hERG run down and resealing

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mvithya
mvithya's picture
hERG run down and resealing

Dear friends
I'm working with automated patch clamp for hERG current measurement in HEK 293 cells
I frequently have problem of run down of current and recently resealing of cells very soon.
Can anyone help me in figuring why this occurs and means to overcome this
Thanks

Fraser Moss
Fraser Moss's picture
I refer you to these previous
Bluejay
Bluejay's picture
 

 
1)  Sometimes replacing 40mM KCl of internal soltuion with 40mM KF helps.   You may wan to vary this amount, i.e. 40mM +/- 20mM to optimize your system. 
2)  In a paper from Roche, they screened over 20 cell lines for hERG and selected the best based on several criteraia, one being less than 20% run down or run up.  Ideally if you are making your own clone, you would pick one with reduced rundown.
3)  I have used cells with as much as 40% rundown, but have to do a large number of duplicates (on the IonworksHT), probably due to the higher variability of cell response with the lower seals.  Yet with enough duplicates, the hERG blockers were comparable with the literature.
4) For some automated systems, particularly the IonworksHT with platic plate, HEK tend to seal poorly compared to CHO cells.  Also HEK cells tend to form grape-like clusters that can cause problems.
5)  It would help if people indicate what technique is used and what type of  automated system since there are at leat 6 different types of autoamated systems in use.

Fraser Moss
Fraser Moss's picture
Bluejay wrote:

Bluejay wrote:

2)  In a paper from Roche, they screened over 20 cell lines for hERG and selected the best based on several criteraia, one being less than 20% run down or run up.  Ideally if you are making your own clone, you would pick one with reduced rundown.

 
Can you post what the reference for that Roche Paper is please?

Bluejay
Bluejay's picture
I believe this was the

I believe this was the reference.  Here's the title from PubMed.
J Biomol Screen. 2005 Dec;10(8):832-40. Epub 2005 Oct 18.

 
A place for high-throughput electrophysiology in cardiac safety: screening hERG cell lines and novel compounds with the ion works HTTM system.
Guthrie H, Livingston FS, Gubler U, Garippa R.

 

lazy
lazy's picture
40 % rundown for how many

40 % rundown for how many minutes?
Thanks.

Bluejay
Bluejay's picture
It takes about 12 minutes

It takes about 12 minutes from initial reading to add drug and take a second reading for a whole, 96-well compound plate.  So the answer to your question is 12 minutes.  (This is a perforated, whole cell system.)

PhilM
PhilM's picture
Have you tried extrapolating

Have you tried extrapolating the fit of the run down during the first 5 min in no drug over the whole experiment during drug application and normalizeing you data to that fit?
I corrects very well for rundown and if you test it with a low affinity drug like Risperidone you can see the washout too.

lazy
lazy's picture
Would you please provide the

Would you please provide the detail of your fitting method?

PhilM
PhilM's picture
I do what frasermoss said to

I do what frasermoss said to do in this post
http://www.scientistsolutions.com/t8657-how+to+measure+real+herg+inhibition%3f.html
see the last entry in the list