 Good morning. We are going to talk today about the, oops, wrong date. I was thinking about Monday. We're going to talk today about the G-protein pathway in neuroscience. It is mostly covered in chapter 11 of Albert's, of Candle, and if you have a copy of Albert's, it's usually in chapter 15 of Albert's. As usual, a concept in neuroscience also occurs in other fields of biology, and so if you've taken by 8 by 9, you may have heard of G-protein coupled receptors. But in neuroscience, really the G-protein coupled receptor pathway is vastly more utilized and more complex and more interactive than in other cell types. So we're going to do a whirlwind version of the G-protein pathway today. It goes all the way from receptors, through G-proteins, to effectors, to intracellular messengers, to kinases, to phosphorylated proteins, and if it stays activated for long enough to gene activation. So this map will appear on most of the images today, or part of the map. In addition, you can see these blue lightning bolts here. Those are places where high-energy phosphate bonds are manipulated or made or broken. Each of the boxes has several sub-boxes, because as usual in neuroscience and in biology, there are several genes that can come into play at each stage in this pathway. Obviously, very complex, obviously very evolved, and about two-thirds of the way through the talk, we are actually going to pose the question, what is the selective advantage of all of this diversity? So let's get started. Back to 1921, almost a century ago, the proof of chemical synaptic transmission, and you may remember that Otto Lerde, the great Viennese physiologist pharmacologist, did this experiment with two frog hearts that shared a bucket of ringer solution, and he stimulated the vagus nerve, the vagus nerve, from the leading to heart A, heart B stopped beating, but later on heart A stopped beating, and so did later on heart B. Clearly, there was a diffusible stimulus going from heart A to heart B. That diffusible stimulus, the so-called vagus stuff, we now know, and we learned very quickly thereafter, is acetylcholine. But it is acetylcholine acting on muscarinic receptors, not nicotinic receptors. So nicotine has no effect on these receptors, and muscarin, a poison from mushrooms, has no effect on nicotinic receptors. So we have also showed you a version of this slide in which some post-synaptic membranes contain G-protein coupled receptors. Some people call them metabotropic because they have a little bit of metabolism involved, rather than ligand-gated channels. Now, G-protein coupled receptors, because of their unique kinetics, and because of their high sensitivity to neurotransmitters, don't need to hang around simply at the synaptic cleft. They can be several microns away from the synaptic cleft as well. There are G-protein coupled receptors for serotonin, for acetylcholine, again muscarinic, and for dopamine, and for dozens of other neurotransmitters. Any questions? Okay, so moving along here, several small molecule transmitters serve as agonists, that is, they activate both ligand-gated channels and G-protein coupled receptors, GPCRs, among vertebrates. The situation gets even more complex for invertebrates, because some neurotransmitters are unique to invertebrates that have the same function, that they activate both types of receptor. Acetylcholine activates nicotinic, acetylcholine receptors, as well as muscarinic. We've said that relentlessly today. GABA activates receptors, very cleverly called GABA A and GABA B receptors. GABA A are ligand-gated channels, GABA B are GPCRs. Glutamate has a slightly different terminology. There are the ionotropic glutamate receptors, and there are the metabotropic glutamate receptors. We've seen lots of examples of the ionotropic, the AMPA, the chionate, and the NMDA receptors, and the metabotropic G-protein coupled receptors are simply called MGURs. Serotonin has an enormous diversity of G-protein coupled receptors, GPCRs, and one lonely ligand-gated channel, actually it has several different subunits, the so-called 5-HT3 receptors, among invertebrates histamine activates ligand-gated channels, and among both invertebrates and vertebrates histamine activates GPCRs, among vertebrates dopamine activates GPCRs, but among invertebrates it also activates ligand-gated channels, we think. So this theme in which an organism has both types of receptors is rather common in general in neuroscience. Now, on a time scale of seconds and also minutes, we are still talking the language of the nervous system. We're still talking electricity, and we're still describing a set of mechanisms that ultimately result in manipulating impulse frequencies, action potential frequencies in individual neurons, but the way we get there from GPCRs is substantially more complex than the way that we get there, that is the way we modulate action potential frequencies using ligand-gated channels. So in general, the G-protein pathway consists of mostly plasma membrane components, although as we'll see there are some intracellular components. Here is the X-ray coordinates of a G-protein-coupled receptor, a G-protein together. This was published in Nature in 2011 and about a year later, partially as a result of this and partially as a result of lots of other triumphs in providing the structure of G-protein-coupled receptors. Brian Kabilka and Robert Lefkowitz shared the Nobel Prize. It's a very basic discovery. So we have a neurotransmitter or a hormone binding to a receptor with seven transmembrane helices. Sometimes the neurotransmitter binds deep inside the membrane to the receptor, sometimes out here. This activates the G-protein. Why it's called a G-protein, as we'll see, is that the G-protein has a cycle. It has three subunits. These three subunits participate in a cycle of binding GTP, that's why we call it a G-protein, hydrolyzing GTP to GDP, releasing inorganic phosphate, and so there is a high energy phosphate bond being broken. For those of you who need a refresher from by eight by nine, you'll remember that we usually think of ATP as the universal energy currency of a cell. Also, GTP is in the soup as well. Typically, GTP exists in lower concentrations than ATP, and the GTP concentration is actually buffered by the ATP concentration. But it, too, is involved in lots of phosphatases. Typically, though, GTP, because of its small concentration, usually is involved in signaling rather than in energy maintenance. Then we have an effector of the G-protein, which is either an enzyme or a channel. You know about both of those. And the G-protein comes over and activates the effector. Sometimes it is the alpha subunit of the G-protein, sometimes it is the beta-gamma subunits of the G-protein, and usually one sees the beta and gamma subunits together. So this pathway, in general, is on the order typically of 100 milliseconds to 10 seconds, rather slower than the ligand-gated channel family. How far does the neurotransmitter or the hormone, well, how far apart are these parts of the G-protein pathway? Typically less than a micron apart. The neurotransmitter or hormone may have diffused in from a greater distance, but the entire family lives modestly close together. So let's talk more about the G-protein coupled receptors. So here we are at the very beginning of the pathway. We're talking about the receptors. Yes, sir. So the question is whether this yellow alpha here on the left is the same as this yellow alpha on the right. The answer is that upon activation by the receptor and upon binding of GTP, the alpha subunit loses its beta-gamma subunit and moves over to activate the effector. How do they move is the question. Typically the alpha subunit is anchored in the membrane by a lipid tail, diffuses within the plane of the membrane to the effector, less than a micrometer. In other cases, actually, the alpha subunit and the beta-gamma subunit are pre-existing in a complex with the effector. We don't know all of the rules and the received wisdom is changing. Any other questions? So the question is, is one micrometer larger than most of the conformational changes? Yes, indeed. So we had a symposium here last month at which Natan Daskal showed his data about G-proteins activating potassium channels. And in some cases, the G-protein and the potassium channel pre-exist in a complex and so the motions are rather small. But in other cases, the G-protein needs to diffuse within the plane of the membrane to find the channel. Any other questions? Alright, well then it's time for me to ask a few questions. Jared, would you distribute the index cards please? Oh, didn't I give them? Oh, Jonathan got them. So sorry. Sorry. This doesn't mean that every time you ask a question, pause. Alright, some generalizations about G-protein coupled receptors. As mentioned, they all have seven alpha helices going through the membrane. There are about a thousand G-protein coupled receptors in the genome. Most of them are orphans, that is their ligands are unknown. Most of them are, in fact, olfactory receptors. And in the human, many of the olfactory receptors have mutated to become non-functional. On the other hand, in dogs and rats and other mammals, they are quite functional. We'll talk about olfaction in a later lecture. So individual receptors respond to low molecular weight neurotransmitters, and we've mentioned serotonin, dopamine, acetylcholine. Some others respond to a short protein, 8 to 40 amino acids, a peptide. Best examples are the endorphins, the enkephalins. A relatively insoluble lipid, such as an anandamide, which is in fact the endogenous ligand for the cannabinoid receptors. Olfactory stimuli, as we mentioned, or light in the eye. So rhodopsin is in the G-protein coupled receptor family. So this is a tremendous variation in stimuli that are transduced. So if we look next at the G-protein, a heterotrimeric G-protein, it is in fact a molecular switch. And as we mentioned, sorry, it has three subunits. So it is a heterotrimeric G-protein. And in this case, the alpha subunit is in fact the subunit that binds the guano nucleotide in this particular image. It is in fact a GDP, which is bound. The gamma subunit, not clear what it does, but a major function is that the two of them have these alpha helices, the beta and the gamma, which stick into the cell membrane and provide anchors. So if we look at, and I'm sorry for the color change here, but here is a three-dimensional diagram of a G-protein coupled receptor. Here is the guano nucleotide bound to the alpha subunit. The beta subunit, I'm going to show you individually in a moment. It's a little clearer. It's in light blue. And the gamma subunit here is purple. It's really quite a lovely structure of the G-protein by itself. Now this does not have the receptor associated with it. If we look only at the beta subunit, it has what we call these days a beta propeller structure. And you can see why. This structure actually sometimes caps the alpha subunit, sometimes moves over and caps the effector. So it plays an important role in actually transmitting the signal from the receptor to the effector. Any questions? Okay. So here we've gone from the receptor to the G-protein. As we are going to see, there are actually roughly four flavors of G-proteins. And here is the beta propeller that I told you about. You can download these PDF, these PDB files for yourself. This stands for Protein Data Bank. But you'll need a viewer. And there are lots of free viewers. I happen to be using Pymol these days, which is free and available. There's no effector in this structure. Now let's go back to the Gigome seal, because patch clamping gave a very important set of insights into the GPCR pathway. You remember we said that the Gigome seal is very tight. So it compartmentalizes a molecule in the pipette outside the cell. It also opens... And so that's an important aspect. It's chemically tight. But the Gigome seal has another remarkable property, which is that it is mechanically tight. And so this means that once you get the seal, you can then dial back on your micromanipulator, move the pipette away from the cell. And a large fraction of the time, the patch comes along and detaches from the cell. So this is called an excised inside-out patch. And if you've been following the topography, it means that the transmitter, that the extracellular surface is inside the pipette, and the cytosolic surface is in the bath. If we do this... And this is important because we are following now the pathway for one of the G-proteins called GI. GI effectors... I in this case means inhibitory. And GI effectors include some potassium channels. So if we have potassium channels in the cell membrane, actually in the patch, and we pull off the patch with no additions, we won't see any channel openings. But if we now add G-beta-gamma subunits to the bath, those G-beta-gamma subunits open a potassium channel. So this tells us right away that we have a channel that's gated by intracellular messengers, in this case by G-beta-gamma subunits. And these would be G-beta-gamma subunits that are liberated from the alpha subunit and from the receptor when the receptor is activated by an agonist. So receptor to G-protein, GI couples to a channel. And so the effectors for G-proteins are in many cases ion channels. In this particular case, the ion channel effector is a potassium channel. It happens to be an inward rectifier potassium channel as well. So not only is this potassium channel gated by the polyion, by the polyamines either spermine or spermidine, as I explained last time, but it also can't be opened unless it has a G-beta-gamma subunit next to it as well. So in terms of our equivalent circuit then for the membrane, we can get a little more complicated than previously. We have the resting potassium conductance. We understand that very well. That's at the Nernst potential for potassium. We have the EPSP, which might give us a synaptic potential. We have the chloride conductance that we're not going to talk about now, but could also be a ligand-gated channel. We have the sodium conductance that underlies the action potential, and then we have variable potassium channels, and the additional potassium channels keep the membrane potential away from threshold by hyperpolarizing it, and therefore they decrease firing. So what we've added today is another kind of potassium channel, the G-protein-gated potassium channel, and it also inhibits the cell, keeps it from firing, and that's one of the reasons why we call the particular G-protein that activates potassium channels GII for inhibitory. Any questions? So when we increase the potassium conductance, as you know, this term dominates, and the result is that the membrane hyperpolarizes and is prevented from firing spikes. As noted, they're inward rectifiers. They latch the cell quiet, but when an excitatory stimulus finally succeeds in depolarizing the threshold, the inward rectifier channels turn off, and they allow the cell to fire spikes. Very handy devices. So as stated, GI-coupled receptors usually inhibit neurons. As we've seen, GI activates some potassium channels. Incidentally, GI also directly inhibits some voltage-gated calcium channels that's also an inhibitory process, and GI also directly inhibits denilyl cyclase, which we will talk about in a few minutes. So all of these actions actually do slow neuronal firing and they decrease transmitter release. So let's talk about another kind of G-protein now, GQ. I don't know where the Q came from. It's lost in time. A lot of these G-proteins were cloned right here at Caltech in Mel Simon's lab. The effector for GQ is not a channel. It's usually an enzyme. So there are two types of effectors, channels and enzymes. Very typically, the enzyme effector for a G-protein makes an intracellular messenger. In this particular example, the intracellular messenger is calcium. And what the enzyme does is to transduce so that calcium in the endoplasmic reticulum becomes calcium in the cytosol. The endoplasmic reticulum is an organelle inside the cell that has a composition very similar to the extracellular solution, that is, it's high in calcium, possibly high in sodium oxidizing. The GQ provokes the endoplasmic reticulum into releasing its calcium by activating an enzyme. So how does that occur? Oh my, well, this is complex. There are a couple of intracellular messengers involved. First of all, we have the, off here, not even shown, we have the receptor activating a GQ. The GQ alpha subunit activates an enzyme, phospholipase C-beta. Phospholipase C-beta in turn hydrolyzes a lipid phosphatidyl inositol 4-5 bisphosphate. There are lots of phospholipids in the membrane. They all have, many of them have inositol as their sugar, and they have various phosphates on them in various positions. This is phosphatidyl, here's the phosphatide, inositol, 4-5 bisphosphate. It's not a biphosphate because in biphosphates the two phosphates are connected to each other. It's bis because the two phosphates touch the inositol ring instead. Well, this liberates part of the phosphatidyl inositol to have just the lipid part, which is diacylglycerol. And so the diacylglycerol can then activate another enzyme, protein kinase C. So there are two parts of this pathway. One is the liberation of the trisphosphate from the phosphatidyl inositol. It becomes inositol trisphosphate, or IP3, and one is activating protein kinase. So the important part now is that the inositol trisphosphate diffuses to the endoplasmic reticulum, where it actually opens an IP3-gated calcium release channel. Which releases calcium from the lumen of the endoplasmic reticulum. On the one hand, oh, and so this is our first example of intracellular ligand-gated channels. So not only are there ligand-gated channels gated by neurotransmitters out here, there are also ligand-gated channels gated by intracellular messengers. And so in addition, the activated protein kinase C is activated concurrently by diacylgucero and by the calcium released from the endoplasmic reticulum, although there are other functions of the released calcium as well. And on the one hand, you can say this is ridiculously complicated. And on the other hand, you can say that it certainly does give employment to lots of biologists and neuroscientists and biochemists and modelers. There are some very nice models of this pathway. Any questions? Okay. So here we have these second messengers, these small molecules. We have PI45P2, phosphatidylenosatol, 4-5-bisophosphate. We have diacylgucero, and we have IP3. This guy turns into these two guys. But if we look further, we have the diagram in candle on page 242 in which we have the phospholipid, we have the diacylgucero part, we have the IP3 part, and there are a couple of phosphatase molecules that cleave at two different places. The phospholipase C, which is the one that interests us most, is producing the diacylgucero and the IP3. So if we look further, it furthermore is true that even PIP2, even the molecule in the membrane, is necessary for keeping some potassium channels open. So in addition to the diacylgucero and the IP3 acting as messengers, PIP2 also acts as a messenger, and GQ activation leads to less PIP2 because it gets hydrolyzed. Some potassium channels close. So here's an example of an experiment in which the control voltage clamp experiment shows potassium currents turning on. And apparently, and then when one adds muscarin to the preparation, acting on muscarinic receptors, fewer potassium channels turn on, and it looks very much as though actually at rest there were some potassium channels producing an outward current because the baseline goes down as well. So here are the voltage clamp pulses. So because these channels were first discovered for muscarinic receptors, we give them the clever name M channels. It's a different muscarinic receptor subtype from the one that opens potassium channels in the heart. So the result of GQ, activating GQ, is that before a slow EPSP, a depolarization to the cell, now we are in current clamp mode, not in voltage clamp mode, a depolarization would produce only one spike because the M channels are open and turn on more and prevent further spikes. But during the activation of the M current by a muscarinic receptor, what we see is that the same stimulus produces an entire train of spikes. So here's an example of how the GQ coupled receptor can excite a cell. Alright, then there is GS. GS got its abbreviation because it is stimulatory. It too activates an enzyme. It too activates an enzyme that makes an intracellular messenger. In this case, the messenger is cyclic AMP. And there is in fact a molecule called cyclase which is activated by GS which takes ATP, splits off two of the phosphate groups and makes cyclic AMP. So these two phosphate groups have left. This phosphate group comes out and has its oxygen attached to another atom on the sugar. So this is cyclic AMP, which is the first discovered in very important second messenger in cells. So here we have the second messenger being made. And a little bit of pharmacology. Caffeine prolongs the lifetime of the intracellular messenger. So typically cyclic AMP is broken down by phosphodiesterase molecules. And caffeine comes and inhibits this breakdown. And this leads to some of the actions of caffeine. So we have here a story in which inside a cell we have an intracellular messenger being made. The intracellular messenger is being made by a cyclase degraded by a phosphodiesterase, which is inhibited by caffeine. So this phosphodiesterase inhibitors prolong the life of the intracellular messenger. There is an analogous pathway inside cells in which not ATP but GTP is cyclized to cyclic AMP. There is also a phosphodiesterase for cyclic AMP that prolongs the lifetime of cyclic AMP. In fact, there's an entire family of these phosphodiesterases. And the question is which drugs inhibit cyclic AMP phosphodiesterase? Anybody want to hazard a guess as to which popular drugs inhibit cyclic AMP phosphodiesterase? What happens when we have made these intracellular messengers, either calcium or cyclic AMP? They themselves activate other proteins, other enzymes, kinases. So we'll follow this other pathway in a little while that a few ion channels get activated directly by calcium or cyclic nucleotides. But for the moment we're going to follow this pathway in which the intracellular messenger activates a kinase, which phosphorylates a protein. So both calcium and cyclic AMP can do this. By a kinase, we mean a kinase is a protein that adds a phosphate group to other proteins. So cyclic AMP gets made. It binds to an inactive protein kinase A. This protein kinase A dissociates into the regulatory subunits and the active kinase subunits. What the kinase does is to take either a serine or in some cases a threonine residue in a protein and add a phosphate to that residue. And that of course is a phosphotransfer reaction. So that takes energy. The phosphate comes from ATP. So the result of kinase is to phosphorylate a side chain on a protein, add phosphate to that side chain. And sure enough, there is a set of enzymes that go the other way that remove the phosphate from those side chains. So we can control either the kinase with cyclic AMP or the phosphatase with other proteins. And here, then, is an example in which Gs by phosphorylating proteins actually excites a cell. So S is stimulatory. In this case, in this experiment, the experimenter is using norepinephrine, which activates beta-adrenergic receptors. The experiment is being done on hippocampal neurons, which we've seen in this course previously. In the control experiment, we add glutamate, we see some firing, and then the firing stops. But if we add norepinephrine to inhibit by phosphorylating a potassium channel, yet another kind of potassium channel, one that has not yet been discussed in this course, we inhibit that voltage-gated potassium channel, we get less opposition to the glutamate depolarization. As a result, we get more impulses. When we wash out the norepinephrine, and every good physiological experiment involves a washout step, we reverse the process. And so here we have a way of stimulating neurons, which the nervous system does, by releasing norepinephrine. So the norepinephrine effect can experimentally be mimicked by agents that mimic or increase cyclic AMP. So in this case, we can mimic the effect of norepinephrine by adding a molecule called 8-bromocyclic AMP. 8-bromocyclic AMP does permeate through cells. It's a cyclic AMP analog that is not hydrolyzed by the phosphodiesterase. And so we get the same kind of stimulation, same longer impulse frequencies. Now, this is a voltage-clamp experiment, rather than a current-clamp experiment. We apply force-colon, which directly activates adenylyl cyclase. And so we see, before we add force-colon, we get this nice after hyperpolarization, which means that the potassium channels have turned on. But when we add force-colon, we get less after hyperpolarization, because we have activated adenylyl cyclase, produced cyclic AMP, activated protein kinase, and phosphorylated the protein, which turns it off. And then everything can be washed out. Well, this is fairly complex stuff. We have receptors. We have several kinds of G-proteins. We have a couple kinds of effectors, and we have two major kinds of intracellular messengers, calcium and cyclic AMP. So what are the selective advantages of such a complex pathway? Well, from the viewpoint of the neurotransmitter, we have actually uncoupled the presence of a neurotransmitter released from a neuron with the actual nature of the response. So there's no direct coupling between the neurotransmitter and the response. We've decoupled from the viewpoint of chemistry. We've decoupled from the viewpoint of speed, because when we activate the G-protein, it stays activated until the GTP gets hydrolyzed, which is typically much longer than the neurotransmitter remaining on the receptor. And to some extent, we have also decoupled localization. The effector and its effects can be as much as a micron away from the receptor. So that's the advantages, but we need to input some energy. So we need energy in the form of these high-energy phosphate bonds. Because there's so much amplification, the typical GPCR pathway is slower than the typical ligand-gated channel pathway. Hundreds of milliseconds rather than a single millisecond. And also, we apparently have lost some cooperativity from the GPCR pathway. The GPCR pathway, when analyzed carefully, consists of a bunch of linear steps. Which is very nice. But the ligand-gated channel pathway actually doesn't work unless two or three transmitter molecules bind to the receptor. So it is a highly cooperative, all-or-none switch. The GPCR pathway is more graded. That has advantages and disadvantages. Any other points that you'd like to make yourselves ideas about advantages, disadvantages of the GPCR pathway? Let's talk about the diversity, at least at the genomic level of the pathway. We have about a thousand G-protein coupled receptors. Each of these G-protein coupled receptors have three subunits. A lot of these three subunits were discovered at Caltech. There are no less than 18 alpha-subunit genes, although they do fall into these four major classes. GI, GQ, GS, and GT. I haven't mentioned GTP, sorry, GT, but we will. There are about five beta subunits and three gamma subunits. There are two major classes of effectors. There are channels. There are about five known potassium channel genes, about four calcium channel genes affected by G-proteins, and there are enzymes. There is an enormous number of enzymes activated by G-proteins in three major classes, and each of those classes has between two and ten members. So this is a very rich, highly evolved pathway. But you could think of this pathway as a 1950s Ford. You look under the hood, and it's got a big engine, a little carburetor, and a lot of horsepower. But actually, the pathway has evolved, and these accessory proteins have made it look more like a honestly programmed 2015 car in which you open the hood, and you see lots of controls and regulators and smog devices and accelerators. And so some accessory proteins have entered the story as well. Here's one accessory protein called, appropriately enough, a regulator of G-protein signaling. And what it does here, the regulator of G-protein signaling, is that it tunes the kinetics of G-protein signaling. The RGS protein, of which there are now a couple of dozen, actually sticks a lysine into the alpha subunit and tickles the GTP binding site to make it hydrolyze the GTP faster, and to make it turn on faster. So here, for instance, is an atrial cell from a heart. The Otter-Lervy experiment, where you add acetylcholine to the heart, it slows down the heartbeat by activating a GERC current. You wash the acetylcholine away, the current goes away pretty quickly. If you try to reconstitute this in a mammalian cell, in a clonal cell line, by expressing the muscarinic receptor and GERC channels, it's a more leisurely process. It takes a couple of seconds to turn on the channels, and a couple of seconds to turn them off. Now you also co-express an RGS protein, and you get back to kinetics. Faster turn on, faster turn off. So what the RGS protein does, and here are the traces amplified here, what the RGS protein does then is to speed up the G-protein cycle by making the GTP binding faster and by making the GTP hydrolysis faster. So there are all of these regulatory proteins that have crept under the hood of the G-protein pathway to make them specialized for various cell types. So we've seen that at a time scale of seconds and minutes, the language of the nervous system remains electricity. Now we're going to go to a longer time scale, and we're going to talk about what happens when a G-protein pathway remains active for hours to days. So we're going to talk about the classical so-called outside-in mechanisms for long-term actions. According, and where did this get started? Well, it actually got started, where do you think it got started, right? With Seymour Benzers, mutants for Drosophila in the 1960s. And he built a very clever gadget using a countercurrent distribution that allowed Drosophila fruit flies to learn to avoid an odorant associated with an electric shock. He discovered a mutant. The Drosophila geneticists, of course, were quite playful with words. So the mutant he discovered was called den dunce, and basically this mutant can't learn to avoid an odorant. And he used this clever apparatus, which I used to bring in here, that had a flexible strip with copper etchings on it that could be connected to the outlet. It really was just plugged into the outlet. And so when you wanted to teach the flies to avoid an odor, you put them in the tube with this odor, and you plugged the copper strip into an outlet, and the flies did not like that. And some of them learned, and you do this several times, and some of them learned to avoid the odor. Naturally, there were lots of controls to avoid the idea that it was just frying the flies. And so there were control odorants, and there were controls without the odorant, et cetera. Clearly, ultimately, Seymour could find a mutant that he called dunce. So this was serial application of an odor for a long time. So what gene, what protein did dunce encode? Dunce encoded a phosphodiesterase in the cyclic AMP pathway. And so the dunce mutant allowed cyclic AMP to build up. Of course, there were other mutants that people found. A lot of them didn't behave very well, so they started being called by vegetables. And so this one is called rutabaga, and it just sits there. It's a cyclase. So what did we just learn? We learned that long-term plastic changes in the nervous system involved with memory and learning actually involved second messengers of the sort that we have discussed today. And that tells us that activating the G-protein pathway repeatedly or for long times changes the activity of neurons, changes phosphorylation of proteins, changes the way neurons encode information. Now, how does all of that occur? Well, there's a part of the cell that I have not showed you yet in this complex pathway. When we activate an intercellular messenger and activate a kinase for a long time and phosphorylate proteins, if those proteins stay around for a while, some of them get into the nucleus. That's the part we haven't showed you yet, the nucleus. Some phosphorylated proteins in the nucleus are actually transcription factors. And so the cyclic AMP and other second messenger pathways can phosphorylate transcription factors. Transcription factors such as CREB here actually sit on DNA and activate or prevent the activation of genes. And so here we have a way now, a very robust way that occurs many times in nature in which activation of a cell surface receptor, in this case a GPCR, leads to activity of new genes. Which can do many, many functions in a cell. So the transcription factor, the many genes have a DNA sequence that's called cyclic AMP calcium response of the element, second messenger response of the element, or CRE. Very cleverly, the protein that binds to CRE is called CREB, cyclic AMP response element binder. Very clever. And the CREB can be phosphorylated to get into the nucleus. So we put a little P in front of it, that means phosphorylated. So here is phospho-CREB binding to CRE, activating a protein. The way we usually test for protein activation, of course, is by putting GFP downstream from the CRE box. So here we have activated protein kinase, getting into the nucleus, phosphorylating inactive CREB, cyclic AMP response element protein, which binds to DNA, to the CRE, which activates a gene, transcribes that gene, ultimately we get translation, and we get entirely new processes inside the neuron. So here we have the entire pathway from GPCRs to gene activation. It is a classical pathway that exists many times in evolution. Part of the pathway takes place in the membrane, part takes place in the cytosol, and if the pathway is active for long enough, the rest takes place in the nucleus. When we include the effects on gene activation, the pathway stretches out. The latter part of the pathway can get activated in as little as 10 seconds, but mostly it takes a couple of days, and CREB itself can move in the cytosol, move into nuclei, so actually these signals can go as far as one meter. Here is a typical activation pathway that you see in textbooks, very much like one in Candel. In this case, there's a mu-opioid receptor. It activates GI or GO. This directly activates a GERC channel. It also directly inhibits an adenylyl cyclase, which acts on cyclic AMP, which decreases protein kinase A activity, changes the phosphorylation of CREB, changes the activation of genes. So we get altered gene expression from repeated activation of this pathway. Many think everybody knows that the mu-opioid receptor is the target for morphine and other painkillers. And many scientists believe that the tolerance and dependence to mu-opioid agonists such as morphine doesn't go by way of the potassium channels, but goes by way of the long-term effects that I have told you on gene activation. That's the so-called outside-in hypothesis for long-term actions. Okay, see you Monday, and I will be at the red door as usual.