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eDay 1 5/11/ Cell biology is based on cell theory ● All living creatures are made of one or more cells ● The cell is the basic structural unit of living things ● On today's earth, cells can only arise by division from preexisting cells ○ At one point living cells did arise from nonliving material ○ Transition from nonliving organic matter to cells, we don't know how many times ○ All the cells are related to a single ancestor There are two basic types of cells on earth ● Prokaryotes: ○ Do not have a nucleus or internal membranes ○ More of them and more successful than eukaryotes, larger mass on earth ● Eukaryotes: ○ Have a nucleus and an extensive external membrane system ○ Three types: protists, animals and plants Basic structure of a eukaryotic cell (animals or protist) ● Plasma membrane ○ Surrounds the cell ○ lipid-bilayer with proteins in it ● Nucleus- ○ DNA is stored, tightly coiled ○ Nucleolus- no membrane separating it, but this is where ribosomal subunits are assembled ○ Separated from the rest of the cell by a double lipid-bilayer, joined by nuclear pore complexes ● ER ○ Where the cell makes membrane proteins- RER ○ From there they are sent to the other membrane bound organelles ○ The space between the nuclear envelope is continuous with the lumen of the ER ● Cytoplasm ○ Solution inside the cell ○ Solution inside the nucleus is called nucleoplasm ● Mitochondria ○ Powerhouse of the cell ○ Double membrane around mitochondria ○ Makes ATP from sugars, fatty acids, amino acids ○ Signaling in some cells, apoptosis (cell death) ● Vesicles ● Lysosome ○ Degradative structure ○ The inside is called the lumen
● Lumen ○ The fluid that surrounds any membrane bound organelle ● Golgi ○ Membrane bound structure that receives vesicles from ER and processes them and sorts it to final destination ● Microtubule cytoskeleton ○ Provides tracks along which vesicles can move and participate in cell division ○ Help cells move and change shape Plants and algae- something extra ● Animal cells don't have chloroplasts ○ Harvest light energy and generate ATP ○ Marine organisms can ingest algae and steal them ○ Have 3 membranes that surround the thylakoids- individual light harvesting organs ■ Need to get proteins from ER across these membranes because mitochondria and chloroplasts get their proteins from cytoplasm ● They have rigid walls ○ Surrounding the plasma membrane is a cell wall made of cellulose ○ Most extracellular matrix ● They have vacuoles ○ Store substances for metabolic purposes There was a single common ancestor cell that gave rise to all organisms today ● All life on earth survives on DNA, RNA and proteins ● All organism use the same core basis and the genetic code is similar from one organism to another ● Take the DNA sequences of currently existing organisms and work backwards ● Current best guess: ○ There was a group of organic molecules on the earth that was created abiotically (non-living processes)- chemical reactions ○ RNA polynucleotides started replicating themselves ■ Encodes info that can be replicated and can form complex 3-d structures and can carry out catalytic activities (like proteins) ○ RNA is fragile and doest surivie under extreme conditions, the information storage function was handed off the DNA, which is more stable ■ Proteins stayed with RNA ○ RNA is intermediate between DNA and proteins ○ At some point lipid bilayer emerged to encapsulate contents ■ Capable or real evolution Presumed evolution of cells ● Believe that prokaryotes evolved first because they are simpler ○ Simpler is not bad ● Eukaryotes evolved from prokaryotes vis symbiosis ○ An ancestral prokaryote somehow ended up with a second prokaryote inside of it (mitochondria of today's cells)
● Light of different colors passes through a filter that only lets one color through, it bounces off a mirror through a lense and fluorescent molecules get excited and give off light at a longer wavelength than the light that excited them and the emitted light is collected in energy ● Not every molecule is fluorescent, when it is it's called a fluorophore ○ Some that will only be excited by blue light, when it shines on the photon, electrons energy level increases, after it is excited, then loses energy through vibration, give off a photon and collapses back to its original state ■ Photon is emitted in a lower energy, more energy emitted than absorbed ■ A blue photon can give if a lower energy green photon or an even lower energy red photon ■ The emitted light is alway a longer wavelength than emitted light ■ All takes place in the span of like a nanosecond ● Multi-colored fluorescent image ○ Labeled with 5 different types of fluorescent molecules each that highlight a different cellular component ○ Blue: DNA binding dye ○ Purple: antibody ○ Green- fluorescent protein ○ Red- fluorescent protein ○ Yellow antibody Immunocytochemistry uses antibodies to visualize proteins in cells ● Antibodies are proteins that are made by the immune system ● Their job is to bind to things that don't belong in the body ○ Typically bind to proteins, but not whole proteins, epitopes : regions of amino acids about 8-12 amino acids long ● Antibodies bind to things in the bloodstream and trigger the uptake by macrophages ● Each B-cell recognizes its own particular epitope ● When you inject the protein into the rabbit you're going to interact with B cells floating in that rabbit bloodstream, anything that recognizes the protein will proliferate and release antibodies ● Attached fluorophores to the antibodies, antibodies will always bind to a certain protein and now its labeled ● You can label the antibody with a fluorophore and it will glow wherever the protein is and mark the protein ● Can use antibodies against antibodies ○ Inject rabbit antibodies into a goat, goat doesn't recognize them and it makes antibodies against rabbit antibodies, then you can collect those and label them with fluorescent dyes ● Problem: ○ If you want to label proteins inside a cell with an antibody you have to get that antibody through the plasma membrane, they can cross the membrane of living cells ○ Want to do this is find a way to poke holes in the cell membrane to let the antibody in ■ Detergents can poke holes in membranes, and some are big enough to let antibodies in and can bind to the proteins they recognize, but the whole can cause thing to leak out
■ Fixatives like formaldehyde- keeps proteins in place, chemically cross-linked to neighbors need to add this first, then use detergent so nothing moves ● Once you do this you kill the cell, will stay the way you fixed it forever ○ Can study living cells by targeting extracellular proteins Antibodies are all around useful molecules ● Immunoblotting (western-blotting) ○ Load samples at the top of a well and add an electric field and proteins migrate based on molecular weight separating themselves ○ Take the polyacrylamide gel and use a stain you would see a ladder, 100s of proteins spread out based on weight, can tell ■ Take the gel and create a sandwich, the gel is placed over a membrane, instead the proteins migrate out of the gel on to the filter paper and cant pass through the membrane they accumulate on the surface, incubate in solution of antibodies and they stick according to the correct protein and then label it fluorescently ■ Antibody lets you figure out what band on the gell corresponds to what protein ● immunoisolation/ immunoprecipitation ○ uses antibodies to pull proteins out of solution ○ All made possible by antibodies and their role in the immune system Fluorescent proteins ● Find a naturally fluorescent protein and clone its DNA sequences they can fuse the two DNA sequences together add GFP, and that would result in tubulin molecules that had the fluorescent protein attached and it would incorporate itself like normal tubulin in microtubules ○ Allows live imaging ● Fluorescent protein comes from a jellyfish A digression: Transfection of cells is a useful cell biology tool ● Transfection: infect a cell with foregin DNA to cause a foregin protein to be expressed in a cell ○ Introduce proteins into cells, infecting the cell with foreing DNA ○ Lets you express proteins in living cells- GFP ○ Or express silencing ● Add molecules that aren't normally present in a cell to see what they do ○ Ex: HeLa cell, cancerous cervical epithelial cells ○ Put a heart cell protein with cervical cell, how many different proteins to put in turn a cervical cancer cell into a heart cell ● Constituently active proteins: ○ Mutant protein that are active all the time, regardless ● Dominant negative proteins ○ Suppress the function of the cells normal proteins ○ Mutant proteins that are inactive and interfere with the cells normal proteins, dominant, inactivate normal cells ○ Similar to knocking a protein out ● Cells can be transfected transiently or stably (RNAi) ○ Transiently: introduce CDNA, study the cells and then they die ○ Stable clones that express the protein of interest forever
“Superresolution” fluorescence techniques that break the resolution of light microscopy ● PALM ● STROM ○ Special kinds of fluorophores that respond only occasionally, fluorophores will blink on and then off, and if you watch this you can calculate what the image is ○ Each of the spots when they are on look like a 200nm sphere, but you watch them turn on 1 by 1 and generate a sub resolution image ○ Reveals the true diameter of the structure ○ Work best for fixed cells that are not moving ○ Ability to resolve ribosomes Electron microscopy ● Smaller wavelengths than light, beam of electrons (wavelength is 0.004nm as opposed to 400-500nm for light) ● The actually resolution is 0.1-2nm, optics are much worse ● CANNOT do either tequinies on living samples! ● TEM ○ Look like a thin section ○ Analogous to light microscopy ○ Have an electron beam instead of light source, magnetic condenser lens, objective lenses that focus the beams and project it onto a camera ○ You have to work in a vacuum because then the electron beam won't work, which means the sample has to be dehydrated does not interact very well with electron beams) allow heavy metals to bind to it in the sample, impregnate the sample with a contract agent that will scatter electrons in the right way, but then the material is too dense to image ○ You imbed your sample in plastic or something else and put them on a slicer and and cut them into very thin section that you put in a microscope and see scattering of electrons by metal ○ How do you tell when everything is: ■ Add antibodies against those proteins to the sample and would bind to specific proteins, attach gold beads to the antibody that are very electron dense ■ Antibodies bind almost irreversible, can only wash unbound antibodies away ● SEM ○ Look like you're looking at the surface of the sample ○ Take a sample and coat it with a heavy metal, create a mask over the top of the sample ○ Shine electron beam on it and the metal will scatter the electrons and you can generate an image based on that, look 3D ○ Resolution is limited to 5/10 nm Gradient diffusion ● Cannot be applied to live cells ● Different cellular organelles have different densities ● If you grind up cells of lyse them, release all their insides, if you take a lysed cell and spin it at a certain speed, the nuclei will purify out (heaviest)
● If you want to study something not as dense then need to pour off the nuclei, then spin it again and the next heaviest will move down ● Isolate fraction based on sequential centrifugation steps ● Turn into an image using animation ● Can create different zones of density of sucrose and cpin it and they organelles go into their zone of density, see bands in the test tube corresponding to the different organelles ○ Can use an antibody to double check, against western blotting or on a TEM Membranes and Proteins Eukaryotic cells have multiple intracellular components separated by internal membranes ● Plasma membrane ○ Separates inside of the cell from the outside world ● Each of the organelles in the cell are separated by at least 1 membrane if not 2 or 3 ○ Mitochondria have 2 ○ Golgi has 1 ○ Vesicles have 1 ■ Separate cytoplasm from the lumen of the vesicle ■ Important because vesicles fuse with other membranes ■ When a membrane vesicle fuses with the plasma membrane that is called exocytosis ● A portion if it membrane remains in contact with the cytoplasm when the membrane fuses, vesicle lumen ends up attached to the outside world, contents of vesicle will be released into the outside world ■ When vesicles are taken up into the cell its called endocytosis ● You grab a small amount of the outside world and pull it inside the cell, so whatever is outside the cell ends up in the lumen of that endosome surrounded by membrane ○ Nucleus has 2 connected to ER ○ All of these membranes are phospholipid bilayers with proteins added to them ○ Lipid composition and protein composition of each of the membranes differentiate functions and organelles Membranes perform several important cellular functions ● They separate one compartment from another ○ Different organelles from each other ○ Cell from outside world ○ Lipid portion is a barrier to the movement of everything except gas, everything else get transported across by proteins ● They provide a scaffold for biochemical activities ○ Advantageous to have different enzymes acting together arranged on flat surface where they can be clustered together ○ Signals have to get across membranes too, they initiate signal pathways ● Plasma membranes mediate some kinds of interactions between cells ● Signal transduction
● Lipid composition affects bilayer thickness and membrane curvature ○ Lipid bilayer made up of a single unsaturated tail will be shorter and more flit, add cholesterol they straighten up and are longer ○ The interaction of water with the hydrophobic tail is important for forming lipid rafts ■ If you have a taller lipid next to a smaller limid, there is exposure of fatty acid chains to the water environment, the neck of the tall phospholipid= energetically unfavorable ■ The cell passively will tend to overtime will cluster all the tall phospholipids together that way water will only interact with one edge of taller raft, all the interiors are protected ○ Different curvature because the head groups of different lipids are or different sizes ■ If the heads are small all the heads tend to go to the center and the tails are wider ○ Almost no region of the cell where lipid composition is the same Membranes are dynamic structures: summary of movement of phospholipids ● Fatty acid chains are waving around (flexion) ● Spin around their axis (rotartion) ● Diffuse within their plane (lateral diffusion) ● ALMOST NEVER: an outer membrane lipid diving across and becoming a inner membrane lipid (flip-flop) ○ Not energetically favorable ○ Specialized membrane proteins that do this they provide a pathway for the head group to be insulated from rest of the bilayer (flipases) Proteins ● Allow selective permeability, transmit signals, conduct biochemical reactions, etc. ● Basic machinery of cells ● Properties: enzymatic, structural and regulatory ● Hierarchy: ○ Primary ■ Linear sequence of amino acids, can be deduced directly from the DNA sequence of that protein or mRNA sequence ○ Secondary ■ Small scale local shape that emerges directly out of the primary structure of that protein ■ Alpha helix and beta sheet based on the DNA sequence ○ Tertiary ■ 3D shape of the whole protein, mixture of all the alpha helix and beta sheet and the turns between them ■ One of the most critical structure for protein to do its job ○ Quaternary ■ Multi-subunit assemblies ■ Regulated process, changes conformation and binds to other proteins, assemble and disassemble in response to signals ● protein folding
○ There is only one right way to fold a protein ○ Some proteins can fold properly on their own, but sometimes they need help ■ Chaperones and chaperonins are proteins that help bind to proteins during or after synthesis and help them fold properly ■ Hsp70- upregulated when you heat shock cell ● Heating a protein unfolds them- dentaure ● Refold the protein and let the cell live ● Bind to proteins and refold them in an ATP dependent fashion ■ Proteins that need EXTRA help ● Chaperonins do this, 2 barrels facing outward and each barrel takes the protein, caps it and when energy is applied the protein is released ● Imported into a giant multiprotein complex and forced to fold in the right way in an energy dependent fashion ○ Ionic bonds, hydrogen bonds and sometimes covalent bonds hold proteins together ■ Cysteine residues tend to undergo disulfide bond formation resulting in permanent covalent bond between cytosine residues ■ Disulfide bonds stabilize protein structures in only oxidizing environments ■ Cytoplasm is NOT oxidizing environment, protein in cytosol will not have disulfide bonds, they are typically found in the lumen of cells or outside world ● Most membrane proteins and secreted proteins are glycosylated via processing in the ER and golgi ○ Oligosaccharides are used as tags to mark the state of protein folding Mammalian PLC is composed of domains that are found in other proteins ● Phospholipase C- PLC ○ Protein that breaks down phospholipids, hydrolyzes them and cleaves the headgroup from the fatty acid tails ○ In a calcium dependent manner, has to bind to the plasma membrane to access the phospholipids ○ Can tell all of this from DNA sequence Quaternary structure ● protein that are binding to each other to form functional units and unbinding to stop function ● dimer = 2 proteins ● homodimer= 2 identical subunits ● heterodimer= 2 subunits are different polypeptides ● Structure is determined by ○ Covalent bonds are very rare, they can disassemble easy ○ Hydrogen bonds, hydrophobic interactions, ionic interaction, polar interaction, polar interaction, van der waals, covalent-disulfide bonds all hold proteins together ○ Sum total interactions that determine if they will form a stable complex or not ■ ***The more interactions, the more likely it is stable, proteins are existing nin supper high concentrations of water and depending on the temperature it was more energy of its warm, so super energetic water molecules are bonbaring into
○ Protein-protein interactions, not covalently inked Most transmembrane regions are hydrophobic alpha helices ● You can identify transmembrane proteins from a DNA sequence ● If you clone a new protein and analyze the DNA sequence, you can calculate the hydrophobicity of different stretches of amino acids ● If there is 1 stretch of hydrophobicity is 20/30 amino acids long means its a single pass transmembrane protein ● If the stretch is 7 peaks of hydrophobicity, each 20/30 amino acids long then it is a multipass transmembrane protein and probably formed a pass for materials to move across the lipid bilayer ○ EX: bacteriorhodopsin (multipass protein) ○ Things pass through the amphipathic, aquehous, hydrophilic pore ■ Can be connected transmembrane proteins, or single transmembrane proteins brought together Beta sheets can also interact with membranes ● Hydrophobic are our toward the bilayer ● The polar are inside ● Forms a pore through the membrane that is a hydrophilic environment The fluid mosaic model of membranes ● The lipid bilayer is a flexible, 2D fluid sheet ● Membrane proteins “float” in this sheet ● Proteins can move laterally in the plane of the membrane ○ Once they are in the membrane they cannot easily leave, takes a lot of energy ○ Once a protein is put into a bilayer it retains its orientation, never gonna flip around in the bilayer ○ The conformation can change, that is when signals pass from outside to inside the cell Many proteins are constrained and cannot move freely within the membrane ● How can we tell if they are free to move in the membrane or not? ○ Method 1: cell fusion ■ Took 2 cells and fused them together ( heterokaryon ), do the proteins from the two cells mix? Or do they stay separated? ■ If they are mobile they will spread out, if not they will only stay in their own separate halves ■ Low temperatures, very few evenly distributed cells and as you raise temp, all the proteins spread out across a surface- because the lipids move in high temp, gel phase of a bilayer ○ Method 2: FRAP ■ A cell that is expressing a transmembrane protein, CD2 fused to YFP ■ I you shine really intense light on a fluorophore, you excite to so much that it dies and when it dies it cannot be fluorescent again ■ If CD2 is immobile in the membrane, the bleach spot will remain ■ If CD2 can move, the bleach spot will fade away with time because the bleached out CD2 will be replaced by new molecules ■ Observing recovery of fluorescence, no recovery= immobile, recovery= mobile
● Fluorescence coming back from another CD2 membrane protein that can move- means its mobile ○ Method 3: single particle tracking ■ CFTR- protein that is mutated and then causes human disease, cystic fibrosis ■ They wanted only a couple of labeled CFT, so they can see the florence of a single quantum dot ■ Recorded the position of the CFTR dot even though you can only see them at 200 nm ■ Latrunculin breaks up actin cytoskeleton ● CFTR was more mobile when they dissolve the actin cytoskeleton, more movement, CFTR free to diffuse in the membrane ■ Connect a fluorescent molecule to a membrane protein (multipass transmembrane protein CFTR) ■ Label the CFTR and track them over time, do you see the CFTR moving? Trace the movements by tracking quantum dots (give off light) ■ Tracking data on the right means mobile protein Lipid rafts ● We have never directly seen a lipid raft because they are below the resolution of light microscopy ● They are many kinds, they are dynamic and they are different sizes ● Fatty acid chains are different, id on of the chains is unsaturated the tails are shorter and more fluid, cholesterol can make the tails more rigid and sphingolipids are more rigid on their own ● Move together to reduce whole much water touched the tails, because they are different sizes ● The bilayer and rafts are self organizing ● Structure is very complicated Membrane transport ● The plasma membrane and its proteins function as a selectively permeable barrier ● Molecules diffuse across the membrane at different rates and some are easier than others ● For those that need help there are transporters in the membrane to control their entry Small uncharged molecules can move across the lipid bilayer by passive diffusion ● Hydrophobic molecules ○ Cross fairly easily, passively diffuse ○ Need no help, driven by diffusion ○ EX: gasses (O2, CO2, N2) and benzene ● Small uncharged polar molecules ○ Go through a little bit ○ EX: urea, glycerol and water ■ The solution on both sides of the membrane is packed so full of water, even a very low probability of any water molecule making it across, by chance some of it can get across ● Large uncharged polar molecules ○ Basically don't get through at all ○ EX: glucose, sucrose ● Ions
○ Potassium rich solution and is highly enriched in biochemicals, all the proteins and nucleic acids that make a cell a cell (has a net charge of ~ -1.2) ● All of these things can cross the membrane except for the biochemical ● All of these biochemicals have to be kept inside the plasma membrane for the cell to be alive ● The total number of + and - charges are equal outside and inside the cell and the total number of particles are the same on either sides ○ Means that the two solutions are isotonic in respect to one another Mainating ICF osmolarity equal to that of ECF ● Isotonic solution ○ The concentration of particles inside and outside the cell are the same ○ The concentration of water is the same as well, no net force for water to diffuse across the membrane ● Hypotonic solution ○ Fatal ○ Fewer particles outside relative to inside ○ Concentration of water is higher outside, water wants to flow inside and the cell swells up ○ If it swells up too much it will explode and die ● Hypertonic solution ○ Higher concentration of particles outside than inside the cell ○ If the concentration of particles is higher outside the cell, then concentration of water is lower ○ Cell will shrink, not fatal Ion and electrochemical gradients ● Uncharged molecules like glucose are uncharged do not send an electrical potential across the membrane ● Charged particles will notice an electrical potential across the membrane ○ Like charges repel, opposite charges attract ● Electrochemical gradients ○ The concentration of + charges are a lot higher outside the cell than inside the cell and that drives a diffusion force for this cation to diffuse into the cell and the rate is encoded in the thickness of the arrow ○ If the membrane has a negative potential inside the cell, opposite charges attract, so the electrical potential will tend to pull the + charge into the cell, the driving force for this catin is higher here because of the electrical potential even though the concentration gradient is the same ○ Electrochemical potential with membrane potential positive inside the cell, the overall force driving the cation in the cell is smaller overall, like repels like ● Electrical potential across the membrane is a very very thin layer of chargers right underneath the membrane ○ Negative and positive charges are equal ○ Except in nm of the bilayer where there is an imbalance of charges ○ The film of these negative charges right underneath the membrane make the potential exist, a thin layer of charges distributed next to the membrane
○ EX: have a simple cell 145K+, 8Cl-, 14Na, 125 biochem ■ Imagine the cell is sitting on a sodium chloride rich saline solution ■ Imagine in the membrane we have a channel that is a protein that allows K+ to flow only in response to a concentration gradient or an electrical potential or both ■ Start off with the channel closed ■ Is there a driving force for K+ to flow? yes , because potassium is 145 inside the cell and only 5 outside the cell, it cannot flow because it cannot cross the lipid ■ Everytime a K+ ion leves, it leaves behind a - charge, now that negative charge doesn't have a balancing + charge, it ends up lining up right underneath the membrane and starts to build a negative membrane potential ■ The more negative charges under the membrane= larger membrane potential ■ As we built up -V inside the cell - membrane potential inside the cell, it decreases the K+ flowing out of the cell, the - charge pulls on the K+ reducing the force to flow, the current gets smaller, as time goes on less K+ flows out of the cell and finally we reach a point the the membrane potential in - enough to balance the concentration gradient, the force is the same and K+ no longer flows even though the channel is still open ○ EX: Instead of a potassium channel we open a sodium channel ■ Measure build up of charge, membrane potential and current ■ There is a driving force for sodium to flow into the cell (140 outside, 14 inside) ■ When the channel is open, sodium flows into the cell and as we do this we build up a layer of + charges in the cell under the membrane, so membrane potential is the build up and the V becomes more + ■ The + charge inside the cell starts counteracting the gradient and eventually come to enough + charges under the cell membrane that they push sodium out of the cell ○ To calculate membrane potential we use the nernst equation ● Multiple kinds of channel open, we are somewhere between the equilibrium potential of those two channels ● What is going on in most cells ○ There are 10x as many potassium conducting pathways than sodium ○ The membrane potential inside the cell is about -75mV, thin film of - charge, but not as negative as no sodium ○ Sodium is always flowing in, potassium is always flowing out ○ When the driving force is 0, there is no flow, when the driving force is not 0 there is flow ○ For every sodium flows in a potassium if flowing out, equal and opposite ○ Sodium-potassium ATPase, constantly maintains the concentration gradient at the expense of ATP ■ Cell shave found a way to use the sodium energy to do work like take up nutrients Passive transport 1- ion channels ● When they are open they allow ions to flow through at a rate of million of ion per second ● They can be closed or open
○ Step 2: the pump is phosphorylated by ATP hydrolysis, which changes the overall conformation of the pump so now the sites are closed off from the inner cell and opened on the extracellular site and 3 Na+ have disappeared= affinity change so sodium comes off ○ Step 3: now high affinity binding sites for potassium, and causes the phosphate to leave and causes another conformational change that results in the outside closing and inside opening and the affinity goes to low affinity ○ This cycle is slower ○ Light driven pumps ● Secondary active transporters ○ Electrochemical gradient established by sodium/potassium ATPase and ion channels in the cell ○ Do not hydrolyze ATP ○ Capture the energy in the driving force for sodium and use it to pump a second substance ○ symporter/ cotransporter ■ The sodium and the second substance move in the same direction ■ EX: sodium/ glucose cotransporter ● Uses the electrochemical gradient for sodium to power the movement of glucose into cells ● Pump a lot of glucose into cells ● GLUT transporter only always glucose concentrations to be equal inside and outside the cell ● 2NA+/ 1 glucose, works against glucose gradient ○ Antiporter (exchanger) ■ Sodium down its electrochemical gradient providing energy to pump a solute out of the cell, opposite directions ■ EX: sodium/ calcium exchangers ● Using the energy of 3Na+ in/ 1 Cl- out ■ EX: absorption of glucose by the small intestine ● It relies on the fact that the apical and basal domains have completely different transporters inserted into them ● Apical side has sodium/ glucose transporters ● basal/ lateral side has sodium/ potassium ATPase and GLUT transporters ● Sodium concentration gradient used to pump glucose inside the cell, it can exit the cell through GLUT transporters ● Na+/ K+ ATPase take the sodium and pumps it out ● Sodium and glucose come in, glucose is transported out, sodium is pumped out and the net result is sodium and glucose ● And osmotic gradient is also created Action potentials
● Stimulus opens sodium channels, huge membrane conductance, new potassium channels open, membrane potential returns to rest ● Cells make action potential to send electrical signals over long distances ● Use these signals to send info at relatively high speeds ● All or none electrical signals ● Sodium channels that open in action potentials are voltage gated sodium conductance, opened by depolarization, you have to depolarize the membrane potential enough the get the sodium channels to open ● Same height at every point along the axon, once you trigger an action potential it will move down an axon and its amplitude will be the same at all points ● The frequency of action potentials encodes the stimulation ● There is no one speed, different kind of neurons have different speeds ● Resting K channels that are open and resting sodium channels ○ Both opened by depolarization of the cell membrane ○ Reach threshold at which sodium channels start to open, they have a gate on the outside and inside that controls if current can flow, both gates have to be open ○ If you make the membrane potential + enough (depolarize), it will pop the gate open, increase sodium conductance and membrane potential becomes more + ○ Then the inactivation gates in the sodium channels close with a delay after activation gates open, sodium conductance is lower and membrane potential drops to initial value, but at the same time voltage gated K+ channels have opened, the membrane potential recovers rapidly, but undershoots its value and overtime the K+ channels close What makes action potentials fire ● Some stimulus that opens channels that let + current cancel out - charges underneath the membrane and depolarize the cell ○ Another nerve ○ A mechanical stimulus (hearing, touch) ○ A sensory stimulus (smell, taste) ● Stimuli initiate opening of gated channel that depolarize the membrane potential ○ Voltage gated ○ Ligand gated (inside and outside) ○ Mechanically gated ● There are also inhibitory signals that prevent neurons from firing ○ Open channels that allow K+ to flow out ● Whether a neuron fires an action potential or not depends on the net sum of the stimulatory and inhibitory inputs Neurons can talk to each other via chemical synapses ● Synaptic terminals ● Terminals have an ion channel that is a voltage gated calcium channel ● Ca concentration outside is ~2mM, inside cells is ~100nM (much smaller) ● Kept low because there are pumps pumping calcium out of the cytoplasm because calcium miles to make crystals with phosphate
