[Neuroscience] Re: measuring resting potential of HEK cells

SJM Guzman jose.guzman at medizin-uni-leipzig.de
Tue Dec 6 07:20:57 EST 2005


David wrote:
> Hope you could help me help me with this question.  I would like to
> know whether expression of a Na channel in HEK cells results in Na
> loading which may be affecting the results of an assay based on a
> membrane voltage dye.
> What is the best way to measure resting potential using e-phys and
> without going whole cell (ie not disrupting the intracellular salt
> concentration)?
> (i)  Is it possible to measure the resting potential in cuurent clamp
> mode with sharp electrodes (0.01 to 0.1 uM tip diameter) and or are HEK
> cells too flat for this?
> (ii)  Would a current clamp of an on-cell patch give me an accurate
> measurement of membrane voltage?
> (iii)  Should I go whole cell but take the current -clamp measurement
> as quickly as possible (before the cell interior is dialyzed out)?
> 
> Thanks,
> 
> David
> 
  Hi David:

I am far away of being an expert, but I would like to share with you my 
suggestions about the question.

(i) A possible solution seems to me to try with sharp electrodes. If 
not, perforated patch-clamp should be a good possibility to study. Just 
ask here if you want to know about it, there're a lot of competent 
people working with this configuration.

(ii) With cell-attached recordings the potential across the patch 
pipette is dependent on the resting membrane potential, which cannot be 
determined!!!

(iii) I would be also possible to read the liquid junction potential 
(the potential between the bath and the pipette solutions) and then 
corrected from the membrane potential recorded under whole-cell conditions.

I hope it helps.

Regards!

-- 
ÿþS


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