[BLANK_AUDIO]. Hi everyone. Welcome to this tutorial on the Molecular Mechanisms of Action Potential Generation. So in the previous two sessions we've been talking about the ionic basis of action potential generation. Now we want to move more towards the biological question of, what are the mechanisms that are responsible for the production of this signature electrical event, that we find in excitable tissue? All of this relates to one of our Neuroscience Core Concepts, the one we've been focusing on. the fact that Neurons communicate using both electrical and chemical signals. So, we continue to talk about the electrical signaling activity of neurons and very soon we'll be moving in to talk about the chemical signals. Well, for today's session we have some learning objectives. I want you to be able to describe the molecular properties of sodium and potassium channels and understand and recognize how these properties explain the voltage and time-dependent permeability changes that underlie the generation of action potentials. And secondly, I want you to be able to describe the molecular mechanisms that establish the chemical gradients for these important ions, sodium and potassium across the neuronal plasma membrane. Well, to begin, let's again reiterate some of our foundational principles, that is, that charges in potassium and sodium permeability underlie the action potential. So we've seen this figure a few times before. It allows us to conceptually understand the resting membrane potential as being a state dominated by the leak conductance for Potassium. the rising face being a phase of action potential generation where there's an explosive increase in the permeability of the membrane for sodium. Such that at the peak of the action potential, the membrane potential comes very close the Nernst equilibrium potential for sodium. Because the sodium permeability of the membrane is so much greater than any other ionic permeability, the ability to predict membrane potential. becomes as a first approximation the Nernst equilibrium equation for sodium. Well, the falling phase of the action potential involves a dramatic reduction sodium permeability, and this is the inactivation of the sodium conductance. We'll talk about the molecular mechanisms for that inactivation in this tutorial. But in addition, we know that the voltage and time dependency of the potassium conductance means that the potassium permeability is rising at the same time that sodium permeability is collapsing. And those two factors together allow us to return to our resting condition where once again the permeability of the membrane due to the fact that there's some leakiness of the membrane to potassium is greater than sodium permeability. And once again the resting membrane potential is established being close but not precisely the Nernst equilibrium potential for potassium. Well this conceptualization allows us to again to understand how changes in membrane permeability lead to changes in membrane potential. And as we discussed last time, and we'll talk about the mechanisms today, changes in membrane potential can in turn have an impact on changes in membrane permeability. Now I would encourage you to, view, again, the animation that's available on the website that supports our textbook. It's animation 2.3 on the action potential. You can click following the hyperlink on your tutorial notes or just navigate on your own to that website. Alright, well, for today what we really want to understand is what are the molecular mechanisms that allow a change in membrane potential to impact membrane permeability? And this drives us to consider the actual integral membrane proteins themselves that constitute ion channels for sodium and potassium. We'll do so in two ways. First we'll consider a functional model for how this feature, change in membrane. Potential leads to a change of membrane permeability, and then we'll consider the actual molecular structure of these proteins and gain some insight as to how this plays out in actual biological terms. So first the functional model. Now, this is an experiment that should be familiar to you now. what we're doing is using our voltage clamp method to apply a step depolarization that is going to be supratheshold, that is well above threshold for the generation of action potentials. And rather than showing you the action potential or the. Voltage and time-dependant changes in conductance, I want to show you our current understanding of what's happening at the level of the integral membrane proteins that constitute permeability channels for sodium and potassium. So let's step through this figure in time and let's look at what happens at the onset of depolarization. So, that would be time 0., when we first depolarize. Now, recall that the inward current is fast, that is the increase in sodium conductance is quite rapid, relative to the increase in potassium conductance, and now we gain some insight as to how that might happen. almost instantaneously, with the depolarization step the, probability of sodium channels reconfiguring from their closed state to the open state is dramatically increased. In other words, sodium channels open at the onset of depolarization. Now notice what's going on for potassium channels. The probability that a potassium channel will go from a close state to an open state, is less than for the sodium channel with this step in depolarization. If we consider the behavior of a single ion channel, which its possible now to do we can understand this behavior in, more principle terms. But on the aggregate what we can say is that the sodium channels respond as a collection of proteins more rapidly. With greater probability of opening with the onset of depolarization relative to the potassium channel. This is why the positive feedback phenomenon is so rapid, and why the repolarization phenomenon lags the explosive rising phase of the action potential. Okay, so let's move a little bit further out in time. Now we are a few milliseconds later. And we see that the sodium conductance is beginning to inactivate. Why? Not because the pore of the channel is pinching down, but because there is a particular mechanism at play here on the inner aspect. That is, the cytoplasmic surface of the sodium channel. There is a gate that we call the inactivation gate that swings shut as the membrane becomes depolarized. So, we'll talk about what exactly that is and why that's happening in just a minute. But for now I want you to note that inactivation of the sodium channel is due to the fact that there is this particular cytoplasmic feature of the channel protein. That swings closed and plugs up the pore of the membrane from the cytoplasmic side preventing any further influx of sodium channels. And this is why that sodium conductance rises so rapidly and then falls equally rapidly because of the development of inactivation. Now at about this time our potassium channels are opening up more consistently. That is the probability of channel opening is greater, and more and more potassium channels are in fact in the open configuration allowing for potassium, to flow out of the neuron. This contributing to repolarization and eventually the undershoot of membrane potential. Now notice the cytoplasmic surface of this potassium channel. There is no inactivation gate, at least on this particular type of potassium channel. So these channels are going to continue to remain in their open configuration provide that we continue to depolarize this membrane, in this case with the voltage clamp experiment. It's only with the cessation of depolarization, that is when we step back down near the resting membrane potential, that we induce a change in both kinds of channels. This hyperpolarization is essential to restore the closed state for both of these channels. For the sodium channel, that means that the pore itself Is closed, and the inactivation gate is open. For the potassium channel it means that the pore simply has closed off preventing any further efflux of potassium ions. Now let me again restate the point about the sodium channel, because it is important. Notice that there's a difference between inactivation and closed. In the inactive state, the pore of the channel is open but the gate has closed on the cytoplasmic surface of the channel. In the closed state, the pore is stopped up preventing the further flow of ion. However, the inactivation gate has been reset and restored. So, when the channel for sodium is back to it's closed state, we are now outside of the absolute refractory period. It's possible for this channel to open up again. That is, it's possible for this channel to reenter a cycle that will allow for channel opening and the influx of sodium. So, the absolute refractory period of the action potential is defined in terms of the molecular structure of the ion channel itself. When these channels are inactive they are incapable of passing current. That inactivation is only relieved with by polarization. So this inactivated state is when we have our absolute refractory period. Okay? Alright, well the relative refractory period, that is a time following sufficient hyperpolarization to reset that sodium inactivation gate. but it's a time when these potassium channels may remain open, and we may still be in the under shoot of the action potentials so it will require stronger stimulus to activate that sodium channel sufficiently to induce that positive feedback circuit that leads to this explosive, regenerative rising phase of the action potential. Okay, now, let's move on from this functional consideration of how sodium and potassium channels work relative to their voltage and time dependencies to consider the actual molecules themselves. Well, these ion channels are very complex. But they do have some features in common, and I want to emphasize their common features, and just highlight some of the differences. So let's begin by thinking about the sodium channel. Well, the sodium channel that we've been talking about is really a complicated, integral membrane protein that contains multiple motifs, in which there are helical segments of the protein that are passing through the membrane. So, we can recognize 4 motifs, with each motif being comprised of 6 transmembrane segments. and these, motifs, fold together in a three-dimensional shape within the membrane. Constituting the voltage gated ion channel that selectively permeable for sodium. Now as this protein establishes its tertiary shape in the membrane it's configured in such a way to establish a pore for the passage of sodium ions, and the electrical... And physical structure of that, ion channel pore account for its selectivity for sodium. I'll also highlight these yellow transmembrane segments that we see in each of these motifs. And these contain positively charged amino acid residues that account for the voltage sensitivity of this protein. Now there are also additional components to this channel. There's something called a beta subunit that aggregates with the the main integral membrane protein that, that forms this channel. So this is the sodium channel. We think there are probably at least ten sodium channel genes that express the proteins that are essential for building these channels. But those genes can have multiple reading frames. And the product of that gene can be modified in various ways. So there may be different capacities to modify this basic structure in interesting ways that will have an impact on its function. And in some cases there may be genetic mutations of these genes that will impair the function of these channels. And that's a fascinating story that you could read more about in a box that's found in chapter four. Okay, so that's a brief look at the sodium channel. We actually know more currently about the molecular structure of potassium channels. One, perhaps surprising fact is that there are a large number of genes that encode potassium channels and potassium channels can have multiple different forms that have different physiological properties owing to their distinct molecular structure. The ion channels that are constituted from these gene products. Typically represent the aggregate of multiple subunits that is not just one integral membrane protein but more commonly four integral membrane proteins that come together and constitute the, voltage gated potassium channel or potassium channel that's gated by some other mechanism. there's a large family of these channel genes and a large number variations on an ion channel that allows the passage of potassium. So far we've been talking about the kind of voltage dependent ion channel that's represented over here on the left hand side of this illustration. this is a channel that is gated by a change in voltage. So it, like the sodium channel, has a voltage sensor, we'll talk more about that in just a moment. But there are other kinds of ion channels that are sensitive to other factors, such as Calcium within the cell. And that is attributable to a cytoplasmic portion of this integral membrane protein that can interact with Calcium. And there's a large variety of other potassium channels that we won't, mention at this point. But together, they contribute to the exquisite regulation of excitability. And the nature of electrical signals that neurons can generate. Now let's back away just a little bit from the more detailed look at the molecular structure of the voltage gated ion channel. And consider again form a more functional perspective how these various components aggregate together to form a functional ion channel. So this is a, model now of a voltage gated potassium channel so we imagine that. These four subunits come together to constitute a channel. And there's something that we'll call a pore loop that forms from the aggregate of these four subunits. And these pore loops together establish the ionic selectivity and the size selectivity for the passage of potassium ions. Now we've already talked some about the voltage sensor that's part of each subunit, and that voltage sensor is illustrated by these wings out to the side of these subunits that carry positive recharged amino acid residues. So in the hyperpolarized state when the inside of the cell is made more negative, these positive charges in these wing-like structures are drawn inward toward that negativity. The conformational change that happens as the subunits pivot allows for a closure of the pore loop regions, and potassium ions are not able to pass outward through this channel with Hyperpolarization. Now imagine what will happen if we depolarize the cell, that is we make the inside of the cell positive. Perhaps by the opening of a voltage gated sodium channel. Now these wings that bear these positive charges are pushed away by electrostatic forces. That pivots the sub units in a way that now opens up a pore in the center of this channel and potassium ions are then able to aggregate and pass out of this pore region. So the function of these ion channels is a reflection of their structure. And the voltage dependency, and even the time dependency has to do with the structure of these voltage sensors. And perhaps the density of charge residues that might contribute to the, to the kinetics of channel opening. Now, as I mentioned, one of the consequences of the genetic encoding from these ion channels is that mutations can occur that have consequences for neurological function and in some cases human health. And you can read more about that topic, if you're interested, in box D in chapter four of our textbook. So what we've been talking about so far are the molecular structures that constitute ion channels, and this is a way of discharging those gradients that are essential for generating electrical signals. Now let's turn to the molecular mechanisms that are responsible for establishing those gradients in the first place. I mentioned when we first introduced this topic, that those gradients are established by ion pumps, and ion transporters. And here, we see a variety of pumps and transporters. Probably the most important that I should emphasize is right over here on the left hand side of figure 4.9. It's the Sodium-Potassium ATPase. This is, arguably, the most important molecule in the nervous system. It seems a little ridiculous to even make such a claim, but if it were possible to identify one molecule of supreme importance, this would be it. The Sodium-Potassium ATPase. It consumes a significant portion of all the energy that is burnt up in the function of the nervous system. And it's one of the principal reasons why the brain requires such a constant, and highly regulated flow of blood to deliver oxygen and glucose the principle substrate for oxidated metabolism in brain tissue. There are other pumps besides the sodium potassium ATPase as we see here. There are pumps for calcium, there are iron exchangers that move sodium and calcium and protons. There are co-transporters that can take advantage of a concentration gradient for one ion to transport another. And we don't need to get into all of this complexity at the present time. Rather, what I want to do is consider in a little bit more detail, the function of our principle path to Sodium-Potassium ATPase. Now the story of how this function works is the result of lot of empirical research that had been done in various ways, one can imagine based on our prior consideration of the action potential. That one can study this pump by removing other sodium or potassium from the solution and asking how these manipulations might affect pump activity. one might also gain some insight by depriving that pump of its supply of energy to find out what's happening. So these kinds of experiments were done and what's shown here in figure 4.10 is an experiment where a tagged sodium molecule was allowed to flux across the plasma membrane due to the activity of this pump. So sodium was loaded up within a cell and the efflux of sodium, which represents the activity of the pump, is measured over time. So, as this experiment unfolds, there is some discharge of sodium ions that can be measured experimentally. And if one removes potassium from the medium outside of the cell, now there's a sudden drop in the activity that's being indicated by the efflux of sodium. So this suggests that there must be some kind of codependency between the influx of potassium ions into the cell and the efflux of sodium. Well when potassium is restored and the fluids around the cell, now the rate of discharge of sodium is restored. But now a key experiment, is imposed upon this model system. the energy that's required to drive this activity is depleted by adding a compound which blocks the synthesis of adenosine triphosphate, which is the main energy currency for active cells. And as this metabolic poison, if you will, is introduced there is a precipitous decline in the efflux of sodium ions providing strong evidence for the energy requirement of this transport. When ATP is restored then there is a, somewhat rapid rise again of the efflux of sodium back towards the rate at which this experiment was run. So taking these two phases of the experiment together we know that there's some kind of codependency between the Influx of potassium and the efflux of sodium. And we also know that this process is energy dependent. So these kinds of experiments have lead to the following model. there is some kind of integral membrane protein that constitutes a pump for sodium and potassium. And this integral membrane protein has enzymatic activity that can cleave ATP. And consume energy in the form of breaking that high-energy phosphate bond. So, we have good reason to believe that there is a configuration of this protein that allows for the binding of sodium ions to the exposed cytoplasmic domain within the central region of this integral membrane protein. And there seems to be a particular stoichiometry. To the transport of sodium and potassium. We think they're likely three binding sites for sodium ions that are available in this configuration. And then with the consumption of energy, in the form of cleaving this high energy phosphate bond from ATP. There's a conformational change such that this protein appears to flip within the membrane. We don't know exactly how this happens, but it seems to change conformation, allowing these three sodium ions now to be discharged on the extra-cellular face of the membrane. And at the same time, this conformational change has exposed, now, binding sites for potassium ions. Now, with the binding of these potassium ions, the unorganic phosphate is lost. And a conformational change occurs. And we are restored to the starting configuration of this protein. And this allows for these potassium ions now to be discharged and enter the extracellular space. So now this protein is ready to return to its starting position in this schema where sodium ions can aggregate. So with the consumption of a high energy phosphate bond, there is a cycle of this pump that transports three sodium ions from inside the cell to outside the cell while at the same time allowing for two potassium ions to travel from outside of the cell to the interior. So as this pump churns and burns energy, we establish a concentration gradient, with higher sodium ions outside of the cell compared to inside. And higher potassium concentrations inside the cell compared to outside. All of this requires energy. Now, notice the stoichiometry. There's more positive charge leaving the cell than entering the cell with each turn of the cycle. From this point of view, we say that this pump activity is electrogenic, that is it has the potential to generate a small current. That current typically is negligible unless we're talking about a very small compartment, where the concentrations of ions might actually change during the course of a high frequency burst of action potentials. Such compartments exist in the smallest of our axons that we find in peripheral nerves. So, in such situations, as ionic concentrations might be altered by a burst of action potential activity, pump activity will increase, more positive charge will leave the cell as a result of the pump, and the inside of the cell will hyperpolarize. So that would be the impact of this electrogenic nature of pump activity. Again, it's typically negligible, except for the very smallest of axons, or the very tightest of compartments where pump activity can have an impact on the resting membrane potential. Well, we can summarize and have an opportunity to review these mechanisms of pump activity by viewing an animation from chapter four. You can follow this animation by navigating to the website that supports our textbook or by clicking on the hyperlink that you have available to you at the bottom of your tutorial notes. Well, I hope this has been helpful. Our next tutorial will focus on the means by which electrical signals can propagate along an axon. And we will return to some of these concepts related to the structure of ion channels and pumps when we consider synaptic neuro transmission, in forth coming tutorials.
