Halo!
...I had saw that most of the times people used EGTA in the solution for the recording pipette in Patch Clamp, so I would like to know for what is that EGTA and if there is so important.

Danke!

EGTA = Ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid

It is a chelating agent useful for the determination/buffering of calcium in the presence of magnesium.

It is NOT to be confused with EDTA - Ethylenediaminetetraacetic
which is a calcium chelator which will also chelate Magnesium and other divalent cations.

EGTA is included in the patch pipette for whole cell patch clamp recording to buffer fluctuations in intracellular [Ca2+] during recording. This prevents contamination of the recorded ion channel signal by Ca2+ dependent second messenger signaling pathways (e.g. calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of certain channels) which could be stimulated by the activation of the ion channel under study. Furthermore, you rupture the cell's membrane during whole cell clamp. It will become very unhappy and release Ca2+ from the intracellular stores in response to the insult. The EGTA will buffer large fluctuations in the intracellular [Ca2+] and make it easier to maintain a good seal during the recording. There are protocols that do not include EGTA in the pipette, but under these conditions it is very hard to maintain the seal.

...it sounds a good explanation, thanks.

Anyway, if we want to see the Ca++ component of some currents, we have to avoid to used EGTA, or not? For example, when we study the currents throught nicotinic receptors, wich are currents for both, Na+ and ca++, and we want to see how much is implicate each ion in the wholle current, then could EGTA introduce some alterations? Or when we want to see the activation of channels that need Ca++ to become open, like BK channles...

Thanks...

As an example for BK patching you can have 10mM EGTA in your patch pipette solution and on the day of recording add a set concentration of fresh CaCl2 (say 0.2mM) to give a final FREE [Ca2+] of 1.9 nM which should remain buffered during your experiment but is sufficient to activate the BK. Av alternative is to use Perforated Patch clamp instead of whole cell patch clamp in which the membrane is not ruptured put punctured with with hundreds of tiny holes by an ionophore like Gramacidin or amphotericin B. This allows the patching of whole cell currents but slows the exchange of cytosol with pipette solution.

For nicotinics you can include 0.9 mM EGTA in your patch pipette and 0.4mM CaCl2. The major source of the Ca2+ observed in your nicotinic whole cell current is going to come from the extracellular solution (where it will be about 2mM). The EGTA is not going to stop this, but it will buffer any large increases in intracellular Ca2+ due Ca2+ flux though the nicotinic channels.

...something else, if EGTA is add to one solution, is normal that the solution become more alkaline? Because EGTA is one acid, so one can think should be the contrary...

Thanks.

You should have 10mM HEPES or an equivalent in both your patch pipette solution and extracellular solution to buffer any potential pH changes

...yes, the solution have 10 mM HEPES, and when I add EGTA, it becomes more alkaline; of course I can adjust it with some acid, is just that I dont understand why it become more alkaline if EGTA is one acid. ... ... ...

Hi ,
Regarding EGTA concentration in BHK cell experiments, what is the highest concentration which you tried?

...in the pipette we used 0.5mM CaCl2 and 5mM EGTA; in the external solution we have 2mM CaCl2. Could be good or something should be chage to get better results?

I'm sorry, can you repost that last message...it was hard to follow with what i believe are a few typo's
Thanks

Kein problem.

...I said, in the solution for the micropipette for whole cell recordings, we put 0,6mM of calcium and 5mM EGTA, and in the external solution (ACSF) we used 2mM calcium. Could be good or something should be chage to get better results?