KCNE4. This dipeptide motif is distinct from neighboring S6 sequences that enable modulation by KCNE1 and KCNE3. Conversely, S6 mutations (S338C and F340C) that alter KCNE1 and KCNE3 effects on KCNQ1 do not abrogate KCNE4 inhibition. Further, KCNQ1-KCNQ4 chimeras that exhibited resistance to the inhibitory effects of KCNE4 still interact biochemically with this protein, implying that accessory subunit binding alone is not sufficient for channel modulation. These observations indicate that the diverse functional effects observed for KCNE proteins depend, in part, on structures intrinsic to the pore-forming subunit, and that distinct S6 subdomains determine KCNQ1 responses to KCNE1, KCNE3, and KCNE4.


Assuntos
Canal de Potássio KCNQ1/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Dipeptídeos/metabolismo , Canal de Potássio KCNQ1/antagonistas & inibidores , Canal de Potássio KCNQ1/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína
17.
J Mol Cell Cardiol ; 44(3): 571-81, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18222468

RESUMO

Life-threatening arrhythmias have been suspected as one cause of the sudden infant death syndrome (SIDS), and this hypothesis is supported by the observation that mutations in arrhythmia susceptibility genes occur in 5-10% of cases. However, the functional consequences of cardiac potassium channel gene mutations associated with SIDS and how these alleles might mechanistically predispose to sudden death are unknown. To address these questions, we studied four missense KCNH2 (encoding HERG) variants, one compound KCNH2 genotype, and a missense KCNQ1 mutation all previously identified in Norwegian SIDS cases. Three of the six variants exhibited functional impairments while three were biophysically similar to wild-type channels (KCNH2 variants V279M, R885C, and S1040G). When co-expressed with WT-HERG, R273Q and K897T/R954C generated currents resembling the rapid component of the cardiac delayed rectifier current (I(Kr)) but with significantly diminished amplitude. Action potential modeling demonstrated that this level of functional impairment was sufficient to evoke increased action potential duration and pause-dependent early afterdepolarizations. By contrast, KCNQ1-I274V causes a gain-of-function in I(Ks) characterized by increased current density, faster activation, and slower deactivation leading to accumulation of instantaneous current upon repeated stimulation. Action potential simulations using a Markov model of heterozygous I274V-I(Ks) incorporated into the Luo-Rudy (LRd) ventricular cell model demonstrated marked rate-dependent shortening of action potential duration predicting a short QT phenotype. Our results indicate that certain potassium channel mutations associated with SIDS confer overt functional defects consistent with either LQTS or SQTS, and further emphasize the role of congenital arrhythmia susceptibility in this syndrome.


Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Mutação de Sentido Incorreto , Morte Súbita do Lactente/genética , Animais , Células CHO , Cricetinae , Cricetulus , Canal de Potássio ERG1 , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go/fisiologia , Predisposição Genética para Doença , Humanos , Recém-Nascido , Síndrome do QT Longo/genética , Síndrome do QT Longo/fisiopatologia , Cadeias de Markov , Potenciais da Membrana , Transfecção
18.
Biochemistry ; 46(41): 11459-72, 2007 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-17892302

RESUMO

KCNE1, also known as minK, is a member of the KCNE family of membrane proteins that modulate the function of KCNQ1 and certain other voltage-gated potassium channels (KV). Mutations in human KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predisposition to potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has been extensively characterized, many questions remain regarding KCNE1's structure and location within the channel complex. In this study, KCNE1 was overexpressed in Escherichia coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings aimed at testing whether recombinant KCNE1 can co-assemble with the channel. Nativelike modulation of channel properties was observed following injection of KCNE1 in lyso-myristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfolded and that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viable cells. 1H-15N TROSY NMR experiments indicated that LMPG micelles are well-suited for structural studies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemical shift data confirmed that KCNE1's secondary structure includes several alpha-helices and demonstrated that its distal C-terminus is disordered. Surprisingly, for KCNE1 in LMPG micelles, there appears to be a break in alpha-helicity at sites 59-61, near the middle of the transmembrane segment, a feature that is accompanied by increased local backbone mobility. Given that this segment overlaps with sites 57-59, which are known to play a critical role in modulating KCNQ1 channel activation kinetics, this unusual structural feature likely has considerable functional relevance.


Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Sequência de Aminoácidos , Animais , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/genética , Clonagem Molecular , Surdez/enzimologia , Surdez/genética , Escherichia coli , Feminino , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/enzimologia , Síndrome do QT Longo/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/fisiologia , Reação em Cadeia da Polimerase , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Soluções , Xenopus/fisiologia
19.
Am J Hum Genet ; 71(3): 663-8, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12148092

RESUMO

Evaluation of candidate loci culminated in the identification of a heterozygous missense mutation (R67W) in KCNJ2, the gene encoding the inward-rectifying potassium current, Kir2.1, in 41 members of a kindred in which ventricular arrhythmias (13 of 16 female members [81%]) and periodic paralysis (10 of 25 male members [40%]) segregated as autosomal dominant traits with sex-specific variable expressivity. Some mutation carriers exhibited dysmorphic features, including hypertelorism, small mandible, syndactyly, clinodactyly, cleft palate, and scoliosis, which, together with cardiodysrhythmic periodic paralysis, have been termed "Andersen syndrome." However, no individual exhibited all manifestations of Andersen syndrome, and this diagnosis was not considered in the proband until other family members were examined. Other features seen in this kindred included unilateral dysplastic kidney and cardiovascular malformation (i.e., bicuspid aortic valve, bicuspid aortic valve with coarctation of the aorta, or valvular pulmonary stenosis), which have not been previously associated. Nonspecific electrocardiographic abnormalities were identified in some individuals, but none had a prolonged QT interval. Biophysical characterization of R67W demonstrated loss of function and a dominant-negative effect on Kir2.1 current. These findings support the suggestion that, in addition to its recognized role in function of cardiac and skeletal muscle, KCNJ2 plays an important role in developmental signaling.


Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/fisiopatologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/fisiopatologia , Músculo Esquelético/fisiopatologia , Mutação de Sentido Incorreto/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Caracteres Sexuais , Sequência de Bases , Condutividade Elétrica , Eletrocardiografia , Feminino , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Síndrome
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