Ca++current too low

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T9
T9's picture
Ca++current too low

Hi everybody!

I do the Ca++ L-type channel recording with whole-cell mode. The problem is that last 2 weeks Ca++current is about 200pA, whereas it was before about 1000-2000pA. Solutions are same, compensation of the cell and pipette is good. Electrodes are fine. Don not get, where I do something wrong:(((Please help to solve this problem!!!!

The FFM
The FFM's picture
 Some questions that need

 Some questions that need answering first so that you can get the best help

1) Expression system?

a) Cultured muscle cells / neurons or brain slices?
OR
b) Which cell line?  and are you using Transient or stable expression?

2) Extracellular Solutions?

a) Are you using Ca2+ or Ba2+ to record the currents through the Calcium channels?

Immediate hunch

For now I'll assume that you're using cell lines and then my first hunch is that if your recording set up has checked out fine, then you have an expression problem.

If you are doing transient transfections, then one of your plasmid preps for the beta or alpha2-delta auxiliary subunit may have gone bad.  if that is the case, surface presentation of the alpha subunit is going to be about 10-100 fold less (and therefore the peak current amplitude and current density will be much less) depending on how bad the deficit in expression of the auxiliary subunit is.

Check the DNA prep A260/280 is still 1.8-1.9 in case it has degraded (>2.0). Also perform a restriction digest to confirm they have not become contaminated or switched with another plasmid prep by accident.  I wonder if your drop in current may have coincided with a new batch of plasmid prep?

Another possibility is whether or not  you have not aliquoted you plasmids into smaller working stocks.  if not,  then perpetual freeze-thawing of the cDNA will kill the quality of the prep and thus expression.

If you are using stable cell lines, then there is a possibility that the line may have stopped expressing one of the Calcium channel subunits.  How many passages are you at is you are using a stable line?  usually people do not go beyond 20 passages post thawing from a young stock stored in liquid nitrogen because the cells begin to "misbehave". Is your selection antibiotic stock still good?

Another possibility is that you transfection reagent has gone off, or you have started using a new lot number which might be a bad batch.  Check back to see if something like this is happened.  Do you co-transfect with soluble GFP to see which cells are transfected for patching and to get a qualitative idea of the transfection efficiency and expression levels?  

You should consider checking expression of your subunits by Western Blot, and even surface presentation of the alpha subunit at the plasma membrane doing biotinylation experiments.  or fix one of your slides and do immunocytochemistry to see if you can visualize the expression. Also if you are using stable lines try some Quantitative real time PCR to establish the expression of the subunit message.

Last Hunch.

If you have been doing pharmacology on your set up one of the drugs you have used in the past might have been absorbed into the plastic of the tubing in your perfusion lines or even your recording chamber.  It might be slowly leaching out into all your present experiments and thus inhibiting the channels  If this is the case change you perfusion lines and do several washes and soaks of your perfusion chamber with lots (liters!) of deionized water.

Good luck!

Hss
Hss's picture
 

 
...I want to ask something; I know is not directly related with your question, even that maybe you know about it. Why is neccesary to compensate? How hight can be the compensation? Can it be 100%?

Regards.

T9
T9's picture
I work with rainbow trout

I work with rainbow trout cardiomyocytes, I actually do not know how high this Ca++ current should be, because when I get 1000pA, we discovered the problem with our bath electrode. I also found an article where they used alos trout cardiac myocytes and they also had something like 200-300pA .About solution: it is Cesium

T9
T9's picture
About compensation it is

About compensation it is imporatnt for correct recording, some do not do the compensation but it is recommended to do. The compensation range depends on the cell shape and size, the bigger cell, the more difficulties you have with compensation. Actually, as I know people who deal with neurons, they do not do any compensation. It is also needed to know more information about cell behaviour during seal formation and patch ruption.