scholarly journals Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain

2016 ◽  
Vol 113 (35) ◽  
pp. 9916-9921 ◽  
Author(s):  
Carol A. Harley ◽  
Greg Starek ◽  
David K. Jones ◽  
Andreia S. Fernandes ◽  
Gail A. Robertson ◽  
...  
Keyword(s):  
Action Potential ◽  
Critical Role ◽  
Herg Channel ◽  
Pas Domain ◽  
Channel Activity ◽  
Single Chain ◽  
Orphan Receptors ◽  
Human Cardiomyocytes ◽  
Channel Function ◽  
Single Chain Variable Fragments

The human human ether-à-go-go–related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.

Functional Modulation of Cardiac ATP-Sensitive K+ Channels

Physiology ◽  
1998 ◽  
Vol 13 (3) ◽  
pp. 131-137 ◽  
Author(s):  
Masayasu Hiraoka ◽  
Tetsushi Furukawa
Keyword(s):  
Action Potential ◽  
Channel Activity ◽  
K Channels ◽  
Channel Function ◽  
Intracellular Atp ◽  
Functional Modulation ◽  
Action Potential Shortening

ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP, but MgATP is necessary to maintain the channel activity. Numerous cofactors modulate channel function. K+ channel openers activate and sulfonylureas inhibit KATP channels. The structure of cardiac KATP channel is a complex of mainly KIR6.2 and SUR2a. Activation of cardiac KATP channels contributes to action potential shortening during ischemia and plays a role in cardioprotection.

scholarly journals Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms

2010 ◽  
Vol 299 (6) ◽  
pp. F1359-F1364 ◽  
Author(s):  
Liang Fang ◽  
Dimin Li ◽  
Paul A. Welling
Keyword(s):  
Blood Pressure ◽  
Critical Role ◽  
Pressure Control ◽  
Surface Expression ◽  
Protein Product ◽  
Sequence Variants ◽  
Channel Activity ◽  
Loss Of Function ◽  
Channel Function ◽  
Channel Expression

The renal outer medullary K+ (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592–599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP2) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP2 depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP2 hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure.

Channel sialic acids limit hERG channel activity during the ventricular action potential

2012 ◽  
Vol 27 (2) ◽  
pp. 622-631 ◽  
Author(s):  
Sarah A. Norring ◽  
Andrew R. Ednie ◽  
Tara A. Schwetz ◽  
Dongping Du ◽  
Hui Yang ◽  
...  
Keyword(s):  
Action Potential ◽  
Sialic Acids ◽  
Herg Channel ◽  
Channel Activity ◽  
Ventricular Action Potential

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating

2021 ◽  
Vol 118 (44) ◽  
pp. e2108796118
Author(s):  
Carol A. Harley ◽  
Ganeko Bernardo-Seisdedos ◽  
Whitney A. Stevens-Sostre ◽  
David K. Jones ◽  
Maria M. Azevedo ◽  
...  
Keyword(s):  
Channel Gating ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
State Dependence ◽  
Herg Channel ◽  
Pas Domain ◽  
Single Chain ◽  
State Dependent ◽  
A Site

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS–CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

scholarly journals Canine Myocytes Represent a Good Model for Human Ventricular Cells Regarding Their Electrophysiological Properties

2021 ◽  
Vol 14 (8) ◽  
pp. 748
Author(s):  
Péter P. Nánási ◽  
Balázs Horváth ◽  
Fábián Tar ◽  
János Almássy ◽  
Norbert Szentandrássy ◽  
...  
Keyword(s):  
Action Potential ◽  
Time Course ◽  
Laboratory Animals ◽  
Delayed Rectifier ◽  
Ion Currents ◽  
Human Cardiomyocytes ◽  
Ventricular Cells ◽  
Repolarization Reserve ◽  
Electrophysiological Studies ◽  
K Current

Due to the limited availability of healthy human ventricular tissues, the most suitable animal model has to be applied for electrophysiological and pharmacological studies. This can be best identified by studying the properties of ion currents shaping the action potential in the frequently used laboratory animals, such as dogs, rabbits, guinea pigs, or rats, and comparing them to those of human cardiomyocytes. The authors of this article with the experience of three decades of electrophysiological studies, performed in mammalian and human ventricular tissues and isolated cardiomyocytes, summarize their results obtained regarding the major canine and human cardiac ion currents. Accordingly, L-type Ca2+ current (ICa), late Na+ current (INa-late), rapid and slow components of the delayed rectifier K+ current (IKr and IKs, respectively), inward rectifier K+ current (IK1), transient outward K+ current (Ito1), and Na+/Ca2+ exchange current (INCX) were characterized and compared. Importantly, many of these measurements were performed using the action potential voltage clamp technique allowing for visualization of the actual current profiles flowing during the ventricular action potential. Densities and shapes of these ion currents, as well as the action potential configuration, were similar in human and canine ventricular cells, except for the density of IK1 and the recovery kinetics of Ito. IK1 displayed a largely four-fold larger density in canine than human myocytes, and Ito recovery from inactivation displayed a somewhat different time course in the two species. On the basis of these results, it is concluded that canine ventricular cells represent a reasonably good model for human myocytes for electrophysiological studies, however, it must be borne in mind that due to their stronger IK1, the repolarization reserve is more pronounced in canine cells, and moderate differences in the frequency-dependent repolarization patterns can also be anticipated.

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