Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging

Chtcheglova, Lilia A.; Atalar, Fatmahan; Özbek, Uğur; Wildling, Linda; Ebner, Andreas; Hinterdorfer, Peter

doi:10.1007/s00424-007-0418-9

Cited by 55 publications

(30 citation statements)

References 57 publications

(83 reference statements)

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“…These antibodies have been used extensively in Western blotting, immunohistochemistry, and localization of hERG channels (Cayabyab and Schlichter, 2002;Wang et al, 2004;Chtcheglova et al, 2008); however, they have not been tested for their electrophysiological effects. We evaluated both antibodies at 100 nM against hERG currents in HEK cells.…”

Section: Resultsmentioning

confidence: 99%

BeKm-1, a Peptide Inhibitor of Human ether-a-go-go-Related Gene Potassium Currents, Prolongs QTc Intervals in Isolated Rabbit Heart

Fang²,

Gao³

et al. 2010

J Pharmacol Exp Ther

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Drug-induced cardiac arrhythmia, specifically Torsades de pointes, is associated with QT/QTc interval prolongation, thus prolongation of the QT interval is considered as a biomarker for Torsades de pointes risk (N Engl J Med 350:1013-1022, 2004). Specific inhibition of human ether-a-go-go-related gene (hERG) potassium channels has been recognized as the main mechanism for QT prolongation (Cardiovasc Res 58:32-45, 2003). This mechanism has been demonstrated for a variety of smallmolecule agents, which access the inner pore of the hERG channel preferentially from inside the cell. Peptide inhibitors of hERG, such as BeKm-1, interact with the extracellular amino acid residues close to the external pore region of the channel. In this study, the isolated rabbit heart was used to assess whether BeKm-1 could induce QTc prolongation like dofetilide and N- [4-[[1-[2-(6-methyl-2-pyridinyl) ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide (E-4031). Five hearts were perfused with 10 and 100 nM BeKm-1 sequentially. ECG parameters and left ventricular contractility were measured with spontaneously beating hearts. Both concentrations of BeKm-1 prolonged QTc intervals significantly and concentration-dependently (4.7 and 16.3% at 10 and 100 nM, respectively). When evaluated for their inhibitory effect in a hERG functional assay, BeKm-1, dofetilide, and E-4031 caused QTc prolongation at concentrations that caused significant hERG channel inhibition. Lastly, two polyclonal anti-hERG antibodies were also assessed in the hERG channel assay and found to be devoid of any inhibitory effect. These results indicated that the isolated rabbit heart assay can be used to measure QTc changes caused by specific hERG inhibition by peptides that specifically block the external pore region of the channel.

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Section: Resultsmentioning

confidence: 99%

BeKm-1, a Peptide Inhibitor of Human ether-a-go-go-Related Gene Potassium Currents, Prolongs QTc Intervals in Isolated Rabbit Heart

Fang²,

Gao³

et al. 2010

J Pharmacol Exp Ther

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“…Applying this technique, VE-cadherin-binding sites on fixed endothelial cells could be identified at a lateral resolution close to 5 nm [4]. In the present study, using living human vascular endothelial cells (EA.hy926 cell line) known to respond to aldosterone [23] and to be capable of expressing the classical intracellular mineralocorticoid receptor (this study), we tried to find out whether specific binding sites for aldosterone were identifiable on the apical cell membranes.…”

Section: Discussionmentioning

confidence: 97%

Aldosterone receptor sites on plasma membrane of human vascular endothelium detected by a mechanical nanosensor

Wildling

Hinterdorfer

Kusche-Vihrog

et al. 2008

Pflugers Arch - Eur J Physiol

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The mineralocorticoid hormone aldosterone acts on target cells of kidney, colon, and the cardiovascular system through genomic and nongenomic pathways. Although the classical intracellular mineralocorticoid receptor plays a key role in mediating both pathways, it is unclear whether there are specific aldosterone receptors located on the cell surface. To search for such sites in vascular endothelium, we used an atomic force microscope (AFM) which measures unbinding forces based on single molecular recognition between an aldosterone-loaded AFM tip and the cell membrane. Aldosterone was tethered covalently via linker molecules to an AFM tip. Human endothelial cells (EA.hy926) were grown in culture and studied in buffer at 37 degrees C. Using the aldosterone-functionalized AFM tip as a mechanical nanoscale indenter, unbinding forces could be measured at randomly chosen sites of the plasma membrane. Sites with strong interactions between AFM tip and cell surface could be identified exhibiting unbinding forces of about 65 pN. The binding probability between the aldosterone-loaded tip and the cell surface at selected membrane sites was 53 +/- 7.2%. Addition of an excess supply of aldosterone to the bath solution blocked the binding of the aldosterone-loaded tip to the cell surface. The binding probability was reduced to 8.0 +/- 1.8% when an excess supply of aldosterone was added to the bath. However, it was not influenced by the addition of spironolactone or dexamethasone. We conclude that aldosterone receptor sites exist on the cell surface of vascular endothelial cells distinct from the classical mineralocorticoid receptors and insensitive to glucocorticoids. Binding of aldosterone to these receptors initiates an intracellular signaling cascade that precedes the classical genomic response and most likely participates in the control of vascular resistance.

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“…Several studies have visualized the localization of membrane proteins such as sodium pump (Jiang et al, 2009) and glucose-transporters (Chtcheglova et al, 2008) on native cell membranes at the single-molecule level using antibody binding. Recognition imaging of cadherin in living cells using an AFM cantilever with a coupled anti-cadherin antibody has shown that individual cadherin molecules form oligomers in the presence of Ca…”

Section: Discussionmentioning

confidence: 99%

Distinguishing between the molecular interactions of synaptotagmin with SNAREs and with lipid bilayer using atomic force microscopy in recognition imaging mode

Takahashi

Henderson

Edwardson

2013

Archives of Histology and Cytology

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Summary. Synaptic vesicle fusion with the presynaptic plasma membrane is mediated by the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins. Assembly of a complex between a vesicle-associated membrane protein, VAMP (a v-SNARE), in the vesicle membrane and a syntaxin 1A-SNAP-25 (t-SNAREs) heterodimer in the target presynaptic membrane drives fusion. Additionally, s y n a p t o t a g m i n a c t s a s a C a 2 + -s e n s i t i v e a n d phosphatidylserine (PS)-dependent trigger protein to initiate fusion. To dissect out the interaction of synaptotagmin with the t-SNAREs, we used atomic force microscopy (AFM) in recognition imaging mode to study its binding to the syntaxin 1A-SNAP-25 complex integrated into lipid bilayers without PS. Using a synaptotagmin-functionalized AFM cantilever, we identified a Ca 2+ -independent binding signal between synaptotagmin and the syntaxin 1A-SNAP-25 complex at the single-molecule level. Within the nerve terminal, this Ca 2+ -independent interaction could potentially locate synaptotagmin close to the t-SNAREs in preparation for recognition of the incoming Ca 2+ signal.

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Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging

Cited by 55 publications

References 57 publications

BeKm-1, a Peptide Inhibitor of Human ether-a-go-go-Related Gene Potassium Currents, Prolongs QTc Intervals in Isolated Rabbit Heart

BeKm-1, a Peptide Inhibitor of Human ether-a-go-go-Related Gene Potassium Currents, Prolongs QTc Intervals in Isolated Rabbit Heart

Aldosterone receptor sites on plasma membrane of human vascular endothelium detected by a mechanical nanosensor

Distinguishing between the molecular interactions of synaptotagmin with SNAREs and with lipid bilayer using atomic force microscopy in recognition imaging mode

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