If we want to understand calcium's essential role of transmitter release, okay? We have to first establish a model system that we can study transmitter release, right? We've got to be able to detect transmitter release from certain preparation. Okay, and then we sort of perturb calcium, and then to see whether the transmitter release is order or not, right? Okay so, how do we do it? We have little reagents, but most importantly in the old days, and still now, we have one of the simple preparation which is the neuromuscular junction. Okay, well the nerve cells will release transmitter to the muscle. And this is a pretty good model system. The reason is that well, it's relatively easy to isolate this nerve with the muscle from frog, from mice. Okay, and one especially advantage is that the muscle cell are pretty big, okay. So, if we want to record from the muscle cell, their electrical response, their post-synaptic response, it's relatively easy. Okay with the smooth cells. Nowadays, it's much easier with the modern technique, but in the old days, much more difficult, okay? So, for example you already have the neuromuscular junction preparation and then you can. You can stimulate using the electrode to trigger the action potential, the nerve, okay? And then you have another electrode in the post-synaptic cell, in the muscle. Once you stimulate and it says, in the regular condition with the regular solution that it has normal calcium in the solution for example, 2 million molar. What you observe is you can record the transmitted release by recording the post-synaptic response to the transmitted release. Well, there is some depolarization and if you can imagine the current that mediates it. The depolarization that EPS sees. Okay then how do you design your experiment to demonstrate calcium is important? Okay, you can remove calcium. How do you remove calcium? >> [INAUDIBLE] >> To engineer buffers so well. You note we are making the buffers. You note its composition, right. So we are making new buffer. You are eliminating calcium. So you can make new buffer without calcium. And sometimes you can add different divalent ions with them if you know calcium is important, well, if you remove calcium you are not only removing this calcium, because you are also changing the ionic composition, where you can just replace, you're adding the similar amount of mechanism. Okay, it's the same concentration, and you do that experiment. Okay, so you can record the nervous bounce, and then you can profuse the cell, without external calcium, and then low and behold you found, there's no transmitter with this, okay? And indeed, people do that experiment, they found there's not transmitter release. And you can even do more sophisticated experiments that, rather than directly removing calcium, you could use the calcium chelator. For example, this is one of them, called EGTA. Now EGTA is a chelator alkalinity that it can selectively chelate calcium, okay. And so you can add enough chelator to chelate the calcium and then you can look at the synapse transmission response again. The synapse transmission might be gone. So why do you want to add the EGTA? Could you add the EDTA? EGTA is another chelator. And it can also chelate calcium. But people for the physiological study of calcium response usually will not use EGTA. Well, indeed EGTA can chelate calcium, but it is not so specific. You can also chelate magnesium, you can also chelate zinc, okay, you can chelate iron, you can chelate a lot of ions. All right, so that's the reason your EGTA might not be so specific. In fact, in the EDTA has already been widely used to stop some chemical reactions. For example if we are digesting some DNA with some restriction enzymes, okay. And then a lot of times you can add EDTA to heating mechanism. Okay because those restriction enzymes rely on the mechanism as a co-factor to be catalytic active okay? So EDTA has been useful for that. But if you want to specific EGTA is one of it. But the EGTA is a slow buffer, meaning that it can chelate calcium, but it binds to calcium relatively slow. And therefore, people, like Roger Tsien invented data, EGTA homolog, it's called BAPTA, that is similar as EGTA, but it has this aromatical rings. That replacing the this carbon chain here okay, and this actually create a small molecule that is much much faster and specific to chelating calcium. Indeed, the reason why Roger Tsien create BAPTA and later is a lot of derivative is Roger Tsien wants to create calcium indicator that can specially recognize calcium to generate some signal changes. For example, in the BAPTA condition if you measure its absorption that with and without calcium, you can see there's a spectral difference in terms of absorbance of calcium. And we are going to further discuss a little bit later how this is more than calcium indicators are derived from BAPTA and it's analog and how does that revolutionize how we started the signalling or second messengers including calcium in the life cells. So that is a very important tool, okay?
