hERG

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Golden ball
Golden ball's picture
hERG

Hello
I am working with hERG current transfected to CHO (very standard cells and whole cell patch condtion).
During recording a nice hERG tail current, sometimes I have resealing trouble. Then I give a slight suction to improve the whole cell configuration. My question is that with this additional slight suction, the nice hERG tail current cannot be no more appeared. Anyone has the similar experience?
Thanks for your input.

Fraser Moss
Fraser Moss's picture
Yes

Yes

You appear to be experiencing a similar problems to myself with the breakdown of the seal between the cell and the pipette once you have gone whole cell.

see this discussion in this forum for more details of what we have discussd in the past.
http://www.scientistsolutions.com/t2475-Anyone+got+a+good+cure+to+stop+access+resistance+creeping+higher%3F.html

Things to try

1) Fire polish all your pipettes

2) Make sure you include ATP, GTP and possibly even PIP2 in the patch pipette solution.

3) switch to perforated patch for longer lasting seals - make sure you fully compensate the series resistance when you do this technique though and sonicate the amphotericin.

littlebush
littlebush's picture
I want to add an alternative

I want to add an alternative tip for the series resistance problem.As for the weapons available for going into whole cell,generally there are just three.1: suction by mouth or by
syringe,also this is the most widerly used but to my experience,this is also the one suffering from the series resistance problem most .The reason is not clear but by conjection,due to the irregularity of the ruptured membrane underneath the pipette tip,they are readily resealing especially ralative high Ca2+ concentration around the site of the access to cytoplasm so high EGTA is needed(commonly 10 mM);The second one to get access into cell is by a Zap function in the amplifier,the appropriate parameters determing a Zap ,size and duration should be tested on your cell by a trial and error way and great variety exists.However,seeming troubsome,after a period of testing,you should find a good range for your cell and getting into cell is very easy at that momemt.In contrst with the suction method,the zap by imagination ,should result in much more regular rupututed membrane and generally they are larger so resealing is more difficult than under the condition of suction way.This pure conjection is not just conjection,it has been validated by the real experiment .By my own experience,using zap can get much more stable series resistance so it is worthy of being tried.However,it seems that the zap efficacy may vary depending on cell type.Some cell line in our lab is not suitble for zap because it almost will make seal loose and unapplicable.The third is the peforated patch mode already had a detailed discussion in a previous post which you can consult.In addtion,with these all three method,another isssue should be considered ,that is ,the shape of the pipette.Generally ,for low and stable series resistance in any whole cell configuration,the taper should be as blunt as possible and the tipe should be as bigger as possible.The geometry given by the pipette's taper and tipe affect a great deal in the following series resistance ,so some previous attentions are worth being paid to the pipette first.

Hope my experience and tip will help you and others.Good luck

Golden ball
Golden ball's picture
Another question, I have. My

Another question, I have. My protocol is: 2 sec to +20 mV, 2 sec to -50 mV, every 10 sec. The outward current by the 1st step looks relatively constant (stable), while the tail current shows a rundown. And finally the tail current amplitude is lower than the outward current. Then the trace of the current does not look nice.
Why this happens?

Bluejay
Bluejay's picture
I've seen the increase in

I've seen the increase in outward current upon repolarization described as 1) the channel desensitized state goes to open state before going to closed state and 2) an unusual difference between the rate of channel opening and closing--slow to close and fast to open? As to the repolarization hump being small, I've seen this attributed to low channel expression levels or low current above leak levels. Would welcome a better explanation.

Fraser Moss
Fraser Moss's picture
hERG tail currents suffer

hERG tail currents suffer particularly badly from rundown as in the whole cell patch configuration the contents of the patch pipette equilibrates with the intracellular milieu.

Steps to avoid this include switching to perforated patch so that you are not diluting the intracellular contents during recording. Of course this comes with its own set of complications because you can wait up to 15 min to establish the perforated patch and the thing can still go whole cell and perforate the entire membrane of the cell. Plus you need to sonicate the amphotericin/gramacidin each time you load the pipette to make sure it stays in solution.

If you stay in whole cell patch you should ensure you have ATP, GTP in your pipette solution and possibly even PIP2.

see the following reference

http://circres.ahajournals.org/cgi/content/full/89/12/1168

Other agents that modulate hERG include 14-3-3 epsilon. This needs the channel to be in a phosphoryalted state to bind (which normally reduces current density) but when it does bind to both the hERG N and C termini there is an acceleration it results in an augmentation of current activation in contrast to the depression of current with phosphorylation alone (Novartis Found Symp. 2005;266:75-89; discussion 89-99. Pubmed 16050263). If you slowly dilute out 14-3-3 in the whole cell configuration this could conceivably be a source of rundown.

On the whole, run down in hERG whole cell patch electrophysiology seems to be an unspoken taboo and people often correct by fitting the rundown with exponentials or straight lines and dividing the peak tail current by the fit for each pulse. This is often not included in the methods of the papers.
See Marrannes et al Comput Methods Programs Biomed. 2004 May;74(2):167-81. PMID: 15013597

Golden ball
Golden ball's picture
Is it correct to think:

Is it correct to think:
the 1st step outward current and the second step outward current (tail current) should be increased or decreased in parallel manner?
Thank you.

Fraser Moss
Fraser Moss's picture
You should study some papers

You should study some papers on hERG gating to understand the somewhat unique mechanism by which the hERG channel opens, inactivates and closes.

A gentle start would be to read

Nature. 2006 Mar 23;440(7083):463-9. by Mike Sanguinetti.

It is a helpful overview and is full of useful references that will give you a more in depth understanding once you've digested the basics.