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Lecture 21: The Structure and Function of the Plasma Membrane How do things get into and out of the cell? How is it transported?Learning objective: ➔ Understand the fluid mosaic model of membrane struc ture★ Fluid mosaic model describes membrane structure. “sea of lipids in which proteins float like...

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Lecture 21: The Structure and Function of the Plasma Membrane How do things get into and out of the cell? How is it transported? Learning objective: ➔ Understand the fluid mosaic model of membrane structure ★ Fluid mosaic model describes membrane structure. “sea of lipids in which proteins float like icebergs” Two layers of lipid in which you have membrane proteins that are embedded and are free to move around in. Thin, 8nm, flexible+sturdy barrier-surrounds the cytoplasm of the cell-barrier that enables partition between inside and outside of the cell. The membrane is 50% lipid & 50% protein and held together by hydrogen bonds. Barrier to polar+nonpolar substances exit and entry.Protein-GATEKEEPERS- regulates traffic Hydrophobic core and hydrophilic ends Lipid bilayer of cell membrane2 back to back layers- 3 types of lipid molecules The lipid bilayer consists of cholesterol and glycolipids scattered among a double row of phospholipids 1. Cholesterol: 2. Glycolipids: 3. Phospholipids:75% of lipids- 2 PARALLEL LAYERS of molecules- Amphipathic molecules(both non-polar AND polar regions in molecule) makeup by far the most of the bilayer. Polar heads and non-polar tails( don’t like to interact with a charged environment). Water will interact with the polar head groups, but will be excluded from the hydrophobic core. ➔ How the molecular structure of the membrane results in selective permeability Membranes are fluid structures and lipids move around within the plane of the membrane leaflet. Lipids rarely flip leaflets therefore the lipid composition of the leaflets can be asymmetric.-how certain molecules can cross the membrane and others cannot. ● Fluidity is determined by: ○ Tail length: Length decreases fluidity, longer the tail=less fluid the membranestiffer the membrane ○ Number of double bonds: more double bonds increase fluidity- introduce kinks in the tail which makes them pass less tightly and gives more fluiditythe membrane is less stable ○ Amount of cholesterol: more=increase fluidity ●

Integral proteins: extend into or completely across the cell membrane (transmembrane protein-span across the lipid bilayer). Amphipathic with each region corresponding with the appropriate phospholipid bilayer region. The hydrophobic region consists of nonpolar amino acids coiled into helices(interacting with the hydrophobic core). The hydrophilic ends interact with the aqueous solution. To remove from membrane- break the interactions between the

hydrophobic amino acids and hydrophobic lipids. Detergent-dissolves lipid, stabilises membrane and allows the integral protein to be isolated. Have to break the hydrophobic amino acids and lipid bilayer interaction. ● ●

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Peripheral proteins: attached to either inner or outer surface of cell membrane and are easily removed from it. Eg. cytoskeleton and associated proteinsMembrane proteins can act as: ○ Receptor Proteins- sense signals from hormones in the blood or neurotransmitters or other peptides and actually bind those receptors and transfer signals inside the cell ○ Cell Identity Markers- sense of self. Express markers that say don’t attack or attack. ○ Linkers-links to other cells, hold cells in place ○ Enzymes-catalyse enzymatic activity-breakdown glucose from diet on the surface of the cell or break down ATP to form cyclicAMP on the inside of the cell to give a signalling pathway. ○ Ion Channels ○ Transporter Protein Membrane proteins that do transport across cell membranes. The laws of diffusion that govern movement across the cell membrane ○ -diffusion Impermeable to large uncharged polar molecules and ions-glucose and amino acids- Na+, K=, Cl-, Ca2+,H=- electrical charge repelled by the non-polar hydrophobic core in the bilayer- another mechanism to get into the cell! Permeable to nonpolar, uncharged molecules-O2, N2, benzene and lipid soluble molecules AND steroids, fatty acids, some vitamin AND small UNCHARGED polar molecules: H2), Urea, glycerol, CO2 Membrane proteins mediate the transport of substances across the membrane that cannot permeate the hydrophobic core of the lipid bilayer:IONS and LARGE UNCHARGED POLAR MOLECULE- glucose, amino acids. Direction of movement is determined by the concentration gradient.high->low Random mixing of particles in a solution as a result of the particle’s kinetic energy – molecules moving from high to low concentration more.- greater the difference, higher the temp.=faster rate of movement across the membrane Increased surface area= increases rate of diffusion Increased distance and size of diffusing substance=slower rate of diffusion

Consequences:The rate of diffusion sets a limit on the size of cells of about 20 µm. To increase diffusion a cell can increase the membrane area available for exchange (diffusion) of a substance. The thicker the membrane the slower the rate of diffusionanything that diffuses across the bilayer is rapid as its only 8nm. Diffusion is very fast over small distances. ● How the compostion of the different fluid compartments act to store energy Gradients across the cell membrane ● There are 2 gradients at play when it comes to diffusion: the concentration gradient(non charged molecules diffuse down their conc. gradient) and the electrical

gradient(ions influenced by membrane potential in addition to their conc. gradient). As ions are charged they will be influenced by both-electrical gradient will go one way and the conc. Gradient will go the other.ocassionally they may go in the same direction. Gradients across the plasma membrane ● Selective permeability enables:difference in concentration of conc. gradient to be established- if the membrane had holes it would not have a concentration gradientthe lipid bilayer being non-permeable to ions is important in establishing conc. Differences across the membrane ● Cells can maintain a difference in charged ions between the inside & outside of membrane (establishing an electrical gradient or membrane potential) in a very similar way to a capacitor 8nm non-conducting hydrophobic core gap separating two solutions of diff. Charge- if you allow ions to flow through the lipid bilayer it will be RAPID and then it will create a chargethe transmembrane protein harnesses the energy created. Ion gradients across the cell membrane ● The gradients of Na+(150 mMol), Cl- (5 mMol)and K+(150 mMol) represent stored energy. Cells use 30% of their energy to maintain the gradients. ● K+ diffuses down its gradient. Slows down as a strong negative charge will mean positive ions(K+) won’t leave, electrochemical equilibrium is eventually reached.positive charge leaves a negative charge behind. ● Cl- membrane potential becomes less negative- DEPOLARISES Cl- enters the cell HYPERPOLARIZES- Cl- leaves the cell. It is the storing of these ions on either side of the membrane, and them being harnessed by different membrane proteins that creates energy to do numerous cellular processes. Cells use approx. 30% of resting energy to maintain concentration and electrical gradients, this represents stored energy- use the energy of metabolism, NaKATPase establish gradients and then put chem. Work in to create these gradients that harness energy. ● Osmosis: Net movement of water through a selectively permeable membrane. High water concentration to low water concentration. Only occurs if the membrane is permeable to water. If an osmotic gradient exists>water moves to eliminate it. ● Water permeability(Pw) is the permeability through a bilayer(Pd) plus through water channels(Pf).Pw=Pd+Pf Pf>Pd ● Water channels (Aquaporins) are large, mercury-sensitive a temp independent. Whereas the bilayer is the opposite(small, mercury insensiitve, temp. dependent) Lecture 22: Transport across cell membranes ➔ The properties driving water movement across cell membranes. ➔ The difference between channel and carrier mediated transport ● Non-mediated transport: No transport protein. Through lipid bilayer eg. O2 CO2 fats- does not involve transport protein. Interacting with the bilayer ● Mediated transport: uses a transport protein ● Passive transport: moves down concentration or electrochemical gradients using only the substances kinetic energy ● Active transport: uses energy to move substances against gradients- have to put

energy in to move it. Vesicular transport: Moves materials across in small vesicles-exocytosis or endocytosis-not discussed. . The difference between passive and active transport of solutes. ● Non-mediated transport:diffusion through the lipid bilayer IMPORTANT- absorption of nutrients-excretion of wastes . The hydrophobic core does not impose a barrier to the molecules. Molecules would have to be non-polar , hydroPHOBIC molecules- O2, CO2, fatty acids, steroids, small alcohols. ammonia and fat-soluble vitamins(A, E, D, K)molecules able to diffuse as they can interact with the hydrophobic core. ● Diffusion through ion channels: interacting with the bilayer and amino acids, charged amino acids create a pore/pathway. (passive mediated transport): The channel forms a water-filled pore that shields the ions from the hydroPHOBIC core of the lipid bilayer: hydrophilic amino acids on the interior and hydrophobic amino acids on the exterior. Very rapid transport as ions do not bind.can get very high flows of ions through these channels. Ion specific, different perm. To different ions. Ionic selectivity. The channels are selective. Specific amino acids lining the pore determine the selectivity of the channel ions- selectivity to particular ions allows them to harness the energy stored in diff ion gradients. Not always open-GATING ● Gating: channels contain gates that control opening and closing of pores eg: IP3 gated Ca2+ channel on rough ER. different stimuli(voltage,ligand binding(molecule from the blood binds to protein), cell volume(stretch), pH(anaerobic metabolism), Phosphorylation)->opening. Gigaseal- patch clamp tech. Focus on one channel. ● Properties of ion channel function:Electrical current: The diffusion of over 1 million ions per second through a channel generates a measurable current (~ 1 0−12 amp). The current flowing through an individual channel can be recorded using the patch clamp technique. The current fluctuations represent the conformational changes in channel structure that are associated with channel gating eg. opening and closing of single ion channels. The current fluctuations represent changes in channel structure associated with gating. ●

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Carrier mediated transport: the substrate directly interacts with the transporter protein. Slower than channels as undergoes a conformational change. Exhibit:Specificity, Inhibition, Competition, Saturation (binding pockets saturated transport maximum). Can be passive or active Saturated- the conc. Of glucose is increased but the uptake is not, uptake is saturated. Facilitated diffusion of glucose-NOT A CHANNEL binding of glucose to the channel protein and then the conformational shape is changed allowing glucose to enter and move down its gradient. Glucose transport occurs until binding sites are all saturated. 1) Glucose binds to transport protein (GLUT) 2) Transport protein changes shape. Glucose moves across cell membrane (but only down the concentration gradient) 3) Kinase enzyme reduces glucose concentration inside the cell by transforming glucose into glucose-6-phosphate Conversion of glucose maintains concentration gradient for glucose entry- important for accumulating glucose in the epithelial tissue ➔ Differentiate between primary and secondary active transport.

Active transport- ball and stairs scenario- requires energy to move molecules and ions against their conc. And electrochemical gradients, ● Two forms of active transport: primary and secondary. ○ Primary energy is directly derived from the hydrolysis of ATP. A typical cell uses 30% of its energy (ATP) on primary active transport Na pump- maintains low conc. Of Na+ and a high conc. Of K+ in cytosol Primary active transporters:Difference in Ion conc. Is important for: ● Maintaining resting membrane potential ● Electrical excitability ● Contraction of muscle ● Maintenance if steady state cell volume- important for Na+ pump removing ions to control the osmolarity of the external solution so that cells are not swollen. ● Uptake of nutrients via secondary active transporters ● Maintenance of Intracellular pH by secondary active transporters- use the energy stored in the ion gradients Pump works continuously as Na and K are continuously leaking lack into cell down their respective gradients. Using the 30% of the energy. Removing of the ions leaked into the cell maintains the ion gradients. ○ Secondary active transporters: Uses energy stored in an ion gradients created by primary active transporters to move other substances against their own concentration gradient. Therefore it uses ATP obtained by hydrolysis indirectly. DO NOT HYDROLYZE ATP DIRECTLY. Powered by the Na+ gradients initially established by the Na pump- good energy source to couple other processes. ● Primary active transport example: Na/KATPase antiporter 1. Na+ binding to binding pocket- changes or allows ATP-> ADP leaves a phosphate on ion channel. Phosphate has a strong neg. charge= changes the conformation of the protein, puts energy to force molecule to change change and this opens the Na+ binding site to open and Na+ leaves to the outside of the cell 2. ATP split/ Na+ pushed out 3. K+ binding/ Phosphate release- K+ binds and phosphate falls off 4. K+ is pushed into the cell 3 Na+ ions removed from cell as 2 K+ brought into cell. Therefore the pump generates a nett current and is electrogenic. Creating a net current of one. Building a high conc. Of Na+ outside the cell and K+ in the cytosol. Other examples-Ca/KATPase(muscle SR) and H/KATpase(stomach)

● Secondary active transporters: example Na+ antiporter or exchangers Na+ ions rush inward Ca2+ or H+ pushed out Na+ symporters or cotransporters:all going into the cell- same direction. Glucose or amino acids in with Na+ ions Lecture 23: Traffic across cells: Epithelial transport of glucose

Epithelial tissue: Cells in continuous sheets- single or multiple layers- cells sit on BM, form the boundary b/twn organs and external body environments, subject to physical breakdown= undergoing constant and rapid renewal process. Skin-stratified squamous epithelium Gut lining- columnar Module 1: EPITHELIAL NOTES ➔ Understand epithelial structure and function:role of tight junctions ● Tight junctions- composed of thin bands- encircle the cell. Make contact with adjacent bands. Below the luminal edge Appearance in EM: membranes fused together in Freeze fracture: interlocking network of ridges in PM ● Tight junctions on the lateral surface act as a barrier to restrict the movement of substances through the intercellular space and a fence to stop Membrane proteins from diffusing through the lipid bilayer. ● Separate epithelial cells into 2 membrane domains: Apical- faces Lumen. Basolateral- adheres to Basement membrane, interfaces with blood This allows different transport proteins to be inserted into either the apical or basolateral membrane. ➔ Understand epithelial structure and function:transcellular and paracellular transport ● Paracellular(governed by diffusion laws and tightness of the junctions: tight junctions like a sieve, how tight the barrier is to the movement of those molecules): Between cells. Diffusion and tightness of junctions. Higher electrical resistance=more tight junctions holding the cell together. The electrical resistance to ion flow through tight junctions can be measured(Ohm's Law). The higher the resistance= the greater the number of tight junction strands= the tighter the junctions. Epithelium can be classified( based on tightness or electrical resistance) into: ● Leaky epithelium – paracellular transport dominates- a lot of ability to move molecules via this pathway . ● Tight epithelium – transcellular transport dominates- no transport via the paracellular as the junctions are so tight, high resistance ● In the GI tract and the kidney, the proximal end is leaky and the distal end is tight. Resistance changes in the proximal to distal direction. ● The tight junction resistance is in a proximal> Distal direction.

Understand epithelial structure and function:how epithelial cells can mediate

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absorption or secretion of a substance Transcellular Primary and secondary active transport often in combination with passive diffusion through ion channels often in combination with passive diffusion through ion channels to produce transport across the tissue. The transport can be either:absorption(transport from lumen to blood) or secretion(transport from blood to lumen).

Rules of transcellular transport: 1) Entry and exit steps: the entry step for absorption is apical but for secretion is the basolateral membrane. 2) Electrochemical gradient: is the entry or exit step passive or active 3) Electroneutrality: movement of a positive or negative ion will attract a counter ion 4) Osmosis: nett movement of ions will establish a difference in osmolarity that will cause water to flow by osmosis ●

Osmosis(movement high>low concentration, water moves to eliminate osmotic gradient) occurs only if the membrane is permeable to water and not to certain solutes!

● Pw( membrane permeability to water) Pf mediated-aquaporins(9 isoforms) Cells have different Pw bc they express different aquaporin isoforms The lipid bilayer : water perm. That is through the bilayer and another through a water channel. Through the bilayer the perm. Is small, blocked, not by mercure (mercury in sensitive), Temperature dependent(lipid fluidity) Through water channels, larger perm. And is mercury sensitive, temp independentaquaporins- allow water to flow through them without any ions. Can dissociate water flow from ion flow by aquaporins or not. Understand Glucose absorption in the intestine and kidney:Glucose/galactose malabsorption syndrome ● Glucose absorption in the Intestine: 1. Tight junctions divide cells into apical and basolateral membrane domains- form a barrier to movement of some molecules via paracellular pathway. 2. Na-pump sets up ion gradients-3Na and 2K for every ATP molecule that is hydrol. Means there is a neg. Membrane potential as 3 Na+ are moved out of the cell. 3. The sodium glucose symporter (SGLT, carrier mediated transport, secondary) uses the energy of the Na gradient to actively accumulate glucose above its concentration gradient, allowing glucose to bind and Na to move down its gradient, more glucose inside the cell than outside- GLUT transporter. 4. Facilitative glucose transporter (GLUT) mediates glucose exit across the basolateral membrane via passive diffusion down its gradient-moving 5. Na taken up via the glucose exits via the basolateral Na-pump 6. The transport of Na and glucose across the epithelium induces paracellular Cl and water fluxes Refer to diagrams on page 175 and 176 Oral rehydration therapy: Sugar, salt, and water solution given to people suffering diarrhea. Ability of glucose to enhance the absorption of Na+ and hence Cl- and

water is exploited in oral rehydration therapy. Glucose-Galactose( carrier, fits the binding pocket) malabsorption syndrome: SGLT(sodium glucose symporter) mutation leading to a high glucose concentration in lumen of the intestine due to sugar being retained (which increases osmolarity) and therefore results in a water efflux. The increased water flow produces diarrhea. Treat by replacing diet with fructose which uses the GLUT5 transporter Understand Glucose absorption in the intestine and kidney:Glucosuria in the kidney Glucosuria: Glucose in urine: Saturation of SGLT in kidney in diabetes mellitus- due to insulin inactivity deficient- blood sugar is high(over 200mg/L). Diabetes SGLT cannot absorb glucose fast enough->appears in the urine. No mutation with the transporter- just too much glucose due to diabetes. Can measure the blood glucose levels and compare it to how much urine is in the urine. Comparing the amount of glucose that is being filtered in comparison to the amount of glucose in the renal ve...