How to measure real hERG inhibition?

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lazy
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How to measure real hERG inhibition?

(I am not sure I attached the figure showing a rundown in hERG recording correctly. I also do not know how to paste the fifure in this space).
The time course of the hERG inhibition (over 20 min) shows a good dose-dependent inhibition, however, the rundown is (I am asure) overlapped. How to get the real inhibition by the drug?
If I measure simply the peak current amplitude without considering the rundown, even the control solution, within a few minutes, can be interpreted as inhibiting the hERG current, which is not true.
As the same, the drug effect, if measured simply from the control value as the reference, must be represented together with the real drug effect and "rundown" effect, which means the value measured should be bigger than the reality.
Previouly, there are topics in this Forum about this issue, but still I cannot figure out what is the best way for the data analysis. And, probably most of people have noticed a wide range of published IC50 values, even in the same experimental condition (temperature, voltage protocol, cell line, etc.). I think this wide range comes from partially the rundown. then, how to interprete the safety assessment of  the cardio-toxicity for drug application to the agent?
Hope this makes sense as a question.
 
 

Bluejay
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1)  One reported solution to

1)  One reported solution to rundown in hERG is to replace about 40mM of KCl in your internal solution with 40mM KF.  You may have to adjust the KF concentration.  It did not affect hERG IC50s in the presentation I saw.
2)  Many researchers screen/select a cell line based on the seal rate and rundown, eliminating cell lines with rundown of more than 20%.
3)  As discussed by this forum and others, rundown or up is generally due to phosphorylation state of the ion channel.
4)  I have used assays with a 40% rundown on IonworksHT.  It adds varability to the results so more samples are necessary, but the results are good.
5)  hERG drug IC50s are subject to a number of variables.  The biggest problem is that they stick to plastic, so use of glass is preferred.  Glass-coated 96-well plates are available.  Make dilutions in DMSO.  For Ionworks, IC50 increases markedly with increasing cell number per well.  Some hERG inhibitors are temperature sensitive, working only at 37C and not room temperature (sotolol, erythromycin).  hERG inhibition is less sensitive in Xenopus oocyte models. presumably due to drug interaction with the egg membrane.
6)  There is a lot of info on the MolecularDevices/MDS website on hERG.  Check out past user group presentations and technical notes.

Fraser Moss
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The simplest way to correct for run-down in a hERG expt is to run the protocol for 3 to 5 min in the absence of drug at the start of the experiment to allow you to fit an exponential to the peak run-down currents in the absence of drug that can then be extrapolated over the complete time course of the experiment.  Call the extrapolated current "Irundown".

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The % block of peak tail current can then be assessed as a function of the peak tail current amplitude (in the presence of drug)/ Irundown at each time point.