Localization of the ergtoxin1 receptors on the vo

Authors: Lilia A Chtcheglova Fatmahan Atalar Ugur Ozbek Linda Wildling Andreas Ebner Peter Hinterdorfer

Publish Date: 2008/02/20

Volume: 456, Issue: 1, Pages: 247-254

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Abstract

The inhibition of the human etheràgogorelated hERG K+ channels is the major cause of long QT syndromes inducing fatal cardiac arrhythmias Ergtoxin 1 ErgTx1 belongs to scorpiontoxins which are K+ channelblockers and binds to hERG channel with 11 stoichiometry and high affinity K d ∼ 10 nM Nevertheless patchclamp recordings recently demonstrated that ErgTx1 does not establish complete blockade of hERG currents even at high ErgTx1 concentrations Such phenomenon is supposed to be consistent with highly dynamic conformational changes of the outer pore domain of hERG In this study simultaneous topography and recognition imaging TREC on hERG HEK 293 cells was used to visualize binding sites on the extracellular part of hERG channel on S1–S2 region for AntiKv111 hERGextracellularantibody The recognition maps of hERG channels contained recognition spots haphazardly distributed and organized in clusters Recognition images after the addition of ErgTx1 at high concentrations ∼1 μM revealed subsequent partial disappearance of clusters indicating that ErgTx1 was bound to the S1–S2 region These results were supported by AFM force spectroscopy data showing for the first time that voltage sensing domain S1–S4 of hERG K+ channel might be one of the multiple binding sites of ErgTx1Schematic representation of the hERG subunit One subunit contains six αhelical transmembrane helices S1–S6 and comprises two functionally distinct modules one that senses transmembrane potential voltagesensor domain S1–S4 and one that forms the K+selective pore S5–S6 S4 domain contains multiple basic + amino acids and acidic Asp residues − in S1–S3 that can form salt bridges with specific basic residues in S4 during gating Movements of the voltagesensor domain enable the pore domain to open and close in response to changes in membrane potential The pore domain contains the highly conserved K+ channel features—the pore helix and selectivity filter which permit selective passage of K+ ionsPeptide toxins can bind to the outer vestibule of the hERG channel 18 19 and thus are very useful tool for understanding the structure of hERG unit Fig 1 They are found in a large variety of venoms such as scorpions snakes bees sea anemone and spiders and have been widely used for structural analyses localization and determination of pore forming regions of voltagedependent K+ channels 1 Toxins modulating K+ channel function are shortchain peptides with a length of ∼30–40 aa and contain three or four disulfide bonds 20 Importantly many of the structural constraints of poreblocking toxins have been gained from the crystal structure of the KcsA K+ channel 21 Unlike the poreblocking toxins which typically bind to the outer pore domain the location and the structural determinants of receptors for gating modifier toxins are poorly elucidated Gatingmodifier toxins typically bind to the voltagesensor domain ie S1–S4 domains and shift the voltage range for channel activation 22 Thus the gating modifier toxins represent ideal probes to examine the structure of gating domains S1–S4 Some hERGspecific peptide toxins have been recently identified peptide 42 aa isolated from the Mexican scorpion Centruroides noxius named ergtoxin 1 ErgTx1 1 toxin 36 aa from the scorpion Buthus eupeus BeKm1 23 ErgTx2 24 peptide 43 aa from the American scorpion Centruroides sculpturatus CsEKerg1 25 and saxitoxin STX 22In this study we focused on the interaction of ErgTx1 which is entirely different from 10 other subfamilies of known scorpion toxins 1 with the wildtype hERG K+ channel Peptide toxins usually block the pore of the channel either directly by occupying the selective filter or by binding to an electrostatic ring surrounding the pore Previously it has been identified that ErgTx1 binds to the outer vestibule of the hERG channel 26 The binding site is probably formed by uncharged residues in S5P and PS6 linkers of the hERG channel 27 A characteristic feature of the action of ErgTx1 on hERG is incomplete block of macroscopic current event at concentrations ordersofmagnitude higher than the K d value Such effect suggests that ErgTx1 is a gating modifier rather than a pore blocker 27 28 In addition it binds near the pore and can not fully occlude the permeation pathway 29 30 The binding site for ErgTx1 on hERG is thought to be formed at least in part by the extracellular linker between S5 transmembrane helix and the pore helix S5P linker 27 which is critically involved in voltagedependent inactivation in hERG 31Generally the information about the structure and function of different voltagegated channels in living cells including hERG K+ channels were gained from patchclamp investigations Here we introduce an alternative way to probe the surface topology of hERG channel by using AFM dynamic recognition imaging and single molecule force spectroscopy Due to continuous progress in the technical aspects of the AFM and tip functionalization procedures the investigations of receptor–ligand interactions on living cells at the singlemolecule level have become possible 32 33 34 To localize receptor–ligand recognition in vitro systems and in living cells fluorescence techniques immunochemistry 35 or single molecule optical microscopy 36 are commonly used However due to the limited resolution from few tens to 200 nm the recognition sites cannot be resolved on the nanometer scale nor can they be correlated to topography features Combination of highresolution atomic force microscope AFM topography imaging with single molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events With the recent development of simultaneous topography and recognition imaging TREC 37 38 39 it becomes possible to quickly obtain the local distribution of receptors on cell surface with unprecedented lateral resolution of 5 nm 40 At present no other microscopic techniques are able to provide directly both structural information of a biological sample and related functional information at such high spatial resolutionIn this study we have used dynamic recognition imaging TREC with AFM force spectroscopy to locally identify a new receptor sites for ErgTx1 in voltage sensing domain of hERG K+ channel with a goal to extend our understanding of microscopic mechanism by which ErgTx1 blocks K+ channelsExperiments were performed either on wildtype human embryonic kidney HEK293 cells or on HEK293 cells stably expressing wildtype hERG abbreviated as hERG HEK293 kindly provided by Professor Craig January University of Wisconsin Madison WI USA HEK293 cells were grown in Dulbecco’s modified Eagles medium DMEM supplemented with 50 U/ml penicillinG 50 μg streptomycin and 10 fetal calf serum FCS in a humidified atmosphere 5 CO2 at 37°C Cultures were passaged twice a week hERG HEK293 cells were cultured as described previously 41 hERG transfected cells were maintained at 37°C in DMEM supplemented with 10 FCS 1 penicillinstreptomycin 2 mM lglutamine 01 mM nonessential amino acids 1 mM sodium pyruvate and 400 μg/ml geneticin G418 Cells were passaged weekly and were not allowed to become more than 80 confluent For AFM studies cells were seeded onto gelatincoated glass slides grown until 40–50 confluence To perform TREC measurements hERG cells were gently fixed with monomeric solution of glutaraldehyde 40a Schematic representation of functionalization procedure of AFM tip with specific antibody via aldehydePEGNHS crosslinker b Scheme of dynamic recognition imaging to visualize hERG K+ channels here binding sites an extracellular epitope between S1 and S2 domains of hERG subunit shown in light grey on a gently fixed hERG HEK293 cell surface

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