Cell Physiology PDF Notes

N.B. Le sbobine sono state disposte in ordine cronologico, tuttavia ci sono degli stessi argomenti che vengono affrontati dalle 2 prof in lezioni diverse. Sono 2 i macro-argomenti del corso; per chi volesse studiare separatamente e più linearmente i due macroargomenti deve seguire queste lezioni: -conoscenza di base della neurofisiologia(etc.): lez. 1-2-3-4-5-10-11-13-14 -compartimenti fluidi del corpo (etc.): lez.6-7-8-9-12 CELL PHYSIOLOGY LESSON I – 15.04.2024 (Ciranna) GENERAL PRINCIPLES All physiological processes are described by equations, some of which can be very complex. However, they can all be summarized into a very general and simple equation, which is the following one: 𝑭𝑭 𝒗𝒗 = 𝑲𝑲 × 𝑹𝑹 Where: v = speed K = conversion factor F = driving force R = resistance opposing the physiological process It means that the speed of any process is: - directly dependent on the driving force F, which means the force that stimulates the process and - inversely dependent on the resistance R, that opposes to the physiological process. For example: An ion current flowing through an ion channel: it is directly depending on the membrane potential (F) and is inversely proportional to the resistance (R) opposed by the membrane itself. Blood flow in circulation: it is directly related to the pressure gradient (F) and is inversely related to vascular resistance (R), which means the diameter of the vessels. These are general examples just to say that each process has a driving force and a resistance opposing the processes. 1 1 REGULATION OF PHYSIOLOGICAL FUNCTIONS 2 In our body there are specific regulation mechanisms: Feedforward regulations: which means that the process reinforces itself, so the final product reinforces its own production. Feedback regulation: that could be either negative or positive. The negative one is when the final product inhibits the process.  This happens in some hormones’ production: a gland produces a hormone that is released in blood; when the concentration of the hormone raises about a certain level, then this performs negative feedback on the gland that is producing this hormone. This negative feedback is widespread in our organism. It is crucial because it acts by inhibiting the first process, and thus by preventing the excess of a specific product. For other hormones there is the positive feedback. 1 Questa foto era molto sfocata e quindi ho riscritto io le cose nei riquadri; la slide è la numero 6 del PowerPoint numero 1 della Ciranna. 2 For greater clarity, this is what the book says about regulation systems: The regulation of processes can essentially occur in two ways: - feedforward: regulation that occurs by independent processes on other processes, so as to give rise to chains of resulting events. For example, anticipatory increases in breathing frequency will reduce the time course of the response to exercise-induced hypoxia. - feedback: regulation that occurs by a product, direct or indirect, of the process itself. In particular: -negative feedback: occurs when the product of a process reduces its efficiency -positive feedback: occurs when the product of a process further stimulates the process itself 2 THE CELL The cell is a very complex system that continuously interacts with the surrounding environment and renews its components and its dynamic balances. For cells, it is crucial to maintain the homeostasis: it comprehends a set of mechanisms that allow us to maintain a relatively stable situation, which we call stationary state, but we have to use energy to maintain it (thanks to an active mechanism). SLIDE: Homeostasis is a dynamic condition, continuously changing in adaptation to both environmental and internal conditions. Importantly, homeostasis must not be considered as an equilibrium state, but as a state of disequilibrium, that is the best state for cells to live and to function: in fact, if there isn’t this disequilibrium the cells will die. For example, think about the intracellular and the extracellular ion concentrations: they are very different! So, there is a disequilibrium between these two parameters, but this disequilibrium is fundamental for cell’s life. For instance, K+ ions are highly concentrated inside the cell, while Na+ ions are highly concentrated in the extracellular side: if this is disequilibrium is disrupted then the cell will die, because the transportation mechanism will fail and also because there will be no more membrane potential. This is a dynamic condition, in fact both the intracellular and extracellular conditions can change because the system must act in order to always recover homeostasis. MAIN FUNCTIONS OF CELLS Energy production: cells obtain energy from molecules that we absorb from diet, in particular from nutrients. This involves a series of catabolic reactions, in which a large molecule is broken down into smaller molecules and then the final catabolic reaction produces water, CO2 and energy through oxidative events. The main molecule that we use for energy is glucose: 1. Firstly, glucose undergoes a series of fast anaerobic reactions that can produce only a small amount of energy. 2. Only later, when it is oxidized in mitochondria, during a process that is not as fast as the anaerobic mechanism but has a higher rate, more energy will be available. 3 Maintaining of the cell structure and function: the cell produces many proteins, with many different functions: structural, transport, defence, catalysis 3. Specific functions: cells are not all equal, but are differentiated and have a specialized function. For example: - Muscle cells have filaments which are able to slide, and this allows the contraction. - Neurons are also very special cells that are able to receive signals, generate signals and transmit them (they’re excitable cells). CELL MEMBRANE 4Cell membrane envelops the cell and consists of a lipid bilayer, which is a thin, double-layered film of lipids. To understand the structure of the plasma membrane we first should remember the conformation of one phospholipid molecule, in which we can distinguish: One end containing the P group: it is hydrophilic and soluble in water. One end consisting of the fatty acid portion: is hydrophobic and soluble only in fats. Since the hydrophobic chains are repelled by water, they have a natural tendency to locate in the internal side of the plasma membrane (=it means that the hydrophobic chains face each other on the two internal sides). Instead, the hydrophilic phosphate portions constitute the two surfaces of the complete cell membrane, in contact with the two aqueous solutions of the intracellular water and the extracellular water. 3 enzymes are involved in the catalysis of many and very different reactions. 4 In questa parte ho integrato qualcosa dal libro 4 Additionally, into the membrane there are many proteins with several different functions. The main functions of the cell membrane are: Separate the extracellular and intracellular compartment, which are really different from each other. It allows exchanges: these exchanges are regulated because not all substances can pass, but only the ones that are useful for the cell. It is important for the communication of the cell.  On the cell membrane there are receptors, which are special proteins that recognize a ligand and then are able to activate a series of intracellular mechanism to reach then the final effect. N.B.: the grey parts represents phospholipids. CELL MEMBRANE PROTEINS In the cell membrane there are many proteins. Integral proteins: which cross the membrane, going from the outside to the inside. There are many types of integral proteins: 1. Ion channel: contain a central pore. 2. Carrier protein: which is a transporter. 3. Receptor: that will bind a ligand, initiating an intracellular chain of messengers which automatically trigger an effect. Peripheral proteins: which do not span the membrane but are associated to the membrane from one side of the membrane. INTEGRAL PROTEINS 5 Overview: In the image below it’s possible to see the structure of an integral membrane protein: In particular, this is the typical structure of a G protein coupled receptor, a large family of receptors (remember that the receptor recognizes the ligand and binds with it). They have a typical structure and span the membrane 7 times. So, they are not channels, but they are receptors activating a signal and then a final effect. Now, we’ll analyze more in detail: receptors, ion channels and carriers. RECEPTORS: The image above shows how a receptor works. The receptor behaves as a lock and the chemical ligand behaves as a key: that key is specifically designed for that lock and can open it. In fact: I. Each ligand has to find its specific receptor II. Then can bind to it strictly III. The ligand-receptor complex activates a cellular response: it will activate some intracellular machineries of messengers (which in turn activate other messengers), and at the end of this cascade there will be a final cellular effect. ION CHANNELS 5 AND CARRIERS: 5 N.B. this is a brief introduction about ion channels, but we’ll have a whole lesson dedicated to them. 6 Ion channels and carriers are also integral membrane proteins. Normally, ion channels are formed by many subunits. - There is a pore inside and, when the channel is open, this pore is a free passage. - However, not all ions can freely pass: ion channels are very specific for a particular ion; in fact, we have sodium channels, potassium channels, and so on. However, there are also some ions channels that let pass more than one ion, but nevertheless they are specific. - When a channel is open, the passage of the ions occurs through diffusion 6. A carrier is a protein with the function of transporter: it binds an ion or a molecule on one side of the membrane, then changes its conformation, and finally releases the molecule on the other side. To sum up: - A ion channel is like a classic door, so when the door is open the passage of ion is free - A carrier acts as a sort of revolving door. ENZYMES Normally, the name of an enzyme immediately tells their function. They are involved in the catalysis of many reactions. In the image below we can observe the adenylate cyclase, which is an enzyme catalyzing a reaction in which cAMP (which stands for cyclic adenosine monophosphate) is formed. 6 Diffusion is a passive mechanism, where ions move from the compartment where they are more concentrated to the compartment where they are less concentrated. We’ll delve into later 7 - cAMP is a really important molecule for the cell because it is an intracellular messenger. - When cAMP is produced, it activates other enzymes, triggering a chain of reactions that ends in a final cellular effect. How is adenylate cyclase activated? It is activated by G proteins 7, that are GTP-binding proteins connected to a membrane receptor. Let’s see the whole mechanism from the beginning: I. A specific ligand binds ITS receptor on the cell membrane and activates it. II. This will subsequently activate the G protein complex. In this image, the involved G protein is a TRIMERIC protein, formed by three subunits: α, β, γ. When the G protein is activated, the α subunit detaches from the other two subunits and exchanges GDP for GTP. III. At this point, this activation will, in turn, lead to the activation of the adenylate cyclase. IV. Then, the activated adenylate cyclase will catalyze the reaction in which cAMP is formed. Another example could be Phospholipase C, which has a function similar to the latter one, because it is activate by G proteins too. I. At the beginning of the process, a signaling molecule, such as a neurotransmitter or a hormone, activates the specific membrane receptor. II. This membrane receptor in turn is connected to a particular G protein called Gq. III. This Gq protein will activate the enzyme Phospholipase C. Phospholipase C breaks down a small number of phospholipids, producing active molecules, that act as messengers:  One such messenger is diacylglycerol (DAG), which activates a kinase.  Another messenger is IP3 (Inositol trisphosphate), that acts on the endoplasmic reticulum. The endoplasmic reticulum stores calcium ions8, that can be released into the cytoplasm upon some signals (one of this is exerted by IP3). 7 In tante altre lezioni se ne parlerà 8 Furthermore, calcium ions are important intracellular messenger; when they are released, they trigger a series of activation or inactivation of some enzymes, and this will automatically lead to a cellular response. 8 HOW DO WATER AND HOW DO OTHER MOLECULES CROSS THE MEMBRANE? WATER TRANSPORT: We already know that each passage is strictly regulated, because the membrane doesn’t allow the passage of all the molecules. Every cell has a specific channel for water molecules: the aquaporins (proteins) that form these pores for the passage of water. Thanks to aquaporins, water can cross the membrane, but this passage depends on a driving force, which is the osmotic pressure. 9Osmotic pressure is generated by two different solutions, that are separated by a semipermeable membrane, that only allows water molecules to pass: water molecules pass from the more diluted compartment to the most concentrated one. At the end, the two solutions will have the same osmolarity. This process also occurs in cells because, as we’ve said, cell membrane is permeable to water. Unlikely water, many solutes cannot freely pass across the membrane! And this is crucial for the cell because the osmolarity must be the same inside and outside the cell. In fact, what happens when the extracellular fluid has a different osmolarity compared to the intracellular fluid? Let’s see the following examples: 9 This concept will be particularly important when we’ll talk about fluid compartments and some mechanisms that occur in this context. 9 Here you can see a red blood cell, which is immersed in a solution: When the RBC is immersed in an isotonic solution: since this solution has the same osmotic pressure of the cell’s interior, there would be no net flow of water molecules and the cell will maintain its shape and its integrity. When the RBC is immersed in a hypertonic solution: since the solution outside is more concentrated than the cell’s interior, then water will go out and the cell will shrink, and eventually die. When the is RBC immersed in a hypotonic solution: since this solution is less concentrated compared to the cell’s interior, water will enter the cell and the cell will swell and will eventually break (=it can “explode”). In general, changes in the osmolarity of extracellular fluids are very critical, and if they are not compensated the cell can die. But luckily our body has a series of mechanisms that maintain the osmolarity of extracellular fluid. For example, kidneys can regulate the quantity of each electrolyte (like ions) in our blood. So, kidneys regulate for example sodium, potassium, and other ions concentrations. CONCENTRATION OF IONS IN THE CELL In an isotonic solution, osmolarity is the same, but the intra and extracellular composition of ions can be different. If we look at the picture, we can see different ion concentrations comparing the inside and the outside of the cell. 10 Cations: Sodium concentration: inside the cell Na+ concentration is very low, while outside the cell is much higher. Na+ concentration in blood is similar to that in the extracellular fluid: so, between blood and interstitial fluid there is not so much difference, but there is a very big difference between the outside and the inside of the cell. Potassium concentration: the distribution of K+ is opposite to that of Na+:  K+ is the main intracellular cation, while Na+ is the main extracellular cation. This concentration is really crucial for the cell, because this is the origin of membrane potential, which is the difference in charges between the inside and the outside of the cell (we are going to explain this better later). Additionally, many cellular transport process rely on the concentration gradient between K+ and Na+. Calcium concentration: it is slightly more concentrated in blood than inside the cell, because calcium ions are linked to blood circulating proteins such as albumin. Inside the cell the calcium concentration is really low. Why is it so low? Because when calcium ions (that are stored in the endoplasmic reticulum) are released, they can activate many mechanisms that lead to different cellular effects. So, in normal conditions, they have to be “sequestered” and not free in the cell. Magnesium concentration: inside the cell the concentration is slightly higher, but there’s not much difference. Anions: Chloride concentration: the main anion is the Cl-, that has a low intracellular concentration and a higher extracellular concentration. A concentration: “A” stands for big protein anions. These are intracellular proteins which have a negative charge, but since they are really big proteins, they cannot go out of the cell and therefore they remain into the cell. That’s why their concentration inside the cell is very high. Keep in mind these different concentrations and then we will explain them better. 11 There is a different concentration of cations and anions between the inside and the outside of the cell, but the total number of molecules is the same (?), resulting in equal osmolarity inside and outside the cell. So, in terms of osmolarity there is no difference, but in terms of ionic concentration there is a big difference. HOW MOLECULES AND ION CROSS THE MEMBRANE Chemical and physical principles regulating the passage of ions and molecules through the cell membrane: PASSIVE PROCESSES: DIFFUSION ACTIVE PROCESSES 1. PASSIVE PROCESSES Passive transport is a process that doesn’t require energy to occur. And it proceeds according to the concentration gradient 10. However, remember that this occurs only for some molecules and substances: For example, a lipophilic molecule can cross the membrane through diffusion, or also oxygen, C02, steroids, because they are soluble in lipids as well. We have 3 main kinds of diffusion: Simple diffusion which occurs through the membrane lipid bilayer and only for lipophilic molecules. Gated diffusion, we know that some ions channels are always open, while others are gated, so they can both be opened or closed. Gated diffusion occurs through gated ion channels (ions only). Facilitated diffusion requires the presence of carriers. So, it’s a carrier-mediated diffusion (it’s a selective process, undergoing saturation). Concentration gradient: Diffusion is a passive process, which does not require any energy source, because there is already a driving force which is the concentration gradient. In the case of ions there is a second driving force, because they are charged molecules ( they are attracted by opposite charges and repelled by the same charges). So, when we think of diffusion of ions we must think of 2 gradients: 1. concentration gradient (chemical) 2. electric gradient: depends on the charges These two forces can go both in the same and in the opposite direction. When the concentration gradient and the electric gradient oppose each other, the ion will move in the direction of the stronger gradient between them. 10 The concentration gradient refers to the fact that molecules will diffuse from the more concentrated compartment to the less concentrated one. 12 Diffusion rate: Moreover, there’s the concept of diffusion rate, which describes how quickly a substance diffuses the membrane under the influence of a concentration gradient. It depends on: -distance: for a long distance there’s a slower diffusion rate, while for a shorter distance the diffusion will be fast -temperature: a higher T will increase the diffusion rate -molecule dimension: it is inversely related to diffusion rate, in fact for a big molecule the diffusion rate is slower rather than a small one -membrane diffusion area: if it is big this will increase diffusion rate -thickness: is inversely related to diffusion rate For ion channels11, the rate of ion diffusion also depends on channel features and on the number of open channels (so, for ion channel diffusion we also have to consider if there are many or few channels and if they are gated, because some channels are always open, while others have a gating mechanism). We have a law that describes all the parameters influencing the diffusion rate: the Fick’s law 𝑨𝑨 𝑱𝑱 = × 𝑫𝑫 × (𝑪𝑪𝑪𝑪 − 𝑪𝑪𝑪𝑪) 𝒔𝒔 J = diffusion rate A = cell membrane area s = cell membrane thickness D = diffusion coefficient C1 = substance concentration in compartment 1 C2 = substance concentration in compartment 2 Brief summary of the concepts related to diffusion: Diffusion does not require any energy source. It occurs thanks to the concentration gradient, while for ion is the electrochemical gradient (that is a double gradient: electric and concentration gradients). Diffusion rate Fick’s law 3 types of diffusion: -Simple diffusion -Gated diffusion -Facilitated diffusion SIMPLE DIFFUSION: 11 As we said before, we’ll delve into this topic in another lesson about ion channels. 13 Simple diffusion occurs only for some types of molecules. For instance: lipophilic molecules, such as steroid hormones, and gases, such as O2. An example of simple diffusion can be the one of the O2 and C02 exchange in lungs. In fact, gases exchanges happen thanks to diffusion. This happens in lungs: oxygen crosses the membrane and goes into the blood and CO2 from the blood goes outside and is released into the air. In peripheral tissues we have the opposite: blood brings O2 to tissues, while CO2 moves from the tissues into the blood capillaries. Also this passage is driven by differences in concentration. There’s a difference in the pressure between the two gases in the different compartments! GATED DIFFUSION: Then we have gated diffusion, which is the diffusion of ions through an active channel. In the picture below you can see a sodium channel: When the channel is open, Na+ ions flow into the intracellular fluid through diffusion. They are, in other words, driven by an electrochemical gradient. 14 FACILITATED DIFFUSION: Book: Facilitated diffusion is also called carrier-mediated diffusion because a substance transported in this manner diffuses through the membrane with the help of a specific carrier protein. That is, the carrier facilitates diffusion of the substance to the other side. An example of facilitated diffusion could be the glucose transporter (Glut), that are a family of transporters that are expressed in different tissues, and one of them is particularly important and is the GLUT 4, which is regulated by insulin 12. So, let’s see how the transporter works: I. It’s a protein carrier that will bind glucose on the extracellular site II. Then the transporter changes its conformation, like a revolving door, and releases glucose inside the cell. It is driven by the concentration gradient: it can only functions when glucose is more concentrated on the extracellular side. When there is a strong concentration gradient the system works. 12 Insulin is a hormone that is really important for our metabolism: it allows glucose uptake by cells in muscle tissue and in adipocytes, that’s why insulin lowers glucose concentration after a meal 15 This is a summary of the three types of diffusion: Simple diffusion, trough the lipid bilayer, that occurs for lipophilic molecules such as steroid hormones, gases like oxygen and C02. This mechanism has no saturation, so if the gradient increases the rate will increase as well without any saturation. Gated diffusion, which is the passage of the ions through ion channels; there is a saturation (which is a limit). When you reach the maximum limit you cannot go further, and this limit depends on the number of ion channels. Facilitated diffusion, which requires a transporter: here there is a much higher degree of saturation, because is a slower process and is strictly dependent on the number of transporters. 2. ACTIVE TRANSPORT Active transport requires energy (ATP), in fact it must be done opposing the concentration gradient: in other words, ions and molecules are transported from the compartment where they are less concentrated to the compartment where they are more concentrated. We have: PRIMARY active transport: because these transporters are able to break down ATP, hydrolyzing it in ADP and phosphate, delivering energy. SECONDARY active transport: these transporters are not able to hydrolyze ATP, but nevertheless they still require energy. They rely on the presence of another primary active transport. PRIMARY ACTIVE TRANSPORT: Book: Primary active transport systems (pumps) are transmembrane proteins that use energy to transfer a substrate against the concentration gradient. The substrate is bound on one side where it is less concentrated and released on the other, where it is more concentrated. Primary active transport systems are specific and selective and are also saturable. We’ll see some examples: 16  Na+/K+ pump This is the most common primary active transport, which is the Na-K pump ATPase. It is located on the extracellular membrane.  It binds 3 Na+ ions in the inside and 2 K+ in the outside.  Then, it pumps Na+ ions out of the cell and K+ ions into the cell. So, this transport occurs against the concentration gradient because Na+ is already more concentrated outside the cell and K+ is already more concentrated in the inside. Remember: the pump is able to hydrolyze ATP. In the picture above, they are shown all the PASSAGES of the mechanism of this pump: I. 2 K+ ions enter the cell. In the meanwhile, 3 Na+ ions bind its internal site on the transporter II. The pump undergoes a conformational change, releasing the 3 Na+ ions outside the cell III. As Na+ is released, 2 other K+ ions bind their specific site on the transporter IV. K+ is released inside the cell and the cycle can start again. 17 Every cell shows this Na-K ATPase, and this is crucial for cell life, because if it doesn’t work, then the concentration gradient is lost and this has really bad consequences on the cell, because then also the secondary active transports cannot work. This concentration gradient is also crucial for the membrane potential. It plays an important role in particular excitable cells, which are cells that are able to receive and send signals. N.B. Irreversible blockers are considered “poison” for the cell, (they kill it) because they block this pump.  Ca2+ ATPases: This is another type of primary active transport (so it’s able to hydrolyze ATP), which is calcium ATPase. There are 2 of them: a) One is located on the plasma membrane and is called plasma membrane Ca-ATPase (PMCA). It pumps calcium to the outside of the cell: it binds calcium inside and brings it outside the cell. b) The other is located on the endoplasmic reticulum, which is the sarcoplasmic endoplasmic reticulum ATPase (SERCA). (Remember that in muscle cells is called sarcoplasmic reticulum, in other cells endoplasmic reticulum).  The Ca-ATPase sequesters and transfers calcium from the cell cytosol into the lumen of the endoplasmic reticulum. That’s why the activity of these 2 transporters make the intracellular cytosol concentration of calcium very low.  Hydrogen ion pump: H+/K+ pump 18 Hydrogen ion pump: there is an exchange between 4 protons (H+) and 4 K+ ions: 4 K+ ions from outside are transported inside, while 4 protons go outside. We can find this type of transporter in many cell types because it is related to the regulation of the intracellular PH. Specifically, this transporter is abundant in in particular cells:  stomach: the gastric secretions depend on the activity of these transporters. You know that gastric secretion is very acidic, and the PH could be very low, because of this transporter, which extrudes protons into the gastric secretion.  We also find it in other organs, such as in our kidneys, which can regulate the amount of protons that we eliminate with urine. Summary of PRIMARY ACTIVE TRANSPORT: Na+/K+ ATPase Ca2+ ATPases H+/k+ ATPase (hydrogen ion pump) SECONDARY ACTIVE TRANSPORT: Book: Secondary active transport systems do not use a direct source of energy but exploit the gradient of another substrate (which in turn must have been produced by a primary transport system). In fact, they do not hydrolyze ATP by themself, but nevertheless require energy. We can distinguish between: - co-transport: when the 2 (or even more) molecules are transported in the same direction. - counter-transport: when molecules are exchanged in two different directions. Distinct sodium-dependent secondary active transporters exist for different molecules: they can work because there is a sodium gradient. It’s like they exploit the concentration gradient for sodium ions. N.B. we’ll also see the K+ - Cl- co-transport (KCC) which doesn’t involve sodium. CO-TRANSPORT, among which we can find:  Sodium-glucose co-transport: the name of this transporter is SGLUT (or SGLT). 19 Na+ and Glu move in the same direction, from the outside to the inside of the cell. I.SGLUT binds Na+ on the extracellular side II.This binding increases the affinity for glucose molecules, because Na+ binding unmasks a binding site for glucose. III. Then there is a conformation change, and the transporter will change like a revolving door. IV. It will release both Na+ and glucose inside the cell. This process happens because sodium has a stronger driving force to enter the cell, because it is low concentrated inside, and outside is very highly concentrated 13: so, there is a concentration gradient for sodium. Where can we find this transporter? INTESTINE: We find it in particular districts where we have to absorb glucose, like in the intestine after digestion: after we digest carbohydrates, glucose molecules are absorbed in the intestine. This process is facilitated by SGLUT located on the intestinal cell membranes, which transports glucose from the intestine into the bloodstream. KIDNEYS: In the kidneys, blood filtration is the first step of urine production. Also glucose is filtered from the blood during urine formation. Normally, almost all filtered glucose is reabsorbed back into the bloodstream through SGLT in the renal tubules. 13 Remember that: the concentration gradient for sodium is created by Na-K pump, which is a primary active transport. 20 This prevents glucose from being lost in urine. In fact, we have to remember that glucose is really important for our metabolism, so we have to recover its molecules: - when glucose enters the tubules then SGLT acts to reabsorb it, - in this way it is not eliminated by urine. In diabetes I type: the patient has no insulin production, and so the concentration of glucose in blood is very high. The SGLT in the kidneys is designed to reabsorb glucose up to a certain limit. When blood glucose levels exceed this limit, the excess glucose cannot be fully reabsorbed and ends up in the urine: this is the reason why patient with this pathology will eliminate glucose by urine and this has a series of catastrophic consequences.  Sodium-amino acids co-transport: it is like SGLUT, but it involves amino acids. Actually, there are different types of Na+-amino acids co-transporters, each specific for different types of amino acids (the most important are four). The mechanism of this co-Transport is: I. Na+ binds to the transporter protein on the cell membrane. II. Then the transporter binds the amino acids III. The binding of both Na+ and amino acids induces a conformational change in the transporter. IV. At the end the release of molecules inside the cell. We find this transport in cells involved in the uptake the amino acids: - cells of the intestine: in the intestine, proteins are digested and broken down into amino acids, which need to be absorbed. Intestinal cells express sodium-amino acid co- transporters to facilitate the absorption of these amino acids. - cells of the kidneys: amino acids are small molecules that are completely filtered out of the blood during the first step of urine formation, known as glomerular filtration. To prevent the loss of amino acids in the urine, these molecules need to be reabsorbed. Tubular cells in the kidney express sodium-amino acid co-transporters to reabsorb amino acids from the filtrate, ensuring they are retained in the body.  Na+-K+-Cl- co-transport (NKCC): co-transport of 1 ion of Na+, 1 of K+ and 2 of Cl-. 21 This transporter moves one ion of sodium (Na+), one ion of potassium (K+), and two ions of chloride (Cl−) in the same direction: from outside the cell to the inside. NKCC is not present in all cell types and is notable because it increases the concentration of chloride ions inside the cell, which is unusual. Where is this transporter expressed? For instance, during the early stages of nervous system development 14, immature neurons express NKCC. This results in a high concentration of chloride ions inside these neurons, and this is important because it changes completely the effect of the neurotransmitter GABA (Gamma-Aminobutyric Acid).  while GABA typically exerts an inhibitory effect in mature neurons,  it exerts an excitatory response in immature neurons. So: GABA is inhibitory in mature neurons, but not in immature neurons. Immature neurons respond to GABA with an excitation. This excitatory response is due to NKCC creating a high intracellular chloride concentration. Remember that there are two types of GABA receptors 15: - GABAA: it is a chloride channel (ion channel). It’s an ionotropic receptor, because when GABA binds to GABAA receptor, Cl- ions flow through the channel thanks to the opening of the central pore and diffuse according to the concentration gradient. Typically, this results in an inward flow of chloride ions in mature neurons (making the inside of the cell more negative). However, in immature neurons expressing NKCC, the high intracellular chloride concentration causes GABAA receptor activation to result in an outward flow of chloride ions (making the inside of the cell less negative), leading to excitatory effects. 14 In the XI lesson, prof. Ciranna will repeat this concept. 15 Also this concept will be repeated in future lessons. 22 Abnormal expression of NKCC during development can lead to diseases then in mature neurons where GABAA receptors produce excitatory effects instead of their normal inhibitory effects. This has been observed in conditions like epilepsy and certain forms of autism. - GABAB 16  K+ - Cl- co-transport (KCC): this is a co-transport but pay attention because it doesn’t involve sodium (we’ll analyze it now since it’s a co-transport!). This transporter operates independently of sodium and facilitates the movement of both potassium (K+) and chloride (Cl−) ions across cell membranes toward the outside. During brain development there is a switch: immature neurons that initially expressed NKCC transporter (as we said) undergo a developmental switch thanks to which they stop expressing NKCC and start expressing KCC receptors which exports chloride ions out of the cell. As a consequence, there is a modification in the Cl- concentration gradient. And this why GABA typically exerts an inhibitory effect in mature neurons. When this switch from NKCC- to – KCC receptors doesn’t occur properly, that can cause some diseases. COUNTER-TRANSPORT, among which we can find:  Na+ - H+ counter transport: it is ubiquitous in expression and plays a crucial role in maintaining cellular pH neutrality. It operates using the sodium gradient, where sodium ions (Na+) move into the cells while protons (H+) are simultaneously transported out of the cells. This process helps regulate and stabilize the pH levels within cells. 16 she didn’t mention this receptor. Some information from internet: GABAB receptors are the G-protein-coupled receptors (GPCRs) for GABA and thus are metabotropic receptors (that are linked via G-protein to K+ channels.) 23  Na+ - Ca2+ counter-transport: it pumps 3 Na+ inside the cell and Ca2+ outside. This keeps a stable concentration of calcium within the cell, which is crucial for regulating various cellular processes since calcium represents a signal. Under resting conditions, the cytosol maintains a low concentration of calcium because this transporter actively moves calcium ions out of the cell. This action is facilitated by plasma membrane calcium ATPase, which is a primary active transport mechanism, as well as by the sarcoplasmic/endoplasmic reticulum, which also plays a role in calcium storage and release. SUMMARY ACTIVE TRANSPORT ACROSS THE MEMBRANE: 24 Moreover, we have to study another specific type of transporter that is independent from Na+:  HCO3-/Cl- counter-transport: It is expressed in erythrocytes (we’ll mainly focus on that), renal tubular cells, gastric parietal cells and pancreatic cells. It exchanges 1 HCO3- (bicarbonate ion) for 1 Cl- ion: as we can see from the image, the bicarbonate is pumped outside the cell and the Cl- inside. The bicarbonate comes from CO2: blood transports CO2 from peripheral tissues, as it is produced during cell metabolism. Then: only a minority of CO2 molecules bind hemoglobin Instead, the majority of them diffuse into erythrocytes through their membrane: I. Inside erythrocytes, CO2 molecules are combined with water to produce carbonic acid (H2CO3) in a reaction catalyzed by the enzyme carbonic anhydrase. II. Then, carbonic acid (a weak acid) dissociates into 1 H+ and 1 HCO3- ion. This is the reaction: Then, the bicarbonate ion is exchanged with 1 Cl- ion across the membrane of red blood cells. This is an important mechanism because it regulates the HCO3- levels on plasma, serving as an important buffer system to maintain neutral PH.  Bicarbonate in the plasma acts as a buffer by neutralizing excess H+, thus helping to maintain the blood’s PH around 7.4. There are also other cells that express this transporter:  Renal tubular cells: renal tubular cells, such as proximal tubular cells. They allow the reabsorption of bicarbonate ions from the filtrate, which helps maintain acid-base balance in the blood.  In this way, bicarbonate is reabsorbed and not expelled with urine. This mechanism allows the cell to maintain the body's acid-base balance by regulating the concentration of bicarbonate in the blood. 25  Gastric Cells: gastric cells produce gastric secretions that are rich in HCl. These cells have a series of transporters: - they use primary active transporter to move protons (H+) into the gastric lumen (stomach) - and they also have this HCO3-/Cl- transporter that exchanges intracellular bicarbonate (HCO3-) for extracellular chloride (Cl-). These Cl- ions are then secreted into the gastric lumen, contributing to the high concentration of HCl in gastric juice. When the stomach produces a large amount of gastric secretion during a meal, bicarbonate is absorbed back into the blood, causing a temporary increase in blood pH after eating.  Pancreatic Cells: pancreatic cells produce secretions rich in bicarbonate thanks to this transporter and this is essential for digestion. The bicarbonate in pancreatic secretions neutralizes the acidic content that enters the intestine from the stomach, creating a suitable environment for digestive enzymes to function. TRANSPORTERS AND DRUGS In physiology, the study of transporters is very important because it can help us to understand some physiological mechanisms and why some diseases exist. In fact, we use and apply the information about transporters also in pharmacological therapies. 26 There are some drugs blocking membrane carriers: Omeprazole: it is a blocker of the H+ pump (proton-ATPase), and this is used in therapy to block excessive gastric acid secretion. Digitoxin: it is a blocker of the Na+/K+ pump, and it must be dosed carefully because it can be lethal. It is used to increase the strength of cardiac strength of contractions and treat heart failure. Digitoxin works by: I. reducing the activity of the Na+/K+ pump, which leads to an increase in intracellular sodium concentration. II. Since the Na+/Ca2+ exchanger relies on the low intracellular sodium concentration to function effectively, when the intracellular sodium levels increase, the Na+/Ca2+ exchanger becomes less effective, resulting in higher intracellular calcium concentration. III. The increased presence of intracellular calcium levels in cardiomyocytes enhances the strength of cardiac contractions. So: Digitoxin, by reducing the activity of the Na+/K+ pump, enhances the strength of cardiac contractions. 27 CELL PHYSIOLOGY LESSON II – 16.04.2024 (Ciranna) REGULATION OF CELL FUNCTIONS Maintenance of homeostasis: Homeostasis is the best condition for a cell to function. As we said, it is a dynamic equilibrium that needs to be maintained using ATP as a power source of energy. It’s dynamic because biological conditions in organisms change constantly. How are cell functions regulated?  Regulation of the Cell Cycle: depends on different substances that allow the regulation, such as growth factors, cyclins, and the transcription factor p53, etc.  Regulation of Cell Functions: is realized by molecules that are chemical messengers (such as neurotransmitters, hormones, and cytokines). They can regulate: - enzymes’ activities, - ion channels - gene expression (transcription, translation, etc.) These chemical messengers work by the activation of membrane receptors, which, in turn, trigger a cascade of intracellular signaling events leading to a final cellular effect. For example: there are the G Protein-Coupled Receptors (GPCRs) that are a large family of receptors. They’re metabotropic receptors17 because they are not ion channels, they do not have any pore but are linked to intracellular signaling mechanisms. They’re associated with G proteins, that are called “G” because they bind GTP (they are GTP-binding proteins), in fact, they’re able to hydrolyze GTP to obtain energy. G proteins18 can be classified according to their structure: trimeric structure with three subunits (α, β, γ) monomeric G proteins (called small). - The GPCR mechanism is activated when a ligand binds to the GPCR. This causes a conformational change in the receptor, which activates the associated G protein. - Specifically, the alpha subunit of the G protein exchanges GDP for GTP and detaches from the other 2 subunits. This activation triggers further signaling steps within the cell. 17 Remember: apart from the metabotropic receptors, there are also the ionotropic receptors  when the messenger binds this kind of receptor, the receptor opens fully and allows ions to flow through it. (we’ll see it better in the future lessons). 18 we already had a brief introduction about this concept in the first lesson 28 Same examples of small G proteins are: - elongation factor (EF, a factor involved in mRNA translation) - Rab3 (involved in membrane fusion process, as in the case of vescicles). N.B. At this point, we solved some QUESTIONS (slides 61-62-62, ppt 1):  Which of the following molecules cross the membrane by diffusion? And through which mechanism? Answer: a) a lipid molecule through simple diffusion; b) glucose through facilitated diffusion (GLUT); c) ion through their open ion channel. 29  For each of the following transport mechanisms, please indicate if the process is either active or passive: a) Fatty acid diffusion through the cell membrane  PASSIVE b) Na+/K+ ATPase  ACTIVE c) ion diffusion through voltage-dependent ion channels  PASSIVE (if the channel is open, the flow is passive) d) GLUT transporter  PASSIVE e) SGLUT (SGLT) transporter  ACTIVE  Indicate the effect (increase/decrease) on diffusion rate of a fatty acid through the cell membrane in the following cases: a) High membrane thickness  DECREASE b) Strong concentration gradient  INCREASE c) Low membrane surface area  DECREASE NEW TOPIC: ION CHANNELS AND MEMBRANE POTENTIAL ION CHANNELS Ion channels are integral membrane proteins formed by several subunits assembled to form a central pore. In the image below, it is possible to see a passive K+ channel and a passive Na+ channel. 30 Properties of ion channels: Selectivity: each channel is specific for one ion or for a couple of ions, due to the specific amino acid composition of the channel. Conduction: which is the ability of the channel to let ions flow through it. Some channels have high conductivity and can allow large ion currents, while others have a low conductivity. Gating: there are various gating mechanisms. It refers to the opening or closure of the ion channel in response to specific stimuli, which control the flow of ions across the cell membrane. In particular, we can distinguish between passive channels (that are always open) and active channels (that have a gating regulation). Types of Ion Channels: Passive Channels (Always Open): they have no gating regulation and are responsible for the resting membrane potential. Non-excitable cells only have passive channels. Active Channels (Gating Regulation): they have a gating mechanism and are responsible for active mechanisms, such as receiving or transmitting impulses. They are expressed only in excitable cells19 like neurons and muscle cells, as they allow changes in the resting potential. Different active channels exist with different gating mechanisms (we’ll see them later in the course, but she said that it is important to have a brief introduction now): o Ligand-gated ion channels: these ion channels are activated by neurotransmitters and are ionotropic receptors. Examples include some receptors for GABA and glutamate. 19 Excitable cells express both passive and active channels 31 o Ion channels gated by intracellular messengers: some channels are opened by intracellular messengers such as cAMP or by phosphorylation. Some messengers, for instance, increase cAMP levels, which help to open the channel. o Ion channels gated by physical stimuli: - Some channels can be activated by membrane stretching; this allows the ions flow through the, that will lead to depolarization (an excitatory signal). These channels are found in stretch receptors, present in the nerve endings that innervate different body districts (such as in muscles). - Other nerve receptors detect temperature; these receptors have channels in their membranes that open at specific temperatures, with different receptors for cold and heat. o Voltage-gated ion channels: these channels open and close in response to changes in membrane potential. Each channel type has a specific activation threshold, meaning there is a critical membrane potential value required to open the channel. For instance, voltage-gated Na+ channels have an activation threshold around -55 millivolts.  The first three families of ion channels —gated by ligands, intracellular messengers, and physical stimuli— are responsible for receiving input signals in neurons. These channels generate a membrane potential that initiates a response within the neuron. The neuron's response to these signals is an event known as action potential. This electrical signal travels along the axon until it reaches the axon terminals, where it synapses with other neurons. The generation and propagation of the action potential are dependent on the fourth family of ion channels: voltage-gated ion channels. This is what is said in the book, I added it for greater clarity: 32 ION FLUX THROUGH AN OPEN CHANNEL When a channel (passive or active) is open, the ion can diffuse, and diffusion is a passive process. This passage is driven by two gradients: - chemical (which refers to the concentration gradient) -electric (it depends on the charge) together, they form the electrochemical gradient. ORIGIN OF MEMBRANE POTENTIAL Book: Information is carried within neurons and from neurons to their target cells by electrical and chemical signals. Transient electrical signals are particularly important for carrying information rapidly and over long distances. These transient electrical signals are all produced by temporary changes in the electric current into and out of the cell; these are changes that drive the electrical potential across the cell membrane away from its resting value. Every cell in our body has a membrane potential: it refers to the separation of charges in the layers immediately adjacent to the cell membrane. (vedi prossimo paragrafo) 33 If we insert an electrode inside the membrane, we can measure this difference in potential. Here’s how to measure it: I. Insert an electrode into the cell. II. The electrode measures a negative membrane potential. The exact value isn’t absolute because it varies depending on the cell type; different types of cells have different resting membrane potentials. For example: - glial cells have a resting membrane potential of approximately -90 mV - and cardiac pacemaker cells have a resting potential of around -60 mV. SEPARATION OF CHARGES ACROSS THE MEMBRANE 34 As we can see from the image: - There is an accumulation of positive charges outside, in the layer very close to the membrane. (the red +) - There is an accumulation of negative charges inside, in the layer very close to the membrane. (the blue -) In fact, the membrane potential is a separation of charges in the layers immediately adjacent to the cell membrane Instead, in the rest of the cytoplasm and the extracellular compartment, there is an equal distribution of positive and negative charges, so they’re electrically neutral. This situation is linked to the presence of many PASSIVE ION CHANNELS on the cell membrane: - K+ channels: there are many of them. Cells have a large number of passive potassium channels, which means that they are very permeable to potassium ions. These channels are expressed primarily in non-excitable cells where the resting potential remains constant. - Na+ channels: they are very few. This means that Na+ ions can’t cross very easily the membrane in resting conditions. - Cl- channels: there are many of them. Hence, we can understand that, in resting conditions, the membrane is very permeable to potassium (K+) and chloride (Cl-) ions but not to sodium (Na+) ions. DIFFERENT ION DISTRIBUTION ACROSS THE CELL MEMBRANE 35 In squid neurons In mammalian neurons N.B. We already discussed these concentrations in the I lesson. 36 In particular, let’s talk about A-: these are intracellular proteins with negative charge that cannot freely diffuse outside the cell. So, they contribute to the negative charge in the intracellular compartment. As a consequence, the cell needs cations (=positive charges), such as potassium ions, to maintain the neutrality of the cytoplasm and thus to counterbalance the intracellular negative charge of A-  Since K+ ions are the only cations 20 that can freely cross (enter and exit) the cell membrane through their numerous passive channels, they are attracted inside the cell by the non-diffusible protein anions (A-). As result, we have a high intracellular concentration of K+ ions! SO: K+ ions are present at high concentration inside the cell because they’re attracted by the A-. Normally, due to their concentration gradient, potassium ions would tend to move out of the cell, because they are already more concentrated inside. However, due to the electrical gradient, they are simultaneously attracted back into the cell by the negative charges inside.  So, there’s a dynamic balance between the chemical force (concentration gradient pushing K+ ions out) and the electrical force (electrostatic attraction pulling them in). The overall result of this dynamic balance is the creation of the resting membrane potential, which represents the membrane potential of a cell at rest. 20 Question: why couldn't other ions enter to maintain neutrality? - Cl- ions: they could move freely but are less attracted, since they are negatively charged: that’s why they remain mainly outside the cell. - Na+ ions: since they have fewer passive channels, they predominantly stay outside the cell, resulting in a higher extracellular concentration. 37 CELL PHYSIOLOGY LESSON III – 17.04.2024 (Ciranna) EQUILIBRIUM POTENTIAL The equilibrium potential (E) is a value that can be determined for each ion, if a channel is open for that ion. For instance, K+ ions have their own equilibrium potential, as do Na+ ions and Cl- ions. When these channels are open, an equilibrium can be established 21. The equilibrium potential for an ion is the membrane potential value at which the flow of that ion is balanced: there is no net movement of the ion across the membrane. It means that the concentration gradient and the electrical gradient for that ion are in equilibrium.  For potassium ions, as we said in the II lesson, these two forces 22 move in opposite directions, eventually reaching an equilibrium point where the net flux of potassium ions will be zero, meaning that is balanced. Specifically, the value of EK (equilibrium potential for the K+ ion) is -90mV  This means that when the membrane potential is at -90 mV, there is equilibrium for potassium ions, resulting in no net flux. In this state, the inward current is equal to the outward current because the two driving forces are equal. How do we obtain this value? We can predict this value with the Nernst equation. NERNST EQUATION The equilibrium potential is influenced by factors such as gas constant, absolute temperature, the quantity of charge carried by each ion, and most importantly, the concentration gradient inside and outside the cell. They are all summarized in the Nernst equation. So, the Nerst equation takes into account different factors that influence this value, one of the most determinant is the concentration gradient (intracellular and extracellular). 21 Each ion has its own unique equilibrium potential value (a specific value). 22 Chemical and electrical gradients 38 Slide: This equation defines the relation between the concentrations of an ion on either side of a membrane that is permeable to that ion and the potential difference (voltage) that will be measured across that membrane under equilibrium conditions.  The Nernst equation can be used to find the equilibrium potential of any ion that is present on both sides of a membrane permeable to that ion and each ion has its own equilibrium potential. EK = the equilibrium potential for K+ ions As we said, the Ek for K+ is: -90 mV. At this value, the net flux of K+ ions is zero, because the inward and outward currents are equal. However, if this value is different from -90mV, there will not be a state of equilibrium because one of the two driving forces will be stronger. The equilibrium potential is also known as the inversion potential 23 because the current changes direction when the membrane potential deviates from -90mV (in the case of K+). This is because 23 The equilibrium potential is also called an inversion potential, because if we go above or below of the zero value, we will have a current but in the opposite direction It means that, if the membrane potential deviates from this equilibrium, a current will flow, but in the opposite direction. 39 currents flow in opposite directions when the membrane potential exceeds or falls below -90mV. In the case of K+ ions:  IF THE MEMBRANE POTENTIAL GOES MORE POSITIVE THAN -9O mV: When the membrane potential is more positive than -90 millivolts (for example -70 mV) potassium ions will flow out of the cell, resulting in an outward current.  IF THE MEMBRANE POTENTIAL GOES MORE NEGATIVE THAN -9O mV: If the membrane potential is more negative (for example -100 mV), potassium ions will flow into the cell, causing an inward current. Moreover, remember that: if the cell is permeable to only one ion, then the membrane potential equals the Nernst potential for that ion when channels are open. Changes in ion concentration lead to corresponding changes in membrane potential.  For example, glial cells possess only passive K+ channels and lack Na+ channels. The membrane potential of glial cells stabilizes at approximately -90 mV, which represents the equilibrium point where K+ ions move out and in at equal rates due to the passive K+ channels. SO: Since glial cells only have passive K+ ion channel their membrane potential is equal to the equilibrium potential of K+. Instead, excitable cells (unlike non-excitable ones) receive various inputs: as result, their membrane potential can change. The change in the membrane potential causes ions to no longer be at equilibrium, leading to a flux of ions either moving inward or outward. To sum up: in the case of excitable cells there is a dynamic equilibrium, because they receive inputs that change their membrane potential and result in fluxes of ions. GLIAL CELLS VS NEURONS 40 GLIAL CELLS: if the cell only possesses passive potassium channels, then the membrane potential corresponds exactly with the Ek for K+ (this happens in glial cells in which their membrane resting potential is about -90mV, that corresponds exactly to Ek). NEURONS: neurons have many passive K+ channels but also a few Na+ passive channels: thus, their resting membrane potential is influenced by both potassium and sodium currents, leading to a more complex equilibrium state. In fact, given the presence of both K+ and for Na+ passive channels, we have to consider the equilibrium potential for both K+ and Na+ ions (not only for K+ as in glial cells). We already know the equilibrium potential for K+, let’s calculate the one of Na+ using the Nernst equation. Equilibrium potential for Na+ ions: By substituting the values for extracellular sodium concentration (about 150 millimolar) and intracellular sodium concentration (below 15 millimolar), we can calculate this equilibrium potential for Na+ ions to be about +55 mV (so, Na+ ions have a positive equilibrium potential). This implies 41 that at +55 mV, there would be no net flux of sodium ions as inward and outward sodium currents would balance each other. However, reaching this equilibrium for sodium is extremely rare, since membrane potential becomes positive at the peak of action potential, which is about +30 mV. Now, neurons express both K+ and Na+ passive channels, with K+ channels being more abundant. As a result, the neuronal resting membrane potential is more strongly influenced by the equilibrium potential of K+ compared to Na+. Let’s see it: NEURONAL MEMBRANE RESTING POTENTIAL Neurons have a resting membrane potential of approximately -70 mV when they are not actively firing or receiving any input. The resting membrane potential of -70 mV in neurons is maintained due to the combined effects of these equilibrium potentials. In fact, we can notice that the neuronal membrane resting potential is different from both the equilibrium potentials of potassium and sodium, that are respectively: - 90mV and +55mV. This indicates that in the resting state of the neuron these ions are not at equilibrium! 24  In fact, at resting potential, an outward K+ current and an inward Na+ current continuously flow through passive channels. Why do we have an inward Na+ current? At first, we have the Ek= -90mV. At this value, also a small number of sodium ions are driven into the cell pushed by two forces: - a chemical driving force (due to the higher extracellular sodium concentration) - an electrical driving force (due to the negative membrane interior that attracts the positive charges of the Na+ ions). These forces work together to drive a small number of sodium ions into the cell, causing the inner side of the membrane to become less negative, typically around -70 mV in neurons.  The influx of Na+ depolarizes the cell, but only slightly from the K+ equilibrium potential, - 70mV. Na+/K+ ATPase: There is a mechanism that opposes the continuous flow of sodium and potassium, and this is a primary active transport: the Na+/K+ ATPase that recovers gradient concentrations (it pumps Na+ outside and K+ inside). The recovery of gradient concentrations for the two ions is very important because otherwise we couldn’t have any membrane potential and therefore any propagation of nerve impulses in cell membranes. Additionally, there are a lot of secondary transport mechanisms that are dependent on the presence of a concentration gradient for Na+ to function effectively. That’s why the Na+/K+ ATPase is very important for cells. 24 (This difference in potentials ensures that neurons are ready to transmit signals when needed). 42 SUMMARY: RESTING MEMBRANE POTENTIAL  DIFFERENT MEMBRANE PERMEABILITY TO DIFFERENT IONS (PASSIVE CHANNELS): Cells have a resting membrane potential due to the presence of various ion channels, BUT NOT for any ion. These include: I. numerous potassium passive channels, II. a limited number of sodium channels, III. numerous chloride channels. Neurons express passive channels for K+ ions (many), Cl- ions (many) and for Na+ ions (very few):  DIFFERENT ION DISTRIBUTION: This creates a very different distribution and separation of ions across the membrane, with: I. high concentrations of potassium inside the cell, II. sodium outside the cell, III. and chloride outside the cell (it doesn’t enter due to its negative charge)  THIS CREATES A SEPARATION OF CHARGES ACROSS THE MEMBRANE This cause the presence of a membrane potential.  ACTIVE MECHANISM OF THE Na+/K+ ATPase: At the resting membrane potential, there is a constant flow of ions through the open channels, but fortunately this is counteracted by the active Na+/K+ ATPase, which helps to restore and maintain the ion gradient. N.B. Remember that each cell has its own resting potential value. THE CELL MEMBRANE CAN BE COMPARED TO AN ELECTRIC CIRCUIT 43 The cell membrane is frequently compared to an equivalent electrical circuit, because there is a comparison between ion currents and electric currents. In fact, they follow the same general rules of physics, for example about: resistance, capacitance, etc. However, of course, they’re different: electric currents are carried by electrons; ion currents are carried by ions, that can be either positive or negative. And, in this context, channels are often represented like current generators as the presence of the channel generates membrane potential. Some properties 25 of channels are: Resistance: when the channel is closed, this makes a resistance to the current flow. Conductance: it’s the ability of a channel to conduct current, i.e. the ability to let the current flow. It’s the inverse of electrical resistance. Permeability: depends on the presence as well as on the number of open selective channels. If there are numerous open channels for a particular ion, the cell will have high permeability to that ion. Conversely, if there are only a few channels, the cell will have a low permeability for that ion. For example, the membrane has high permeability to potassium ions at rest, while a low permeability for sodium ions. So, this is a picture showing three potassium channels which are present on the membrane, and they all participate the resting membrane potential: 25 We’ll introduce them now, just to understand the concept of permeability which is important for the Goldman equation. But we’ll delve into them later in the lesson. 44 HOW DOES EACH PASSIVE CHANNEL CONTRIBUTE TO RESTING MEMBRANE POTENTIAL? This is calculated with the Goldman equation: This equation states that: each passive channel type contributes to resting membrane potential; the amount of contribution is directly proportional to channel abundance (=it depends on the number of ion channels). In fact, permeability (indicated by the letter P) depends on the presence as well as on the number of open selective channels. - Permeability is very high for potassium (it contributes more). -Sodium permeability is very low. -Chloride ions also contribute but they are highly distributed outside, since they can’t enter inside the cell due to the presence of negative proteins inside. N.B. however, Cl- concentration can change if the cells express a chloride transporter. For example, remember that in the previous lesson we talked about the NKCC and KCC transporters. At this point of the lesson, we solved some QUESTIONS: 1. Can you tell the difference between passive and active ion channels?  Passive channels are always open, while active channels have a gating mechanism. 2. Indicate the physical mechanism by which ion flow through a passive channel and through an active channel.  Through diffusion in both cases, since the ion flow occurs only when channels are open. Remember that the difference it that passive channels are always open, while active channels are not. 3. Which driving forces influence the amplitude and the direction of ion currents?  The chemical force due to the concentration gradient and the electric force. 4. Does resting membrane potential depend on either passive or active ion channels?  Only on passive channels. 5. Is resting membrane potential value the same (-70 mV) in all cell types?  No, it depends on the cell type. 6. Do non-excitable cells possess a resting membrane potential?  Every cell has a resting membrane potential because every cell has passive channels. 7. Consider these standard neural conditions: 45 - Intracellular K+ concentration 150mM and extracellular K+ concentration 5,5mM. - Ek (K+ equilibrium potential): -90 mV. - Resting membrane: -70mV. 7.1 If extracellular K+ concentration is temporarily raised, will Ek change?  Yes, because (according also to the Nerst equation) it will change. In this case, it will become more positive. 7.2 If extracellular K+ concentration is temporarily raised, will resting membrane potential change?  YES. In fact, if K+ ions concentration is raised outside the cell, they will tend to enter the cell and they will bring positive charges inside. As result, the membrane will depolarize because it will become less negative. Remember this concept because it’s very important in medicine: if K+ concentration is raised outside in the blood or in any extracellular fluid, this will change the membrane potential and this will have consequences on excitable cells.  Potassium disequilibrium in the blood will cause involuntary contractions. And so, our body has physiological mechanisms that keep potassium concentration in the blood constant at the right level. 26 She made an example to better understand this concept (of the 7th question): - What happens if membrane potential is depolarized to -60 mV? In which direction will potassium move? Since the equilibrium potential is -90mV, at more positive values, potassium ions will go outside and there will be an outward current. - And what happens if membrane potential is hyperpolarized to -100mV? They will go inside. And this also happens in some physiological conditions when a neuron is strongly hyperpolarized, and the membrane potential goes very low. The neuron has some “protection” mechanisms to go back to the resting membrane potential. And one of these protection mechanisms is the presence of some particular potassium channels. CELL MEMBRANE PROPERTIES Membrane passive properties influence the ability of a neuron to “sum up” all the different stimuli that come from different inputs. In other words, these passive properties influence the ability of a neuron to make summation of many inputs. These passive properties are: Capacitance: 26 (Moreover, she’s an electrophysiologist and during electrophysiology measurements of currents and nervous transmission they use a very classical protocol to depolarize a neuron: they apply a solution with a high potassium concentration. As result, the neuron will depolarize, and it will discharge the action potential). 46 It’s the ability to keep charges separate. It’s similar to capacitance in electrical circuits: capacitance is a property of systems with two conducting solutions separated by an insulator to form a capacitor (so, charges are kept separated on the two sides). The cell membrane, similarly, consists of lipid layer acting as an insulator separating the extracellular and intracellular ionic solutions, which behave like conducting solutions. This configuration allows for the separation of charges on either side of the membrane. In some regions of the membrane we have ion channels, but in other regions no: in these regions where ion channels are absent, charges are kept separated resembling the behavior of a capacitor. The membrane's capacitance property causes a delay in the change of membrane potential in response to an electrical impulse. Why do we have this delay?  While in regions with open ion channels: the flow of ionic current through these channels results in a rapid change in membrane potential, in the areas without channels, the capacitance of the membrane slows down the response to an applied stimulus, leading to a delayed change in the overall membrane potential. Membrane capacitance changes among different neurons: bigger neurons, since they have a larger membrane, have a higher membrane capacitance with respect to smaller neurons. When bigger neurons receive an input current they will change the membrane potential with a larger delay:  This big delay has an important consequence: this high membrane capacitance will slow down any change in membrane potential and favor summation of stimuli arriving at different times: it’s the so-called temporal summation. In fact, the membrane of a larger neuron remains depolarized for a longer duration, which facilitates the integration of inputs at various time points. 47 N.B. capacitance influences nervous transmission by impacting the speed at which membrane potential changes in response to current input. The time constant, measured in milliseconds, represents the time required for the membrane potential to reach its final value after receiving a current pulse. In addition to capacitance, other passive properties include resistance and axial resistance, which further play a role in shaping neuronal function. Resistance: it’s the resistance of the membrane to a current. If there are many open channels the resistance is low, if there are a few open channels the membrane has a higher resistance level. Axial resistance in axons and dendrites: it’s another type of resistance. We should already know the structure of a neuron: it has a cell body, a certain number of dendrites (that receive the inputs from the synapses with other neurons) and an axon, which is a relatively long projection along which the signal can travel. 48  As the signal travels along the axon, it encounters axial resistance, which is resistance in the longitudinal direction. In fact, initially (at the very beginning), the stimulus spreads passively through passive channels 27. However, the current decreases in amplitude as it moves away from the point of stimulation due to axial resistance. The distance that the current can propagate before being completely abolished depends on the axial resistance, which is influenced by the diameter of the axon. Specifically, the axial resistance is inversely proportional to axonal diameter. A larger axon will have less axial resistance and allow the current to travel a longer distance before being abolished. A smaller axon will have a higher resistance and thus the stimulus will travel for a shorter distance. This variation in axial resistance affects the summation of inputs, known as spatial/ space summation. Space summation refers to the summation of stimuli arriving at different spatial points at the same time. SLIDE: a low axial resistance allows passive current spreading to long distances, thus will favor summation of stimuli arriving at different space points: spatial summation. 27 passive spreading 49 RECAP OF CAPACITANCE AND AXIAL RESISTANCE AND OF TEMPORAL AND SPATIAL SUMMATION Nerve transmission is strongly influenced by membrane passive properties:  Capacitance: a high membrane capacitance will slow down changes in membrane potential, thereby favoring the summation of stimuli arriving at different time points: temporal summation.  Axial resistance: a low axial resistance allows passive current spreading to long distances, thus will favor summation of stimuli arriving at different space points: spatial summation. In other words: axial resistance determines how long a current can travel before being extinguished: if the current travels for a longer distance, the different stimuli arriving at different space points will be summed. This is important if we consider a neuron with a complex dendritic tree that receives multiple synaptic contacts: in this case, the integration of signals from up to 10,000 synapses is crucial! The ability to integrate these signals is influenced by passive membrane properties. And, generally speaking, larger neurons have a greater capacity for signal integration. ACTIVE CHANNELS Up to now, we’ve talked about passive channels. Passive channels are present in all the cell types 28! However, in the case of an excitable cell we don’t only have passive channels, but also ACTIVE CHANNELS. Active channels are located in the receptive areas of neurons: dendrites and soma. Why are they called “active”? they’re called “active” because they have a gating mechanism: they can be open or closed. 28 And on them it depends the resting membrane potential of a cell 50 The presence of active channels makes cells excitatory because they are able to receive messages from other neurons and to generate signals. The presence of active channels generates changes in membrane potential, which are graded based on the intensity of the stimulus: A high stimulus results in a large amplitude potential A low stimulus produces a smaller potential FOUR CLASSES OF ACTIVE CHANNELS As we said during the II lesson, there are four classes of active channels. 1) Ligand-gated active channel (the ligand is a neurotransmitter): when the neurotransmitter binds the binding site on a receptor, the receptor opens. - Ligand-gated channels are also called ionotropic receptors for neurotransmitters and they are located in synapses 29 (specifically, on the post synaptic membrane). - When ligand-gated receptors are open, they generate a synaptic potential, called post- synaptic potential, which can be either excitatory or inhibitory, depending on the neurotransmitter and receptor type involved. Moreover, this post-synaptic potential is a graded potential. It means that the amplitude of these potentials is dependent on the number of channels that are opened, and on the amount of neurotransmitter released. 29 N.B. a synapse refers to a contact (not physical) between two neurons where communication occurs via the release of neurotransmitters. In a chemical synapse, a neurotransmitter is released by the presynaptic terminal of one neuron and binds to these ligand-gated receptors on the postsynaptic neuron's dendrites and cell body. 51 A higher number of open channels and increased neurotransmitter release result in a larger amplitude of the postsynaptic potential Fewer activated receptors lead to a smaller potential. 2) Activated by phosphorylation: are activated by intracellular messengers. These channels can be activated through phosphorylation or other intracellular mechanisms. These mechanisms are triggered by membrane receptors, which then initiate a series of intracellular events that modulate the channel. These channels are also located in the receptive areas of neurons. 3) Activated by physical stimuli: are activated by physical stimuli, such as stretch or pressure. An example is represented by the tactile receptors in the skin. These channels are connected to the membrane through a protein ridge, and opening occurs when the membrane is stretched. As we said, these types of channels are specific to neurons that function as stretch receptors; they are in: SKIN (pressure receptors) MUSCLES: stretch receptors in muscles play a vital role in regulating reflexes that trigger muscle contractions in response to stretch. INNER EAR: we have acoustic and vestibular receptors. In particular, we have ciliated cells that detect stimuli through the bending of cilia. This causes the opening of these receptors and causes the cell to depolarize. When these receptors are activated, they generate a potential called receptor potential, (indeed, the change in membrane potential is due to the influx of current through the open channels). Also this receptor potential is graded, meaning that the strength of the stimulus determines the size of the potential: - a stronger stimulus results in a larger potential - a weaker stimulus produces a smaller potential [these three class of receptors are all located in receptive areas!!!!] 4) Voltage-gated: they mediate the output of neuron, which is the response of a neuron. They open as result of a change in the membrane potential, but each case is different according to the specific voltage-gated channel, because they’re all different. Let’s analyze the following image, which represents a very general situation: 52 These channels remain closed at the resting membrane potential, where the outside of the membrane is positively charged (+), and the inside is negatively charged (-). However, when the membrane potential changes, these channels open based on their specific threshold potential. The threshold potential is specific for each ion and it corresponds to the membrane potential value at which the channel will open. The opening of the channel allows the flow of ions. It is important to note that these channels can also become inactivated, meaning they are no longer permeable to ions. It’s different from the closing mechanism, because a different portion of the channel is involved in the inactivation (as you can see from the image). After being inactivated, the channel cannot be open even if depolarization occurs! This inactivation serves as a protective mechanism to prevent continuous flow of ion currents. For example: voltage-gated sodium channels open rapidly but also inactivate quickly, within 1-2 milliseconds. In fact, each channel type has its own unique timing for opening and inactivation, some are very fast and some very slow. ACTION POTENTIAL (SPIKE) This concept is quite hard to understand at first: you can follow this all mechanism by following the red line in the image. You just have to imagine it step by step. 53 Voltage-gated channels are responsible for the output signal from a neuron. It is the spike in the membrane potential, also called action potential. Action potential depends on VOLTAGE-GATED Na+ CHANNELS and VOLTAGE-GATED K+ CHANNELS. Let’s analyze the image above step by step, since it illustrates the mechanism of action potential:  There is the red line that represents the membrane potential of a neuron.  At the beginning, at value of about -70 mV there is the resting membrane potential.  Then, if we send a stimulus (such as injecting artificially a current with an electrode) to this membrane, the membrane potential will depolarize (= it will become less negative). We can apply stimuli of different amplitude:  where there is the number 1 in the image: it’s a current of depolarization of small amplitude; so, the membrane will depolarize just a little bit, but nothing happens.  where there is the number 2 in the image: we increase the stimulation current; the membrane depolarizes a little bit more, but also in this case nothing happens because we haven’t reached the activation threshold.  where there is the number 3 in the image: we apply a stronger stimulation and reach the activation threshold:  The threshold is the threshold for the opening of the voltage-gated sodium channels [these channels are open when the membrane potential reaches -55mV (around -50 mV)]  When the membrane potential reaches this threshold, voltage-gated Na+ channels open. When many Na+ voltage-gated channels open, sodium ions will flow into the cell (according to the chemical and electric gradients), bringing inside many + charges.  This inward flux of + charges will cause the depolarization of the membrane potential. The membrane will strongly depolarize: in fact, the membrane potential spikes up to around +30mV during this phase. SO: the positive peak of active potential is due to open the voltage-gated Na+ 30 ions channel. 30 don’t make confusion between voltage-gated and passive Na+ channels 54  After this peak is reached, it will stop, because the sodium channels will become inactivated, leading to a refractory period 31 where the membrane is unable to respond to further stimuli.  This means that the potential will gradually go back to its typical negative value: in other words, the membrane will start to repolarize. This happens thanks to the voltage-gated K+ channels: Following the inactivation of the sodium channels, voltage-gated K+ channels open. N.B. don’t make confusion between voltage-gated and passive K+ channels!!! Voltage-gated potassium channels are distinct from passive potassium channels, as open in response to reaching a specific membrane potential threshold. The threshold for voltage-gated potassium channels is typically around -20 millivolts. Thus: when the membrane potential reaches the threshold of -20mV, voltage-gated K+ channels open and allow K+ ions to flow out of the cell, resulting in the repolarization of the membrane. (In fact, given the positive membrane potential at this point, both the electrical and chemical gradients drive potassium out of the cell!).  As a result of this outward flow of potassium ions, the membrane potential becomes more negative, leading to membrane repolarization. However, it repolarizes even more: it becomes hyperpolarized, meaning that it reaches a value even more negative with respect to the typical resting membrane potential value of about -70 mV. In fact, it reaches a value of about -90 mV.  Then, once voltage-gated K+ channels have been open for a certain period, they will also undergo inactivation, similar to sodium channels but with a different rate of inactivation.  At the end, following the inactivation of voltage-gated potassium channels, the membrane potential returns to its resting state of -70 mV. SO: this return to rest is attributed to the inactivation of these channels, which leads to a decrease in the membrane's permeability to potassium ions. The following diagrams show this mechanism: 31 Vedi pagina 65 55 the last image shows the kinetics of the two types of channels: VOLTAGE-GATED Na+ channels: have a very fast opening and a fast inactivation VOLTAGE-GATED K+ channels: have a very fast opening and a slow inactivation And remember that voltage-gated Na+ channels are involved in the DEPOLARIZATION of membrane potential, while voltage-gated K+ channels are involved in the REPOLARIZATION of membrane potential. N.B. in the following lessons, we’re seeing different types of voltage-gated channels. TO RECAP: 56 CHANNELOPATHIES There are mutations of genes coding for ion channels that can give severe pathologies. A mutation can either modify ion current amplitude or modify channel inactivation rate. A reduced inactivation rate will increase ion current duration: it means that the channel will remain open for a longer time and this can cause some problems. The consequences: channelopathies can cause serious alterations in:  Neuronal excitability (like in epilepsy, in which some neurons are more excitable)  Muscle excitability (can cause cardiac or back problems) It’s important to know these mechanisms related to channels because, when they are disrupted, many health problems can arise. And it’s also important because many therapies used in the medical field are based on these mechanisms related to channels. 57 CELL PHYSIOLOGY LESSON IV -18.04.2024 (Ciranna) VOLTAGE-GATED ION CHANNELS We will see each of them in detail and notice that they are very different. Nowadays, we have discovered many of them because of the new techniques that allow us to find out their exact structure. For each ion channel, we now know their primary, secondary, tertiary and quaternary structure: so, we know how the proteins are assembled in space and which subunits form the channel, also considering how they are assembled in the membrane. Main features of different ion channels: - Structure: they are formed by several subunits and, depending on which subunits are expressed in a tissue, the channel in that specific tissue will have different features. - Ion selectivity: each channel is selective for one ion. This is true for voltage-gated ion channels, (in the next lecture) we will see that, instead, ligand-gated channels are selective for two or three ions.  For example, glutamate and NMDA receptors are ligand-gated channels that are permeable for Na+, K+ and Ca2+, and are still selective because other ions can’t pass through them. For now, talking about voltage-gated ion channels, we know that one channel has a selectivity for one ion only (so we talk about voltage-gated Na+ channels, K+ channels, Ca2+ channels and Cl- channels). This ion selectivity depends on the specific structure of the channel: there is a portion of the protein that is recruited for this function. - Single-channel conductance: every channel has its conductive properties. o If a channel has a large conductance, the current passing through it is large in amplitude o If a channel has a small conductance, it collects a very small current. When we talk about a single channel current, the range of amplitude is between 10 and 30 pA (picoampere). These features were discovered thanks to an important electrophysiology

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