Diploma thesis is based on monitoring the succes of transfection of cell linie HEK293. In theoretical part are described principles of transfection methods, cell lines, vectors and reporter genes. HEK293 cells EBNA1 were used for practical part. It was studied the difference between GFP and EGFP plasmids. As well as using various transfection reagents under different culture conditions.

Efficient production of the recombinant proteins (r-proteins) must be based on previous testing of an expression of a small amount of the r-proteins. This work focuses on optimizing the expression of the r-proteins in 12-well plates. It includes testing of an appropriate speed of shaking, production and transfection volume. It compares all the current testing vessels (it compares a 50-ml centrifugation tube to new tested plates that can substitute the unsuitable tubes). It also compares these new tested plates to production square bottles in order to compare the r-protein expression in the plates to the r-protein expression in the bottles. It monitors effects of carbon dioxide on a number of vital cells, their viability, a relative frequency of positive cells on GFP in various cultivation vessels (plates, tubes, bottles), and pH of HEK 293 cellular cultivation during the 4-day cultivation process as well. On the basis of the results and statistical processing of the results, we have set the optimal agitation speed of 230 rpm for the 12-well plates. We have also set the appropriate production and transfection volume of 2 and 0.5 ml for the 12-well plates. In order to evaluate variables and compare cultivations in all the vessels, the tubes could be substituted by the plates. There is a statistically significant impact of carbon dioxide on the number of cells, their variability, relative frequency of cells (positive on GFP) and pH of the cellular HEK 293 cultivation in the cultivation vessels. There is the strongest r-protein expression in carbon dioxide conditions. The results of this work allow to employ the 12-well plates when we aim to test the expression of the r-proteins in a small amount and in carbon dioxide conditions. On the basis of the findings, the expression of the r-proteins in the 12-well plates and carbon dioxide conditions can substitute the expression of the r-proteins in the production bottle and in carbon dioxide free conditions.

One of the key components of the innate immune system are natural killer (NK) cells. The task of these cells is to induce apoptosis in target cells (e.g., cancer or virally infected cells). The target cells are identified by their interaction with surface receptors of the NK cells. On the surface of the NK cells, there are activating and inhibiting receptors. One of the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands of this receptor have been identified, one of them being the epithelial adhesin Epa1 of yeast Candida glabrata. The invasive candidiasis caused by this yeast is a feared complication for patients with haematological diseases. The use of the NK cells in immunotherapy includes bispecific fusion proteins which can bind to the NK receptor with one part and to tumour antigen with the other part. This work focuses on recombinant preparation of the NKp46 protein. To facilitate a study of the effects of O-glycosylation on the binding of the ligands, a mutation of the glycosylation site NKp46 T225A was prepared. A stably transfected HEK293S GnTI- and HEK293T cells had been prepared and these proteins were then extracellularly secreted. The Epa1 protein had been produced in E. coli bacterial expression system and purified. The binding ability of the Epa1 protein...

Monoclonal antibodies are well-established as a therapeutic in the biopharmaceutical market, targeting a variety of diseases and with 79 approved products by the United States Food and Drug Administration in December 2019. Therapeutic monoclonal antibodies are commonly produced as recombinant proteins in mammalian cell lines, due to their capacity of post-translational modifications, most notably glycosylation. Furthermore, an identified bottleneck within the production of recombinant proteins is the translocation of nascent proteins from the cytosol into the lumen of the endoplasmic reticulum. The signal peptide, which is located at the N-terminal of nascent proteins, plays a central role in the process of protein secretion. Several studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems. The aim of this study was to evaluate the expression of three human recombinant antibodies in Human Embryonic Kidney 293 (HEK293) cells by evaluating 16 different signal peptide combinations, consisting of eight heavy chain (HC) and two light chain (LC) signal peptides. The impact goal was an efficient secretion of recombinant antibodies, and thus lower production cost of recombinant antibodies in HEK293 cells. First, 16 HC and LC signal peptide plasmid constructs were generated for each of the three recombinant antibodies. Thereafter, transient gene expression in HEK293 cells were performed at three independent experiments. Finally, the antibody titers were quantified using Biacore concentration analysis. The produced antibody titers for the three studied recombinant antibodies were highly dependent on the used signal peptides. Interestingly, the evaluated HC and LC signal peptide combinations resulted in 3 times higher and 2 times higher antibody titers compared to the original signal peptides used by the Drug Discovery and Development platform at Science for Life Laboratory, for two of the studied antibodies respectively. The results presented in this report further demonstrates the necessity to evaluate signal peptides in order to achieve increased expression of recombinant antibodies in mammalian systems.

No / T-type Ca2+ channels are a distinct family of low voltage-activated Ca2+ channels which serve many roles in different tissues. Several studies have implicated them, for example, in the adaptive responses to chronic hypoxia in the cardiovascular and endocrine systems. Hydrogen sulfide (H2S) was more recently discovered as an important signalling molecule involved in many functions, including O2 sensing. Since ion channels are emerging as an important family of target proteins for modulation by H2S, and both T-type Ca2+ channels and H2S are involved in cellular responses to hypoxia, we have investigated whether recombinant and native T-type Ca2+ channels are a target for modulation by H2S. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS, selectively inhibits Cav3.2 T-type Ca2+ channels heterologously expressed in HEK293 cells, whilst Cav3.1 and Cav3.3 channels were unaffected. Sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ using TPEN prevented channel inhibition by H2S. H2S also selectively inhibited native T-type channels (primarily Cav3.2) in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca2+ channel Cav3.2. Results have important implications for the proposed pro-nociceptive effects of this gasotransmitter. Implications for the control of cellular responses to hypoxia await further study.

La possibilité de programmer une cellule dans le but de produire une protéine d’intérêt est apparue au début des années 1970 avec l’essor du génie génétique. Environ dix années plus tard, l’insuline issue de la plateforme de production microbienne Escherichia coli, fut la première protéine recombinante (r-protéine) humaine commercialisée. Les défis associés à la production de r-protéines plus complexes et glycosylées ont amené l’industrie biopharmaceutique à développer des systèmes d’expression en cellules de mammifères. Ces derniers permettent d’obtenir des protéines humaines correctement repliées et de ce fait, biologiquement actives. Afin de transférer le gène d’intérêt dans les cellules de mammifères, le polyéthylènimine (PEI) est certainement un des vecteurs synthétiques le plus utilisé en raison de son efficacité, mais aussi sa simplicité d’élaboration, son faible coût et sa stabilité en solution qui facilite son utilisation. Il est donc largement employé dans le contexte de la production de r-protéines à grande échelle et fait l’objet d’intenses recherches dans le domaine de la thérapie génique non virale. Le PEI est capable de condenser efficacement l’ADN plasmidique (vecteur d’expression contenant le gène d’intérêt) pour former des complexes de petites tailles appelés polyplexes. Ces derniers doivent contourner plusieurs étapes limitantes afin de délivrer le gène d’intérêt au noyau de la cellule hôte. Dans les conditions optimales du transfert de gène par le PEI, les polyplexes arborent une charge positive nette interagissant de manière électrostatique avec les protéoglycanes à héparane sulfate (HSPG) qui décorent la surface cellulaire. On observe deux familles d’HSPG exprimés en abondance à la surface des cellules de mammifères : les syndécanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si l’implication des HSPG dans l’attachement cellulaire des polyplexes est aujourd’hui largement acceptée, leur rôle individuel vis-à-vis de cet attachement et des étapes subséquentes du transfert de gène reste à confirmer. Après avoir optimisées les conditions de transfection des cellules de mammifères CHO et HEK293 dans le but de produire des r-protéines secrétées, nous avons entrepris des cinétiques de capture, d’internalisation des polyplexes et aussi d’expression du transgène afin de mieux comprendre le processus de transfert de gène. Nous avons pu observer des différences au niveau de ces paramètres de transfection dépendamment du système d’expression et des caractéristiques structurelles du PEI utilisé. Ces résultats présentés sous forme d’articles scientifiques constituent une base solide de l’enchaînement dans le temps des évènements essentiels à une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possède un profil d’expression des HSPG qui lui est propre, ces derniers étant plus ou moins permissifs au transfert de gène. En effet, une étude menée dans notre laboratoire montre que les SDC1 et SDC2 ont des rôles opposés vis-à-vis du transfert de gène. Alors que tous deux sont capables de lier les polyplexes, l’expression de SDC1 permet leur internalisation contrairement à l’expression de SDC2 qui l’inhibe. De plus, lorsque le SDC1 est exprimé à la surface des cellules HEK293, l’efficacité de transfection est augmentée de douze pourcents. En utilisant la capacité de SDC1 à induire l’internalisation des polyplexes, nous avons étudié le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggèrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gène, les HSPG sont essentiellement étudiés dans leur globalité. S’il est vrai que le rôle des syndécanes dans ce contexte est le sujet de quelques études, celui des glypicanes est inexploré. Grâce à une série de traitements chimiques et enzymatiques visant une approche « perte de fonction », l’importance de la sulfatation comme modification post-traductionnelle, l’effet des chaînes d’héparanes sulfates mais aussi des glypicanes sur l’attachement, l’internalisation des polyplexes, et l’expression du transgène ont été étudiés dans les cellules CHO et HEK293. L’ensemble de nos observations indique clairement que le rôle des HSPG dans le transfert de gène devrait être investigué individuellement plutôt que collectivement. En effet, le rôle spécifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivité à l’expression génique demeure encore inconnu. En exprimant de manière transitoire chaque membre des syndécanes et glypicanes à la surface des cellules CHO, nous avons déterminé leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant à l’effet de cette surexpression sur l’efficacité de transfection. Par contre, lorsqu’ils sont présents dans le milieu de culture, le domaine extracellulaire des HSPG réduit l’efficacité de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprimé de manière stable dans les cellules CHO, seulement une légère modulation de l’expression du transgène a pu être observée. Ces travaux ont contribué à la compréhension des mécanismes d'action du vecteur polycationique polyéthylènimine et à préciser le rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293. / With the aim to express a protein of interest, the transfer of exogenous genetic material into host cells was established in early 70s with the development of genetic engineering. Approximately ten years later, insulin was the first human recombinant protein (r-protein) produced at large scale in Escherichia coli and commercialized. Challenges associated with the production of more complex and glycosylated r-proteins brought the pharmaceutical industry to develop mammalian expression platforms. Thus, the expressed r-proteins are correctly folded and biologically actives. As a means to transfer genetic materials of interest into mammalian cells, the synthetic vector polyethylenimine (PEI) is probably the most popular due to its efficacy, ease of use, cost-effectiveness and stability in solution. Consequently, PEI is largely employed for the production of r-proteins by large scale and extensively studied in the context of non-viral gene therapy. PEI is capable to efficiently condense plasmid DNA (expression vector containing the gene of interest) to form small nanoparticles termed polyplexes. The latter must circumvent several steps to deliver the gene of interest to the cell nucleus. When formed at the optimum conditions, polyplexes exhibit a net positive charge which can interact electrostatically with negatively charged heparan sulfate proteoglycans (HSPG) located at the cell surface. There are two major families of HSPG that are largely expressed at the surface of mammalian cells: the syndecans (4 members, SDC1-4) and the glypicans (6 members, GPC1-6). Although it is generally accepted that HSPG are involved in the binding of polyplexes, their individual role toward polyplex binding and the subsequent phases of gene transfer need to be confirmed. Following optimization of the mammalian CHO and HEK293 cells transfection conditions, we undertook an in-depth study of polyplexes uptake, internalization kinetics, as well as transgene expression kinetics with the aim to better understand the mechanisms underlying gene transfer. We observed several contrasting differences between the two cell lines and the type of PEI used. Our results presented as a scientific article, establish strong basis of the gene transfer process over-time. Every cell type possesses its own expression profile of HSPG which can display individual potency toward gene transfer. Indeed, a preliminary study conducted in our laboratory showed that SDC1 and SDC2 have distinct features with regard to gene transfer. While both are capable to bind polyplexes at the cell surface, the expression of SDC1 enhances polyplexes internalization whereas the expression of SDC2 drastically inhibits it. Furthermore, when SDC1 is expressed at the surface of HEK293 cells, the transfection efficiency is increased by twelve percent compared to control cells. By using the ability of SDC1 to mediate efficient internalization of polyplexes, we have studied the intracellular traffic of SDC1 / polyplexes complexes. Our conclusions lead to new insights concerning the path by which polyplexes can mediate efficient transfection. In the context of gene transfer, HSPG have been essentially studied in their entirety. Although the role of syndecans is the subject of some studies, that of glypicans is unexplored. Thanks to a series of chemical and enzymatic treatments leading to « loss of functions », the importance of sulfation as post-translational modification, the effect of HS chains and of glypicans on the attachment, internalization of polyplexes as well as transgene expression were investigated in CHO and HEK293 cells. Taken together, our observations indicate clearly that the role of HSPG should be investigated individually instead of collectively. Consequently, the individual potency of each HSPG member regarding gene transfer remains to be defined. We demonstrated that, in fact, the transient expression of some HSPG in CHO cells have a beneficial effect on polyplexes uptake while others have a negative effect. Unfortunately, this method did not allow concluding about their effect on transfection efficacy. However, when present in the culture medium, the extracellular domain of HSPG decreases transfection efficacy of CHO cells without inducing polyplexes dissociation. Strangely, when each HSPG is stably expressed in CHO cells, only subtle modulations of the gene expression level were observed. This study contributed to a better understanding of the mechanisms underlying PEI mediated gene transfer in CHO and HEK293 cells and clarify the role of HSPG in gene transfer.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-03-28T13:31:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Giseli_Ritterbusch.pdf: 1007939 bytes, checksum: 6829555e6aeec4b59e73c9a7f0f29087 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-28T20:59:38Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Giseli_Ritterbusch.pdf: 1007939 bytes, checksum: 6829555e6aeec4b59e73c9a7f0f29087 (MD5) / Made available in DSpace on 2017-03-28T20:59:38Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Giseli_Ritterbusch.pdf: 1007939 bytes, checksum: 6829555e6aeec4b59e73c9a7f0f29087 (MD5) Previous issue date: 2015-01-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O vírus da bronquite infecciosa das galinhas (VBI) é o agente etiológico da Bronquite Infecciosa (BI), uma enfermidade altamente contagiosa que causa grandes perdas econômicas na avicultura. O VBI é um vírus envelopado, que possui genoma constituído de RNA fita simples, que codifica 4 proteínas estruturais, dentre elas a Nucleoproteína (N), que é produzida em grande quantidade na infecção viral e é reconhecidamente imunogênica. O controle da BI se faz com a imunização das aves através da aplicação de vacinas vivas atenuadas, seguidas de vacinação utilizando antígeno inativado, sendo o sorotipo Massachusetts o único liberado para uso no Brasil. Um dos objetivos do presente trabalho foi realizar um estudo exploratório, afim de conhecer a opinião de diferentes segmentos da avicultura sobre a situação atual da ocorrência de BI nos planteis brasileiros e os custos que ela representa. Diante disso, surge então a necessidade do desenvolvimento de vacinas alternativas e seguras para controle da BI, entre elas a utilização de vacinas vetoriais. Dessa forma, com o objetivo de desenvolver uma vacina efetiva no controle da BI, amostras variantes de VBI foram clonadas em adenovírus humano recombinante e utilizadas para transfectar células HEK293, originando adenovírus recombinantes carreadores do gene N do VBI. Estes vírus foram purificados e utilizados como vacinas recombinantes para imunização de aves SPF. Com base nos dados obtidos, observou-se que apesar das diferentes estratégias de vacinação, a BI ainda é considerada uma doença de alta prevalência que continua causando significativas perdas econômicas na produção avícola de corte e postura no Brasil. Os resultados obtidos demonstraram que a vacina recombinante não induziu uma resposta sorológica detectável pelo teste de Elisa comercial utilizado, bem como não reduziu os escores de lesões nos tecidos das aves vacinadas e desafiadas. Assim, a vacina recombinante carreada por adenovírus defectivo expressando o gene N do VBI foi construída e caracterizada, porém se mostrou ineficaz e não induziu suficiente proteção às aves experimentalmente imunizadas frente ao desafio com VBI. / The infectious bronchitis virus (IBV) is the etiologic agent of Infectious bronchitis (IB), a highly contagious disease that causes great economic losses in the poultry industry. The IBV is an enveloped virus that has RNA single strand genome, encoding four structural proteins, among them Nucleoprotein (N), which is produced abundantly in viral infection and is known immunogenic. The IB control is done by immunization of birds by applying live attenuated vaccine, followed by vaccination using inactivated antigen, wherein the Massachusetts serotype is the only released for use in Brazil. One of the goals of the present work was to conduct an exploratory study in order to know the opinion of different segments of the poultry industry on the current situation of the occurrence of BI in Brazilian squads and the costs that it represents. Therefore, the development of alternative and safe vaccines to BI control is necessary, including the use of vectors. In order to develop an effective vaccine to IB control, samples from IBV field variants were cloned into recombinant human adenovirus and used to transfect HEK293 cells, resulting in recombinant adenovirus carriers of the N gene of the IBV. These recombinant viruses were purified and used as vaccines to immunization of SPF chickens. Based on the obtained data, it was observed that despite the different vaccination strategies, IB is still considered highly prevalent disease that causes significant economic losses in Brazilian poultry industry. The results here obtained showed that the recombinant vaccine does not causes detectable positive serological responses by commercial Elisa test in vaccinated chickens and does not reduce the tissues damage in vaccinated and challenged chickens. Thus, the recombinant vaccine carried by defective adenovirus expressing N gene of IBV was constructed and characterized, but seemed to be ineffective and did not induce sufficient protection to experimentally immunized chickens against IBV challenge.

From a physical perspective biological cells consist of active soft matter that exist in a thermodynamic state far from equilibrium. Not only in muscles but also during cell proliferation, wound healing, embryonic development, and many other physiological tasks, generation of forces on the scale of whole cells is required. To date, cellular contractions have been ascribed to adhesion dependent processes such as myosin driven stress fiber formation and the development of focal adhesion complexes. In this thesis it is shown for the first time that contractions can occur independently of focal adhesions in single suspended cells. To measure mechanical properties of suspended cells the Optical Stretcher – a dualbeam laser trap – was used with phase contrast video microscopy which allowed to extract the deformation of the cell for every single frame. For fluorescence imaging confocal laser scanning microscopy was employed. The ratio of the fluorescence of a temperature sensitive and a temperature insensitive rhodamine dye was utilized to determine the temperatures inside the optical trap during and after Optical Stretching. The rise in temperature at a measuring power of 0.7W turned out to be enough to open a temperature sensitive ion channel transfected into an epithelial cell line. In this way a massive Ca2+ influx was triggered during the Optical Stretcher experiment. A new setup combining Optical Stretching and confocal laser scanning microscopy allowed fluorescence imaging of these Ca2+ signals while the cells were deformed by optically induced surface forces, showing that the Ca2+ influx could be manipulated with adequate drugs. This model system was then employed to investigate the influence of Ca2+ on the observed contractions, revealing that they are partially triggered by Ca2+. A phenomenological mathematical model based on the fundamental constitutive equation for linear viscoelastic materials extended by a term accounting for active contractions allowed to quantify the activity of the measured cells. The skewness and the median of the strain distributions were shown to depend on the activity of the cells. The introduced model reveals that even in measurements, that seemingly are describable by passive viscoelasticity, active contractililty might be superimposed. Ignoring this effect will lead to erroneous material properties and misinterpretation of the data. Taken together, the findings presented in this thesis demonstrate that active processes are an essential part of cellular mechanics and cells can contract even independently of adhesions. The results provide a method that allows to quantify active contractions of suspended cells. As the proposed model is not based on specific assumptions on force generating processes, it paves the way for a thorough investigation of different influences, such as cytoskeletal structures and intra-cellular signaling processes, to cellular contractions. The results present an important contribution for better mechanical classification of cells in future research with possible implications for medical diagnosis and therapy.

N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...

Yes / Introduction:The human ether-a-go-go related gene (hERG) encodes the pore forming protein which mediates the rapid delayed rectifier K+ current in the heart (IKr). Together with other ion channels hERG determines the cardiac action potential and regulates the heart beating. Dysfuction of the hERG ion channel will lead to acquired long QT syndrome (LQTS). Therefore, new drug candidates must pass the test for a potential inhibitory effect on the hERG current as a first step in a nonclinical testing strategy. Arrhythmias in patients with LQTS are typically triggered during physical or emotional stress, suggesting a link between sympathetic stimulation and arrhythmias. It is well known that potassium level can affect the QT interval through affecting IhERG both in vivo and in vitro.In this study, we try to find out whether the trigger effect still exist when K+ changes violently in a short time period. In other words, whether the risk of TdP aggravate when patients suffer from acute water electrolyte balance disorder, which is a common symptom in hot weather. Methods: HEK293 Cell line stably expressing hERG channel were cultured in DMEM supplemented with 10% of fetal bovine serum.Whole-cell patch-clamp method was applied for ionic current recordings. The compositions of pipette was (in mM) 125 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 5 Na-ATP adjusted to pH 7.2 with KOH. The bath solutions for recording the IhERG currents was 136 NaCl, 4 KCl, 1 MgCl2, 10 HEPES-Na, 1.8 CaCl2 and 10 glucose, pH 7.4 with NaOH. The low extracellular K+ solution was 115 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 10 Na-ATP adjusted to pH 7.2 with NaOH. Patch-clamp experiments were performed at room temperature (22 ± 1°C). The recording of low K+ current was carried out immediately after the original normal K+ solution has been totally replaced. Isoproterenol (ISO) 100nM was added into both kinds of K+ solution to apply the effect of β1-AR stimulation. Results: We found that low K+ solution increased IhERG from 907.39±18.68to 1620.08±249.44pA(n=30,P<0.05); Low K+also shifted the I-V curve to the left. IC50 in control is 10.31±5.52 mV, low K+ is -6.15±1.58 mV. When adding ISO 100nM to extracellular solution, same effects were shown for both groups.ISO decreased Imax for both group. In control group, Imax reduced from 907.39±18.68to493.16±54.41pA (n=30, P<0.01), while in low K+ group, I max decreased Imax from 1620.08±29.44to 488.48±81.87pA(n=30,P<0.05). At the same time, ISO shifts the I-V curve to the right for the control group and shift the curve to the left for low K+ group. IC50 in control when added ISO is 22.25±3.80 mV, while IC50 in low K+ group after adding 100nM ISO is -31.00±5.73 mV. Conclusion: The results from this study is contradict to those in our previous study where low K+ combined with ISO can lead to temporarily increase of QT interval in vivo.It is reported that an increase in net outward repolarizing current, due to a relatively large increase of IKs, is responsible for the changes of QT interval in response to beta-adrenergic stimulation in vivo(2). Therefore future studies need to co-transfect IKs channel to confirm this. References: 1. Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, et al. Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. The Journal of clinical investigation. 2009;119(9):2745- 57. 2. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. Journal of the American College of Cardiology. 2000;35(3):778-86.