That it was long identify so the behaviors of because of flies somehow will be easy to paralyze. That shaking itself. Okay, and that's when it's called shaker. And then when people painstakingly map the mutations and then they identify. It looks like a potassium channel because, just by looking at its primary sequence, people found that it looks like, in sequence, very similar to sodium channel, except sodium channel has this four repeats of this 20 transmembrane region, and potassium channel if you look at a private sequence. It only has one of the repeats, but in reality full analysis that people actually found in this potassium channel that it will [INAUDIBLE]. So, even though it has only one, six transmembrane but In reality, four of them come together that has this 24 transmembranes together that they can mediate loss response. So after identifying the genes or the primary sequence for this ion channel, then we have the ability to understand what might be the structural basis for these unique features of those ion channels, and we talk about a little bit right. The generation of action potentials requires those ion channels has these unique properties. One is they have this wattage dependence okay. And the other is they have a very special selectivity. Okay, and for the wattage selectivity sodium channel it also has this unique features that you will inactivate. Meaning that even after the wattage deploys, over time actually you will somehow non permeable to sodium, so called inactivation, which is different than simply closed. Because people define closed as when you change in voltage you can open, people call that in a closed state. But if you change in voltage you can no longer open. It's a different confirmation, okay? People called it inactuation, okay. So how do you understand the basis of this unique features? First, what is your sensing? How does, after I identify the primary sequence of this ion channel, how does this ion channel sense voltage? What would be your source? What might be the molecular basis? How can it sense voltage? Okay, so the Italians said, well, this protein might have the ability to sense voltage, right. And the way to sense voltage well, charge will move in an electrical field, okay. So the best way to sense voltage is to have the charged particle. And for this big protein that are encoded by the composition of 20 amino acids. The most likely, maybe, that the protein has certain rescues that has or carries charges either positive or negative. And then, a point to the electrical field changes across the membrane. It makes sense, right? So where does this charge reduce my residing? Well, we relate localize in those in this protein that we identify. So, now voltage sensor need to sense the voltage, okay, and where does these voltage changes happening inside the cell. It's electrical field changes so it's voltage. It's not just a pressure okay. Is just pressure right. So it actually can feel the changes. So where does this, it can actually feel change near the membrane. Actually it's across the membrane right. We have already started a cell membrane potential. Well, this is a cell and we said inside is -70 millivolts and outside we define as zero, okay? During action potential, that what happens is there will be an action potential just like this. And initially [INAUDIBLE] and then to complete [INAUDIBLE] is close to [INAUDIBLE] What does this wattage change happening? Well, it's actually across the membrane so if you have the voltage sensor only in the intracellular part of the membrane. That's just the membrane it has the bilayer. So you have a protein for example, this protein. It has the transmembrane region. It has this extracellular region. And intracellular region. If you have the wattage censor only inside. The cell, in the intercellular region. Where does this membrane change happen in? Where is actually is across this membrane? Okay, in the intercellular part, it's not actually in the electrical field, okay? So, so even when the membrane changes, it might sense a little bit of this charge, the accumulation near to the interface but it's too small. The most of the water you drop is across this membrane, right. So most likely this water gee sensor if let is going to sense the water. It needs to be in the high electrical field that has the highest changes. Right, so then you can auto is confirmation and in fact this is how people identify. So were people looking at the structure or the primary sequence of this protein for example the shake of potassium channels, okay? And is the relatively easily to identify that in my half six transmembrane, region. Okay, and immediately indeed, immediately people recognize this one transmembrane domain. It's unique, this is called s4 domain. So, this is 1,2,3,4,5,6. This [INAUDIBLE] domain is so unique. There really is most of the transmembrane region, how do you know they are transmembrane? How do you know? Well most of the transmembrane region, because they are buried inside the membrane, they have this unique property of amino acids that are hydrophobic. Okay? So people can determine that transmembrane region if they have quite a number of hydrophobic residues all together. Okay? And in this S4, is unique not that it has and this is a puddle of sequence, that he has quite a field or in fact may need positive charges if you count this number, one two three four five six seven. There are seven positively charged inside of this region. And by comparison, you know, S1 has 0, S2 has a zero. 2 and S3 has 1. Okay, all the regions almost don't have this positive charges or this charges that are really electrostatic, that can interact with other electrostatic residues. So, this [INAUDIBLE] the clues, that this might be the region that we'll sense the electrical field, and is in a profitable location. It's just in the transmembrane region where the electrical field are changing the most. In fact if your looking at sodium channel of calcium channel, as I mentioned those sodium channel, what you get your sodium channel, or calcium channel they have 25 transmembrane regions. But they can separate into, again, as if four sub unit, and into sub unit while you observe in their corresponding [INAUDIBLE] domain has this positive charges, okay? Even though sold in China is slightly different, some has more than the other, but they always have these kinds of [INAUDIBLE] with these positive charges, okay. So people are speculating, those might be the senses that can sense electrical field and then mediate the voltage dependency open and close of this ion channel. Then here comes the question. This indicates this might be. How do you know? How do you design an experiment to prove of this proof. This is indeed the voltage sensor. First, if you think that doing the voltage change this thing has a confirmation change, does it really convince you this are the voltage sensor? Well it may be, it may not be because you don't know whether this is primary or secondary. May there's additional wattage sensor is dragging this thing to move so even if it has the conformational change it does not prove. That you will be the sensor. It could be secondary. It would be the sensor. It's something else changing this one. So the experiment that you design may not completely demonstrate that as well. Is it a working sensor? Great, so she said since you have a very prediction of these positive charges then you know we just specifically make mutation. We only change those positive charges, okay? And then to see, first, your prediction, well if we If they are indeed the voltage sensors. Those ion channel open at a different voltage. They sense the electrical field. So if you are changing those things then the voltage dependence of those ion channel might be altered. That's one of your predictions. Indeed, people have done those experiments and they found mutated salt and will dramatically change in the wattage dependency of those ion channels. Okay. There are more precise measurements other people have done. Well as new Mention that this ion channel the can sense wattage, and because of this wattage sensor if they are indeed a wattage sensor they are in a electrical field. Sensor in wattage, well from a [INAUDIBLE] we know that if you have a charged particle in the electrical field. Whenever it move, the charged particle in the electrical field will move according to the direction of the electrical field. But whenever you have a charged particle movement, you actually create current. Okay? In fact, the definition of currents, the electrical current, is charged particle movement is electrical current, okay? So the prediction will be that if they are the motion sensors and they are in electrical field, if they are indeed advance the can sense this electrical view, they will move. And if they are able to move, the current mediated by these charged particles, and we are mutating some of those charged particles. You will correspondingly seeing the reduction of the currents. Okay? So, the moving on the charged particles on the wattage sensor in that electrical field. That's what we call gating currents. Okay. Those are different. Then the currents that are passing through the pore ridges between S5 and SF6. So this iron channel has a big hole that's once it's open you will allow sodium for example go in through the sodium channel so those are the sodium ions. Going through the port, forming by S4, S5 and S6. We are going to see the crystal a little bit later, but these formed mainly by S5 and S6 region has this pore region that are forming the pore. Okay, and those currents are different than this current. First, the sodium current going through this port will be much, much, much larger than the current generated by a movement of this small antigen or [INAUDIBLE] resting, why? Because you could have a huge concentration difference across the membrane. And when's an ion channel open and close? As long as it's open, it will allow all the ions move under the electric chemical gradients. Okay, so you have more than just one copy of these ions to move. But for this gating current measurements, how many protein you have? And only in that protein you have this teeny, tiny movements, okay? So the gating current will be much, much smaller. Or, and so but indeed people deal with measurements again initially in [INAUDIBLE]
