 Is cholesterol important for any function? You bet. You will hear more about many different membrane proteins, I hope. But this is a particular love story of my ligand-gated iron channels. They are imperative in the nervous system where when a nerve signal reaches the end of one nerve cell and has to jump to the next one, this happens by releasing small compounds in neurotransmitters that diffuse over, say, 10 micrometers or so, and then they bind to another receptor. If the right neurotransmitter finds the right receptor, this ligand-gated ion channel, that is a channel responding to a ligand, will open up and create a new nerve impulse in the next nerve cell, and we get nerve signal propagation. For a long time, we hardly knew anything about these receptors, but today they're fascinating because they're kind of Dr. Jekyll and Mr. Hyde of receptors. They can be either anionic or cationic channels. No, anionic and cationic. They can bind any of a bunch, like a dozen different small neurotransmitters, and depending on what receptor I have, I will either hyperpolarize or depolarize the membrane. In addition to that, they're very sensitive to small compounds such as anesthetics, alcohol. It's in fact the main targets of those compounds, but they don't compete with this ligand. In fact, these compounds can't open the channel, they can only amplify or dampen this property. I won't take you through all the details of them, but I will show what they do with cholesterol. So normally, under normal circumstances, one of these channels, say the 5-HD3 serotonin receptor, it's going to work great. We can measure that in the lab by adding a bit of the molecule opening it, and then you see this current. After a while it will close, and then we add a bit of the compound again, and it's repeated. If I now take the same channel and do repeat the experiments, but in a membrane deficient in cholesterol, this is what happens. Do you see? There's almost no current at all remaining. So this is a channel that is not just that cholesterol can somehow influence the channel. It's a channel where the normal functionality will not work at all, unless you have cholesterol present in the membrane. And that's kind of interesting, because this is a nerve system channel that will only occur in vertebrates. Vertebrates will always have cholesterol expressed, but then we have bacteria, and bacteria do not have cholesterol expressed, which is correlated, again, with not having a nervous system. Twenty years ago we didn't have any good structures of these channels. Today we do. The first structures were determined by Nigel Anouin, and they were very low-resolution cryeum results by today's standards. It was amazing then. We were thrilled when this came out. So this is the two-dimensional class just of the transmembrane section, and what made us ecstatic here is that we could see that it's a pentamer, five different subunits, and each subunit has four helices going straight to the membrane. There is one helix in each subunit pointing towards the pore, the red part there, and then one helix pointing furthest out from the membrane. We couldn't see any cholesterol there, though. That's the acetylchol receptor, by the way. But armed with that, there were several groups that had a really interesting hypothesis where the cholesterol might bind. It could go perhaps between the helices in different places, maybe in the outer parts, maybe attached on the surface. And Grace Branigan and Michael Klein did a beautiful set of simulations where they just made attempts placing cholesterol in different places, and then they tried to reason what would that lead to, and then comparing that to Nigel Anwin's structure. I'm not showing you this because it's right. In fact, it turned out to be wrong, but it's still a beautiful hypothesis, and I think this is how we should develop science. Come up with a hypothesis, test it against experiment, discard the ones that are already incompatible, and as new experimental data comes out, then we might have to discard more of our models, or maybe even all of our models, but it's a role model for how science should be done. Why am I mentioning this? Well, because today we have structures where we see that cholesterol. Remember that fourth helix, the one furthest away from the pore? A few years ago, we started getting more and more functional indications that if you do mutations in that helix under some circumstances, it appears that we influence how the channel works, and that could be related to cholesterol binding. And then just a year or two ago, colleagues of ours, Chris Ulens and John Benziger, they found out that there is a cholesterol binding site in this fourth helix in the transmembrane domain. Beautiful work that confirmed all of these things that we had thought a long idea for a long while. In fact, we too have been able to determine structures of this in collaboration with Ryan Hipsgrub, where Yu-Huan Chang and my team has done simulations of the structure. This is an acetylcholine receptor, just as Nigel Angwis was, and in this particular receptor, we have cholesterol bound here in the transmembrane where it says CHS, cholesterol, hemisuxetate. Not only do we see cholesterol binding, we see a whole range of other molecules bound in particular ellipid, but not where you think it might bind. In this particular structure, we have an intracellular domain inside the cell here that is somehow responsible for keeping all five subunits apart. And what Yu-Huan and Ryan's team noticed is that we likely have a density with a lipid stabilizing that. So this is a single lipid located outside of the bilayer because it's interacting. The lipid is stabilized by the protein, but the protein is also likely stabilized in its pentameric conformation by this lipid. Pretty cool. And here too, we have overall stabilization of this channel, either in open or closed states, depending on whether we have all these compounds present or not. I could talk an hour about that, but I won't in the interest of time, but they're amazing channels. But I will show you one last thing about them. A relative of these channels is the GABA receptor, and it's arguably the most important target for anesthetics. So the GABA receptor itself, it was only two years ago we started having structures of them. One important, not anesthetic, but sedative, that binds to GABA is diazepam. You might have never heard of diazepam, but you might have heard of Valium, which is the market name for diazepam. What Jochuan and Ryan's team showed that if you take GABA and bind diazepam to it, diazepam will rigidify the entire channel and cause it to be slightly narrower. You probably see here, right, going to the state. And that likely explains some of the effects that will have an urinary system. We're changing the conductance in these channels because we now have a diazepam bound. If you do this experiment and happen to take too much diazepam, Valium, that can be pretty severe, and then you might end up in the emergency room. And then they have to give you an antidote. And the antidote to this would be flomatzinil, which is another small compound. GABA plus flomatzinil, when we're binding flomatzinil to the same protein, do you see here how flomatzinil appears to have the opposite effect? It makes the entire transmembrane domain slightly looser and pushes the subunits apart. So it makes sense. Flomatzinil will have an opposite effect and counter the effect of diazepam. So this was merely small sedatives and everything, but this works for anesthesia too. We know less about anesthetics, but the mechanisms are likely exactly the same.