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BIO241 Lecture 4

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Jennifer Harris

Thursday, January 15, 2009 - We will look at the ABC transport proteins, then examples of ion channels that are a distinct category from transport proteins but also involved in moving small molecules across membranes and we’re going to look at how these ion channels contribute to the membrane potential in animal cells and also look at the different categories of ion channels in terms of gated ion channels. - Here is an overview of the different types of transport proteins that we’ve looked at so far. We looked at passive transport carried out by uniporters, active transport carried out by coupled transporters and particularly, the symporters and antiporters. Then we looked at ATP driven pumps, these also carry out active transport and in this case, it is primary active transport. - We also looked at the P-type ATPases that are phosphorylated, V type ATPases that mainly pump protons across membranes, F-type ATPases that are involved in ATP synthesis and then today we’re going to look at the final category of transport proteins: the ABC transporters.  Dimerization of ATP binding domains  Dissociation  Conformational changes in transmembrane domains - Here is an overview of what ABC transporters look like. They’re called ABC transporters b/c they have 2 ATP binding cassettes (domains). ATP binding cassettes would be ABC so it says domains but they’re also called ATP binding cassettes & in this overview, there are 2 ATP binding cassettes that are found on the cytosolic side of the membrane. Those can be the ATP binding domains, then there are two transmembrane domains in these proteins. - They’re found in both bacterial and eucaryotic cells, the main difference b/w these 2 figures is that in bacterial cells, they’re mainly carrying out nutrient uptake from the environment & in eucaryotic cells, they’re typically carrying out the transport of toxins & waste products outside of the cell. This is not exclusively true, there are some bacterial ABC transporters that will also export toxins outside of the cell but one of the main functions is to uptake nutrients. - How is this done? One of the first steps is ATP binding. On the left is the original conformation of the ABC transporter, a small molecule, which could be a nutrient from the environment, will bind to the ABC transporter since this pocket here where the molecule binds is exposed to the outside of the cell. - Then the ABC transporter will bind ATP, there are 2 ATP binding domains, so this ABC transporter will bind 2 ATP molecules and this will induce a conformational change in this protein. This is important, again this is a transporter protein and one thing that unifies these transporter proteins is that they all undergo conformational changes as they’re transporting small molecules across the membrane. - You have ABC transporter there, ATP binds & induces a conformational change, then the ATP is hydrolyzed to ADP & that exposes this small molecule now to the cytoplasm of the cell & it will be released in the cytoplasm. That is how basically the overview of how ATP binding & ATP hydrolysis induces this conformational change. - Recap: ATP binding induces the dimerization of ATP binding domains, so you can see that those are the two ATP binding domains, they aren’t associated with each other, when they bind to ATP, they dimerize together inducing a conformational change. Then the ATP is hydrolyzed shown in the slide and this causes the dissociation of the small molecule from the ABC transporter and ATP binding and ATP hydrolysis induces these conformational changes in the transmembrane domains as shown in the slide. This exposes the binding site of small molecules, it goes from being exposed to the extracellular space and then the conformational change induces it to be exposed to the cytosol of the cell. The substrate binding site goes from being exposed on one side of the membrane, to the other and that induces transport. - Now when the eucaryotic ABC transporter that would be exporting toxins out of the cell, the original conformation would have substrate binding sites on the cytosolic side, the small molecule can bind, ATP binding to the ABC transporter would induce dimerization of these domains, and induce a conformational change of the transmembrane domains. ATP hydrolysis would induce the dissociation of the small molecule now in this case to the outside of the cell.  Sequester toxins – removed by leaf shedding - In bacteria, these ABC transporters are important for importing nutrients into the cell but they’re also involved in exporting toxins and they can actually be very important in developing antibiotic resistance in bacteria since some of these ABC transporters can actually transport antibiotics outside of bacterial cells and then these bacteria can become more tolerant to antibiotics. So they can be involved in import and export in bacteria. - In plants, they are involved in transporting toxins from the cytosol to the vacuole & this is done to store these toxins in the vacuole & then they can be removed by leaf shedding. So the toxins become sequestered in the vacuole by the ABC transporters & then plant can get rid of these toxins by shedding its leaves. - In animals, they’re also used to export natural toxins & also waste products & also drugs. One of the very famous examples is called multi-drug resistant protein 1 or MDR1. This protein can export drugs from cancer cells and there is over-expression of MDR1 in numerous types of cancers and this can lead to resistance of these cancers to chemotherapeutic drugs. Since the cancer overproduces these ABC transporters called MDR1, the chemotherapeutic drugs are pumped out of the cancerous cell & are much less efficient at treating cancer. - This is a diagram of MDR-1 and normally it's expressed in liver, kidney and intestinal cells. It is involved in excreting natural toxins or waste products into the bile, urine or feces but it is also able to export drugs so its ability to excrete toxins also make it capable of exporting drugs and it is overexpressed in many cancer lines following treatment with chemotherapeutic drugs. This leads to resistance of cancers to chemotherapy. - ABC transporters play very important roles in numerous drug resistance, antibiotic resistance and we’re not going to cover it in detail but it is also the mutation of ABC transporters that can lead to cystic fibrosis. So they play important role in diseases and drug resistance. - That is as far as we will cover the transporter proteins, so we just finished the ABC transporters and now we’re going to move onto a second category of proteins that move small molecules across membranes: channel proteins. All channel proteins carry out passive transport and unlike transporter proteins, channel proteins do not undergo conformational changes as they’re transporting their small molecules across the membrane.  Weaker interactions with solute  Faster transport by channels  Several molecules pass through when open - Here is a diagram of a channel protein and basically what it does is it creates a hydrophilic pore across the membrane, so the grey is the lipid bilayer, then there is the channel protein in green and it creates this hydrophilic pore through which these small molecules can cross the membrane. - Most channel proteins are selective meaning they will transport one particular type of ion, so you’ll have potassium channels, you’ll have sodium channels, you’ll have calcium channels and they will be specific for that type of ion. We’re going to go mainly into different ion channels. These are the major examples we’re going to cover. - They all carry out passive transport, now they create a weaker interaction with their solutes than transporter proteins, so you’ll recall that the glucose uniporters also carried out passive transport but they bind to one molecule of glucose, undergo a conformational change, release the glucose on the other side of the membrane and then change conformation back to be able to transport another molecule of glucose. You can see that that it would be much slower than transporting a molecule just allowing it traverse the membrane through a channel. They have much weaker interaction with the solute, and they carry out faster transport than transporter proteins that carry out passive transport. There is much faster transport by channels than by transporter proteins. - Another important feature is that several molecules can cross ion channels simultaneously. When the channel is open, multiple small molecules can cross through the channel. This is in contrast to the uniporter which binds to one molecule and transports one molecule at a time across the membrane. - Ion channels are found in animals and in plants and in microorganisms so they are found throughout the different kingdoms and there are two major types. - There are the non-gated ion channels that are always open. The major example we’ll cover is the potassium leak channels & what we’re going to cover is that they play a major role in generating the resting membrane potential in animal cells. These channels are non-gated channels and they’re always open. - The 2 category are gated ion channels and these are not always open, they need to be opened by an external stimulus such as a chemical or an electrical signal which is required for this channel to open.  Always open  K moves out of the cell - Now the first example we’re going to go through are the non-gated ion channel and particularly the potassium leak channel. - The potassium leak channel is found in the plasma membrane of animal cells and they’re always open. These are the non-gated channels. - As you’ll recall, potassium is in higher concentration in the cytosol relative to the outside of the cell so what happens is the potassium moves out of the cell down its concentration gradient. - Remember that the Na-K pump is pumping Na outside of the cell and K inside of the cell and there is a buildup of K inside of the cell relative to the outside. There is a higher concentration inside and these channels allow K to move down their concentration or electrochemical gradient outside of the cell. - They’re found in the plasma membrane of animal cells and these are the major contributors to the membrane potential across animal plasma membranes.  Difference in electrical charge on two sides of membrane - So if you’ll recall, the membrane potential is the difference in electrical charge on the two sides of the membrane and in animal cells, the plasma membrane is positively charged outside relative to the inside. Now this membrane potential is very important, the positive ions on the outside can be used as we saw by symporters and antiporters to carry out secondary active transport in both animals and plants. We looked at the Na-glucose symporter in lecture 2 as an example of using these positive charges outside to move glucose against its concentration gradient. - In nerve cells, this membrane potential also play an important role in generating an action potential so basically this is the movement, depolarization of this membrane potential that moves along the nerve cell that eventually leads to the stimulation of another cell downstream so it’s used by a nerve cell to transmit an electrical signal. Membrane potentials play a very critical role in generating action potentials in nerve cells of animals.  Major role in membrane potential  Net: 1 (+) ion pumped out  ~10% of membrane potential - How are these membrane potentials generated in animal cells? Well he mentioned that the K leak channel is the major contributor to the membrane potential in animal cells & this is due to the outward flow of K through these channels, down their electrochemical gradient. As K is moving down its electrochemical gradient, it’s building up positive charges on the outside of the membrane. - Then the second contributor is this sodium potassium pump. Recall that this results in a Na gradient where there is a low Na concentration on the cytosolic side because the pump is pumping Na outside of the cell and you result in a high cytosolic K concentration because K is being pumped into the cell. However, this is electrogenic, meaning that there aren’t equal charges being transferred across to each side of the membrane. There are three Na ions that are being pumped out for every two Ks that are pumped into the cell. This results in a net positive charge being pumped out of the cell relative to inside of the cell, so you can see how that can contribute to a positive charge outside of the membrane. Surprisingly, this only contributes 10% of the membrane potential. The rest is majorly contributed by the potassium leak channels. So you have a net positive charge of one being pumped out by the Na-K pump and in addition to that, you have K ions leaking out to the outside down their electrochemical gradient and that also contributes to a positive charge outside of the membrane and this is the major contributor.  Each ion needs a counter-ion  More (+) on outside (Na , K ) -  More (-) on inside (Cl and fixed anions) - What are the principles behind this? One thing that is important to realize is that ions come in pairs. We talked about the sodium ion in isolation as being positively charged but there are always negative ions that are also present in the cell to counterbalance these positive charges. Each ion needs a counter-ion. So on the outside the cell, there would mainly be sodium and chloride ions, these would be counter-ions of each other. In the cytosolic side, you have mainly K, also chloride ions, and you have a lot of fixed anions – anions are negatively charged and many of these are in the form of proteins so proteins are mainly negatively charged, nucleic acids such as RNA are also mainly negatively charged and other macromolecules all contribute to having this negative charge inside of the cell. These are all counter-ions to the positive K ions inside the cell. + +  More (+) on outside (Na , K )  More (-) on inside (Cl and fixed anions) - What’s happening is ions are diffusing from high to low concentration through these K leak channels, so K is moving out of the cell across the
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