Chapter 4-.pdf

BIOTECH 2CB3 Dr. Uzma Nadeem Chapter - 4 The Structure and Function of the Plasma Membrane Gerald Karp, 9th edition Learning Outcomes:  Properties and history of studies on Plasma Membrane (PM)  Lipid composition of membranes (Phosphoglycerides, Sphingolipids and Cholesterol)  Fluid Mosaic...

BIOTECH 2CB3 Dr. Uzma Nadeem Chapter - 4 The Structure and Function of the Plasma Membrane Gerald Karp, 9th edition Learning Outcomes:  Properties and history of studies on Plasma Membrane (PM)  Lipid composition of membranes (Phosphoglycerides, Sphingolipids and Cholesterol)  Fluid Mosaic Model  Membrane Proteins (Integral., Peripheral and Lipid anchor)  Membrane lipids and membrane fluidity  Dynamic nature of the PM  Ways of solute movement across cell membrane  Membrane potential (Resting Potential, Action Potential)  Propagation of AP as an impulse  Neurotransmitter and its mechanism of action  Action of drugs on synapses Introduction to the Plasma Membrane Outer edge of a differentiated muscle cell grown Fig 4.1 Erythrocyte (it was stained with heavy metal called osmium)-dark staining seen by Electron microscopy The trilaminar appearance of membranes as revealed by electron micrograph of the plasma membrane and sarcoplasmic reticulum. • Plasma membrane: The outer boundary of the cell that separates it from the world is a thin, fragile structure about 5 – 10 nm thick. • Not detectable with light microscope need electron microscope. • The 2 dark-staining layers in the electron micrographs correspond primarily to the inner & outer polar surfaces of the bilayer. • All membranes examined closely (plasma, nuclear or cytoplasmic) from plants, animals or microorganisms have the same ultrastructure. Introduction to the Plasma Membrane An Overview of Membrane Functions 1) Compartmentalization 2) Scaffold for biochemical activities 3) Selective permeability barrier 4) Transporting solutes 5) Responding to external signals 6) Intercellular interaction 7) Energy transduction Fig 4.2: A summary of membrane functions in a plant cell. Introduction to the Plasma Membrane An Overview of Membrane Functions 1) Compartmentalization: Membranes form continuous sheets that enclose the cell and intracellular compartments. (hydrolytic enzymes, acid hydrolases are sequestered in a membrane-bound vacuole). 2) Scaffold for biochemical activities: Membranes are themselves compartments. They act as scaffolds. (site of enzyme localization, e.g CO2 fixation in the outer surface of the chloroplast). 3) Selective permeability barrier: Membranes have Gated channels that control the movement of selected molecules (e.g. water molecules can penetrate rapidly through the PM) 4) Transporting solutes: Membrane proteins facilitate the movement of substances form one side to another side. Typically against a concentration gradient (e.g. H+ ions against con. gradient) 5) Responding to external signals: Membrane receptors transduce signals from outside the cell in response to specific ligands. (e.g. plant hormone Abscisic acid, a stress hormone, controls the Calcium efflux from the internal sources and close stomata) 6) Intercellular interaction: Membranes mediate recognition and interaction between adjacent cells.(e.g. openings between adjoining plant cells called plasmodesmata allow the flow of material from one cell into its neighbors). 7) Energy transduction: Membranes are involved in the processes by which one type of energy is converted into another type. (e.g. Conversion of ADP to ATP in the inner membrane of the mitochondrion) How did we discover the composition of plasma membrane? 7 Introduction to the Plasma Membrane First insights of the composition of the outer layer of cells came from:  The realization that like dissolves in like  For stuff to enter a cell, it must dissolve in the outer layer  The more lipid-soluble a solvent is, the more readily it would enter the cell; therefore the layer must be made of lipids  A Brief History of Studies on Plasma Membrane Structure Q) How do we know that PM is lipid bilayer? Overton placed plant root hairs into hundreds of different solutions containing a diverse array of solutes. The more lipid‐soluble the solute, the more rapidly it would enter the root hair cells He concluded that the dissolving power of the outer boundary layer of the cell matched that of a fatty oil. How? Observe under microscope the changes in the protoplasm’s volume. Tested 500 compunds Introduction to the Plasma Membrane A Brief History of Studies on Plasma Membrane Structure In early 19 century, two Dutch scientist, E.Gorter and F. Grendel proposed that cell membranes might contain a lipid bilayer. How? Calculating the surface area of a lipid preparation They used RBCs to determine the PM chemical nature. Why? • Using red blood cells, their lipid components can cover approximately twice the cell’s surface area Lipid bilayer • Ratio = surface area of water covered by the extracted lipid/surface area of the extracted RBC • The lipid bilayer accounted for the 2:1 ratio of lipid to cell surface area • The most energetically favored orientation for polar head groups is facing the aqueous compartments outside of the bilayer. Langmuir animation: https://www.youtube.com/watch?v=j8yqyRr2VQg Fig 4.3: Bimolecular layer of phospholipids with water soluble head groups facing outward Introduction to the Plasma Membrane • Cell physiologists determined that there must be more to the structure of membranes than simply a lipid bilayer:  Lipid solubility was not the sole determining factor as to whether a substance could penetrate the plasma membrane  Surface tensions of membranes were calculated to be much lower than those of pure lipid structures - Why? • • These could be explained by the presence of proteins within plasma membranes The proteins themselves can be single proteins, or part of complexes •Remember: the membrane is fluid, and thus dynamic Fig 4.4: Organization of proteins embedded in the lipid bilayer The Lipid Composition of Membranes • Membranes are lipid–protein assemblies held together by noncovalent bonds (fluid mosaic model) – The lipid bilayer is a structural backbone and barrier to prevent random movements of materials into and out of the cell – The proteins determine the specialized activities of that cell type. • The ratio of lipid to protein varies, depending on the type of cellular membrane, the type of organism, and the type of cell: Fig 4.5: Electron micrograph of a nerve cell axon The Lipid Composition of Membranes The Lipid Composition of Membranes Membrane Lipids • Membrane lipids are amphipathic (both hydrophilic and hydrophobic regions) with three main types: – Phosphoglycerides – Sphingolipids – Cholesterol 1. Phosphoglycerides • Lipid containing a phosphate group is called phospholipid • They include the glycerol-backbone and called Phosphoglycerides.  Phosphoglycerides or Glycerophospholipids are diglycerides; two hydroxyl groups of glycerol are esterified to fatty acids; the third is esterified to a hydrophilic phosphate group Fatty acid • The kinds and relative proportions of phospholipids vary greatly among types of membranes Q) Can you identify the difference between triglycerides and phosphoglycerides? 1. Phosphoglycerides  Most phosphoglycerides have a small hydrophilic group linked to phosphate: choline, ethanolamine, serine or inositol – This group, together with the negatively charged phosphate, forms a highly water-soluble domain, called the head group.  Fatty acyl chains are hydrophobic, unbranched hydrocarbons approximately 16 to 22 carbons in length. A fatty acid may be fully saturated, or unsaturated (monounsaturated, or polyunsaturated). Q) What about fats and oils? (saturated, unsaturated?)  Phosphoglycerides often contain one unsaturated and one saturated fatty acyl chain. 2. Sphingolipids • Sphingolipids are less abundant class of membrane lipids and are derivatives of sphingosine, an amino alcohol that contains a long hydrocarbon chain • Sphingolipids consist of sphingosine linked to a fatty acid by its amino group (which is then called a ceramide) • Substitutions (subs) to the terminal alcohol of ceramides can create new molecules: Sphingomyelin: the only phospholipid that has no glycerol backbone and composed of ceramide + phosphatidylcholine (e.g. found in nerve tissues, RBCs and ocular lenses) Glycolipid: the molecule is a carbohydrate Cerebroside: the molecule is a simple sugar Ganglioside: small cluster of simple sugar and sialic acid  The fatty acyl chains of sphingolipids are longer and more saturated than Phosphoglycerides  Nervous system is rich in glycolipids Fig 4.6: 2. Sphingolipids  Cerebrosides are neutral glycolipids; each molecule has an uncharged sugar as its head group  A ganglioside has an oligosaccharide head group with one or more negatively charged sialic acid residues • People who cannot synthesize ganglioside (G M3) suffer from a serious neurological disease and results in severe seizures and blindness  Cerebrosides and gangliosides are especially prominent in brain and nerve cells  Human ABO blood group is an example of glycosphingolipids and they have an enzyme that adds N-acetylgalactosamine to the end of the RBC membrane glycolipids Review Slide of chemical structures of membrane lipids Polar P Glycerol Fatty acid chain The chemical structure of membrane lipids Fig 4.6: Summary Slide for Lipids serine Sphingosine Fatty acid OH R NH2 Sphingosine+Fatty acid= Ceramide) choline OR (an 18 carbon amino alcohol with an unsaturated hydrocarbon chain) Sphingolipids (Ceramide) NH2 Sphingomyelin (no glycerol backbone) Ceramide+Phosphatidylcholine =Sphingomyelin Summary Slide for Lipids OR NH2 Sphingolipids (Ceramide) choline Sphingomyelin Cereboside Ganglioside 3. Cholesterol • Cholesterol is a smaller and less amphipathic lipid . • Can make up to 50% of animal membrane lipids • The hydrophilic -OH groups are oriented toward membrane surfaces • Carbon rings are flat and rigid; interfere with movement of phospholipid fatty acid tails • Plant cells contain cholesterol-like compounds (sterols) that fulfill a similar function like membrane fluidity and permeability Fig 4.7: Cholesterol molecules (green) oriented with their small hydrophilic end facing the external surface of the bilayer The Lipid Composition of Membranes The Nature and Importance of the Lipid Bilayer The Lipid Composition of Membranes • Cells membranes have distinct lipid compositions, differing in lipid types, head groups, and species of fatty acyl chain(s) – Lipid composition can determine the physical state of the membrane and influence membrane protein activity • The entire lipid bilayer is only about 5-10 nm thick; and since hydrocarbon chains are not exposed to the aqueous environment, membranes are always continuous, unbroken, and very dynamic (fluid/flexible) structures • All these physical traits allows the cell to function, examples are: – Cell division and the formation of secretion vesicles – Maintaining the proper internal environment (i.e keeping chemical and electrical gradients, amongst other things) The Lipid Composition of Membranes The Nature and Importance of the Lipid Bilayer • An important feature of the lipid bilayer is its ability to selfassemble- HOW? • In vitro, we can add phospholipid molecules and they would assemble spontaneously to form liposomes, with a bilayer structure • Liposomes are vehicles to deliver drugs or DNA within the body; they can be linked to the liposome wall or contained at high concentration within its lumen. The walls of the liposomes are constructed to contain specific proteins (hormone, antibody) and take them to the target cells. • To protect the liposomes from the immune destruction, stealth liposome is created that contains a polymer called polyethylene glycol (PEG). • Liposomes are good to treat some medicines such as chemotherapy or fungal infections that have toxic effect on the healthy cells or could damage organs. https://www.youtube.com/watch?v=vUqwIL5lgS8 Fig 4.9: Liposomes: synthetic vesicles Question Are the various lipids in a membrane randomly distributed between the two monolayers of what makes up the lipid bilayer? The Lipid Composition of Membranes The Asymmetry of the Membrane Lipids • The lipid bilayer consists of two distinct leaflets that have a distinctly different lipid composition- How do we know? • (experiment: treated intact RBC with lipid digesting enzyme called phospholipase) • https://figures.boundless-cdn.com/20013/large/0302phospholipid-bilayer.jpeg -found that 80% of PC, 20% PE and 10% PS of the membrane is hydrolyzed • The different lipids exhibit different properties, therefore providing distinct functionalities to each leaflet • For example, glycolipids are found on the exoplasmic side, while proteins responsible for transmitting signals from the membrane to the inside of the cell are in the cytosolic side • Phosphatidylethanolamine is in the inner leaflet and tends to promote the curvature of the membrane Fig 4.10: • Thus, the lipid bilayer is composed of two semi-stable, independent monolayers having different physical and chemical properties SM: sphingomyelin PC: phosphatidylcholine PS: phosphatidylserine PE: phosphatidylethanolamine PI: phosphatidylinositol Cl: cholesterol Membrane Proteins: The Mosaic Part of the Fluid Mosaic Model • Mosaic: all membranes are lipidprotein constructs • Strong support for this model came from freeze-fracture experiments • Freeze-fracture technique divides the phospholipid leaflets of the membrane. Membrane Proteins • Membrane proteins have different affinities to the core (lipophilic) of membranes, and so they differ in the way they interact with the lipid bilayer • Membrane proteins are grouped into 3 classes:  Integral  Peripheral  Lipid-anchored Membrane Proteins: Integral Proteins • Integral proteins: Penetrate and pass through lipid bilayer (i.e. transmembrane); make up 25 -30% of all encoded proteins and 60% of current drug targets Spanning segment • These proteins act mostly as: – Receptors – Channels (for ions and other solutes) – Agents for electron transport • These proteins are amphipathic: Fig 4.13a: – Hydrophobic domains anchor them in the bilayer (van der Waals with fatty acyl chains); this creates a seal which maintains the permeability barrier – Hydrophilic regions (mostly globular proteins) form functional domains outside of the bilayer Membrane Proteins: Integral Proteins • Hydrophobic transmembrane domains make integral membrane proteins difficult to isolate in a soluble form • Their isolation from membranes requires detergents like ionic SDS, which denatures proteins, or nonionic Triton X-100, doesn’t alter a protein’s tertiary structure (diagram) – Detergents are amphipathic and can substitute for phospholipids in stabilizing and solubilizing integral proteins • After purification, the protein’s amino acid composition, molecular mass, and amino acid sequence can be determined Ionic Detergent https://www.bio-rad.com/ Fig 4.16: Solubilization of membrane proteins with detergents Non-Ionic Detergent Can you quantify protein or see its expression after isolating it from the cells? •PLoS ONE 8(3):e58181 Membrane Proteins: Integral Proteins • Some of the problems facing scientists studying these proteins are: (1) They are present at low numbers per cell (2) They are unstable in the detergent-containing solutions required for their extraction (3) They are prone to aggregation (4) They are heavily glycosylated and cannot be expressed as recombinant proteins in other types of cells Membrane Proteins: Integral Proteins Identifying Transmembrane Domains • Protein segments embedded within the membrane, or transmembrane domains, have a string of about 20 mostly nonpolar amino acids that span the lipid bilayer as an a helix • Fully charged residues can be found in transmembrane helices, but they tend to be near one of the ends of the helix close to the polar environment Fig 4.18: Glycophorin A, an integral protein with a single transmembrane domain with a Gly-X-X-X-Gly sequence Q)How do scientists know the structure of a membrane protein and its orientation within the lipid bilayer? Membrane Proteins: Integral Proteins Identifying Transmembrane Domains • By knowing the amino acid sequence of an integral protein, we can identify transmembrane segments using a hydropathy plot • Hydrophobicity of amino acids can be determined from their lipid solubility or energy required to transfer them from a nonpolar into an aqueous medium – Transmembrane segments are peaks in the hydrophobic side of the spectrum • Orientation of the transmembrane segment within the bilayer? – The flanking side that is more positively charged (amino acids) is typically extended toward the cytoplasmic side! Fig 4.20: Hydropathy plot example Membrane Proteins: Peripheral Proteins • Peripheral proteins: Attached to the membrane by weak electrostatic interactions and are located entirely outside of bilayer on either the extracellular or cytoplasmic side (noncovalent bonding to the polar head group of the lipid bilayer and/or to an integral membrane protein) • They are easily solubilized and extracted with highsalt solutions • Peripheral proteins typically have a dynamic relationship with the membrane, being recruited or released as needed • They can act as: – – – Mechanical support for membranes Enzymes (Electron Transport Chain) Factors that transmit signals Fig 4.13 b: Peripheral proteins Membrane Proteins: Lipid-anchored proteins • Lipid-anchored membrane proteins are covalently linked to a lipid molecule that is situated within the bilayer • They are distinguished both by the types of lipid anchor and their orientation: • – On the outer-leaflet, lipid-anchored proteins are mostly associated via an oligosaccharide to a phosphatidylinositol – While inner-leaflet proteins are anchored to membrane lipids by long hydrocarbon chains They act as: – – Adhesion molecules Enzymes Lipid-anchored proteins Fig 4.13 c: Review  Properties and history of studies on Plasma Membrane (PM)  Lipid composition of membranes (Phosphoglycerides, Sphingolipids and Cholesterol)  Fluid Mosaic Model  Membrane Proteins (Integral., Peripheral and Lipid anchor)  Factors affecting fluidity Q) Why membrane fluidity is so important? Q) Is it easy to move things in highly ordered and organized structure? Q) Can a non-viscous environment hold structure? Q) What should be the best? Membrane Lipids and Membrane Fluidity • The physical state of membrane lipids can be described by their fluidity (or viscosity) • At higher temperatures ( e.g. 37oC), the lipid of membranes exists in a relatively fluid state, and is described as a two-dimensional liquid crystal – Molecules retain a specified orientation; the long axes are parallel, yet individual lipids can rotate around their axis or move laterally within the plane. • If the temperature is slowly lowered to the transition temperature, the lipid is converted to a frozen crystalline gel and movement is greatly restricted Fig 4.23: Structure of the lipid bilayer depends on the temperature: a) above and b) below the transition temperature. Membrane Lipids and Membrane Fluidity http://www.nutrientsreview.com/wp-content/uploads/2014/12/Trans-cis-fatty-acid.jpg • • Saturated fatty acids resemble a straight, flexible rod. Unsaturated fats could either be cis or trans fats, cis-unsaturated fatty acids have crooks in the chain at the sites of a double bond. Cis fats are beneficial and promote good cholesterol while trans fats are harmful to cardiovascular health (especially from unnatural sources) • Factors affecting membrane fluidity: – Saturated chains pack together more tightly than those containing unsaturated chains – The greater the degree of unsaturation of the fatty acids of the bilayer, the lower the temperature before the bilayer gels – The shorter the fatty acyl chains, the lower its melting temperature. • Cholesterol abolishes sharp transition temperatures and creates a condition of intermediate fluidity: dampens temperature effects Membrane Lipids and Membrane Fluidity Q) Can you explain the transition temperature for two membranes? The first membrane has a transition temperature of 7 oC and the other has 32 oC. Hydrocarbon chain? Degree of saturation? Less hydrocarbons- more fluidity More double bonds-more fluidity Membrane Lipids and Membrane Fluidity Q) Keeping in mind the lipid contents, do you think phospholipid contents will be changed in summer or winter if you analyzed the membrane by taking tissue samples from the organisms? WHY? You collected membrane samples from the same animal in winter and in summer and analyzed the lipid contents. What would be the lipid composition of membrane when you analyze it in winter versus summer. Think in terms of hydrocarbons and unsaturation https://www.google.com/imgres?imgurl=https%3A%2F%2Fmedia.istockphoto.com%2Fid %2F152 https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.dreamstime.com%2Fphotos-images% In winter -less hydrocarbons and unsaturated (lower degree of saturation) than the membrane in summer. Membrane Lipids and Membrane Fluidity The Importance of Membrane Fluidity • Membrane fluidity provides a compromise between a rigid, ordered structure lacking mobility and a completely fluid, non-viscous liquid lacking structural organization and mechanical support • Molecules can come together, carry out a necessary reaction, and move apart • Membranes arise from preexisting membranes, and their growth occurs by the insertion of lipids and proteins into the fluid matrix • Cellular processes, including cell movement, cell growth, cell division, formation of intercellular junctions, secretion, and endocytosis, depend on the movement of membrane components and would probably not be possible if membranes were rigid, non-fluid structures Membrane Lipids and Membrane Fluidity Maintaining Membrane Fluidity • Internal temperatures of most organisms fluctuate with the external temperature, so cells respond by altering phospholipid composition. If the temperature is lowered, cells can remodel membranes to make them more cold resistant (i.e. keep them fluid at colder temperatures) • Remodeling is accomplished by: – (1) desaturating single bonds in fatty acyl chains to form double bonds, and – (2) reshuffling chains between different phospholipid molecules to make ones that have two unsaturated fatty acids • Desaturation is catalyzed by desaturases • Reshuffling is accomplished by phospholipases, which split the fatty acid from the glycerol backbone, and acyl- transferases, which transfer fatty acids between phospholipids • In addition, the cell changes the types of phospholipids being synthesized in favor of ones containing more unsaturated fatty acids at colder temperatures The Dynamic Nature of the Plasma Membrane • A phospholipid can move laterally within the same leaflet with considerable ease – A phospholipid can diffuse from one end of a bacterium to the other end in ~ 1 sec., but it takes hours to days to move across to the other leaflet • To flip-flop, the hydrophilic head of the lipid must pass through the internal hydrophobic sheet of the membrane  not too favourable! • Flippases are enzymes that move certain phospholipids from one leaflet to the other • Visualizing the fact that membrane proteins can move became the cornerstone of the “fluid mosaic model” Fig 4.25: The possible movements of phospholipids in a membrane The Dynamic Nature of the Plasma Membrane Q) Are proteins stagnant or mobile in the plasma membrane? The Dynamic Nature of the Plasma Membrane The Diffusion of Membrane Proteins after Cell Fusion Cell fusion to reveal mobility of membrane proteins: fusion of human and mouse cells • Cell fusion is a technique whereby two different types of cells, or cells from two different species, can be fused to produce one cell with a common cytoplasm and a single, continuous plasma membrane – • Fig 4.26: Cell fusion be induced by certain viruses, electric shocks, or with polyethylene glycol Labeled proteins have shown that membrane proteins can move between fused cell The Dynamic Nature of the Plasma Membrane Restrictions on Protein and Lipid Mobility • Proteins can be labeled and tracked by fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) • If the labelled proteins are mobile, then random movements of these molecules should produce a gradual reappearance of fluorescence (rate) • The extent of recovery provides a measure of the percentage of molecules that are free to move • It turns out: Measuring the diffusion rates of membrane proteins by FRAP: variable nature of fluorescence recovery is dependent upon the protein examined – 1) in live cells, movement is slow; – 2) only 30-70 % are mobile In single‐particle tracking (SPT) , individual membrane protein molecules are labeled, usually with fluorescent molecular tags that emit light under a microscope Fig 4.27: The Dynamic Nature of the Plasma Membrane Control of Membrane Protein Mobility • Proteins can be: – A: Randomly mobile (A), – B: Immobile- because of underlying skeleton – C: Mobile in a directed manner as a result of interaction with other proteins • Protein movements are limited by interactions with: – D: Interaction with other integral proteins – E: The cytoskeleton (restricted by fences formed by proteins of the membrane skeleton – F: Restricted by extracellular materials Fig 4.28: Patterns of movement of integral membrane proteins The Dynamic Nature of the Plasma Membrane Q) Do lipids move freely or their diffusion is restricted like proteins? The Dynamic Nature of the Plasma Membrane Membrane Lipid Mobility • Just like membrane proteins, phospholipid diffusion is also restricted within the bilayer (even though they are smaller) • Phospholipids are confined for very brief periods to certain areas and then hop from one confined (by “fences”) area to another • Fences restricting motion are constructed of rows of integral membrane proteins bound to the membrane skeleton by their cytoplasmic domains Fig 4.29: Experimental demonstration that diffusion of phospholipids within the plasma membrane is confined The Dynamic Nature of the Plasma Membrane Membrane Domains and Cell Polarity • Most membranes vary in protein composition and mobility (i.e. they do not have a homogenous plasma membrane) • Example: Epithelial cells of the intestinal wall or kidney tubules are highly polarized whose surfaces carry out different functions – The apical plasma membrane absorbs substances from the lumen and possesses different enzymes – The lateral plasma membrane interacts with neighboring epithelial cells – The basal membrane adheres to an underlying basement membrane Fig 4.30: Differentiated functions of the plasma membrane of an epithelial cell. The Dynamic Nature of the Plasma Membrane SEM of human erythrocytes and membrane ghosts SDS–PAGE of membrane proteins Fig 4.32: • The Red Blood Cell: An Example of Plasma Membrane Structure – Homogeneous preparation of membrane “ghosts” can be prepared by hemolysis. – Membrane proteins can be purified and characterized by fractionation using SDS-PAGE electrophoresis. © 2013 John Wiley & Sons, Inc. All rights reserved. Review The physical state of membrane lipids can be described by their fluidity (or viscosity) Factors affecting membrane fluidity: Saturated chains versus unsaturated chains The greater the Degree of unsaturation of the fatty acids of the bilayer, the lower the temperature before the bilayer gels The shorter the fatty acyl chains, the lower its melting temperature. Cholesterol abolishes sharp transition temperatures and creates a condition of intermediate fluidity: dampens temperature effects Less hydrocarbons- more fluidity More double bonds-more fluidity Membrane Fluidity Maintained by (Remodeling) Desaturating bonds (- to = bonds) by reshuffling enzymeschains between different phospholipid molecule (desaturases, phospholipases and acyl-transferases) Flippases are enzymes that move certain phospholipids from one leaflet to the other Are proteins mobile? Cell fusion experiment How can we label and track proteins? Fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) How are proteins located in membrane? a) Randomly mobile b) Immobile c) Mobile in a directed manner d) Interaction with integral proteins e) Cytoskeleton (restriction by fences) f) Restricted by extracellular materials Solute Movement Across Cell Membranes There are five different ways by which substances cross cell membranes: 1. 2. 3. 4. 5. Simple diffusion through the lipid bilayer Simple diffusion through an aqueous channel Diffusion of ions through ion channels Facilitated diffusion (binding is involved) Active transport 1.Simple Diffusion Through the Lipid Bilayer • Diffusion requires both a concentration gradient and membrane permeability • Lipid permeability is determined by the molecular size and polarity of a solute Q) How we measure the polarity?  Polarity is quantified by the partition coefficient, or the ratio of solubility in a nonpolar solvent to that in water. It shows the ability of a drug to cross the cell membrane • Small molecules penetrate the lipid bilayer more rapidly than larger ones • Polar molecules, like sugars and amino acids, have poor membrane penetration • The greater the lipid solubility, the faster the penetration Fig 4.34: The relationship between partition coefficient and membrane permeability. Questions Q) Which substance has more permeability? Sucrose or Caffeine? Caffeine? Q) How polar molecules (sugar and amino acids ) cross the cell membrane if they have poor permeability? They have special mechanism that mediates their entry into the cells Q) Molecule S1 and S2 have same partition coefficient but S2 is little smaller than S1. Based on this information which one you think is able to penetrate the lipid bilayer more rapidly? Why? S1 because it is smaller 2.Diffusion Through the Lipid Bilayer via Aqueous Channels Fig 4.35: The effects of differences in the concentration of solutes on opposite sides of the plasma membrane • Diffusion of water through a semipermeable membrane is called osmosis where water diffuses from areas of lower solute concentration to areas of higher solute concentration • Cells swell in hypotonic solution, shrink in hypertonic solutions, and remain unchanged in isotonic solutions. 2. Diffusion Through the Lipid Bilayer via Aqueous Channels • Plants utilize osmosis in different ways as they are usually hypertonic compared to their fluid environment • There is a tendency for water to enter the cell, causing it to develop an internal (turgor) pressure that pushes against its surrounding wall • In hypertonic solutions the plant cell undergoes plasmolysis, and the plant loses its support and wilts • A family of small integral proteins, aquaporins, allow the passive movement of water across the plasma membrane in plants and animals  It’s central channel is lined primarily by hydrophobic amino Fig 4.36: The effects of osmosis on a plant cell acid residues and is highly specific for water molecules  Aquaporins are prominent in kidney tubules or plant roots https://www.science.org/doi/10.1126/science.aao2440 Aquaporins in kidney https://www.researchgate.net/figure_fig2_303416704 3.The Diffusion of Ions through Membranes • Ions cross membranes through ion channels – Ion channels are selective and bidirectional (two directions), allowing diffusion in the direction of the electrochemical gradient • Superfamilies of ion channels have been characterized by patch-clamping experiments • The voltage across the membrane can be maintained (clamped) at any value, and the current originating in the small patch of membrane surrounded by the pipette can be measured • Fig: shows a glass micropipette is enclosing a patch of the membrane containing a single ion channel. Pipette is wired as an electrode (a microelectrode), a voltage Fig 4.38: can be applied across the patch of the membrane enclosed by the pipette and the responding flow of ions through the membrane channel can be measured. Measuring ion conductance by patchclamp recording https://www.youtube.com/watch?v=mVbkSD5FHOw Revision Is water transport active or passive? –why? Passive- concentration gradients What kind of proteins are involved in water transport and found in the PM? Aquaporins – an integral protein Ability of a drug to cross the cell membrane is called? Partition coefficient 3. The Diffusion of Ions through Membranes • Most ion channels can exist in either an open or a closed conformation, and are called gated. The three major categories of gated channels are: • 1. Voltage‐gated channels: Conformational state depends on the difference in ionic charge on the two sides of the membrane. (e.g, local anesthesia) https://www.youtube.com/watch?v=wLSSf2NRbqk • 2. Ligand‐gated channels: Conformational state depends on the binding of a specific molecule (ligand), which is usually not the solute that passes through the channel. Ligand-gated channels can either open or close after ligand binding to the outer or inner surface of the channel. (e.g, General anesthesia, GABA, nicotine) • 3. Mechano‐gated channels: Conformational state depends on mechanical forces (e.g., stretch tension) that are applied to the membrane. E.g., Specific cation channels can be opened by stereocilia movement on the hair cells of the inner ear in response to sound or motions of the head. Fig: https://www.news-medical.net/health/Importance-of-Ion-Channels-in-the-Body.aspx What is the practical application of ion channel in medicine? Procaine and Novocaine (sodium channel blocker and act by closing ion channels in the membrane of sensory cells and neurons) Closing of theses ions channels results in inhibition of action potential to the affected cells (Skin or teeth) and brain will not get the information of the stimululs. 4. Facilitated Diffusion • In many cases, the diffusing substance binds selectively to a membrane-spanning protein, called a facilitative transporter. • Solute binding triggers a conformational change to expose it to the other membrane surface and diffuse down its concentration gradient. • Facilitated transporters can mediate the movement of solutes in both directions and depends on the relative concentration of the substance on both sides. • Facilitated diffusion is similar to an enzymecatalyzed reaction since it is specific for the molecules transported. Fig 4.44: Schematic model of facilitated diffusion. The alternating conformation of a carrier that exposes the glucose binding site to either the inside or the outside of the membrane 4. Facilitated Diffusion • Transporters can be regulated and exhibit saturation-type kinetics • It is important in transporting polar solutes, like sugars and amino acids, that do not penetrate the lipid bilayer • The glucose transporter (GLUT) is an example of facilitated diffusion: – When insulin levels are low, responsive cells contain relatively few glucose transporters (GLUT), GLUT4 (isoform of GLUT), on their plasma membrane Fig 4.45: Kinetics of facilitated diffusion compared to simple diffusion – Rising insulin levels stimulates the movement of transporters to the cell surface where they can bring glucose into the cell Fig 15.26:Regulation of glucose uptake 5. Active Transport • Cells maintain an imbalance of ions across the plasma membrane, which cannot occur by either simple or facilitated diffusion – Equilibrium means death for cells! • Gradients are generated by active transport, which depends on integral membrane protein “pumps” to bind a solute and move it across the membrane in a process driven by changes in the protein’s conformation • Coupled energy input is needed like ATP hydrolysis, absorbance of light, electron transport, or the flow of other substances down their gradients. Revision: Solute Movement Across Cell Membranes 1. Simple diffusion through the lipid bilayer- partition coefficient 2. Simple diffusion through an aqueous channel- aquaporins 3. Diffusion of ions through ion channels-voltage, ligand and mechano-gated channels 4. Facilitated diffusion (binding is involved)- GLUT4 and glucose transport 5. Active transport – gradient and coupled with energy Lipid permeability is determined by the molecular size and polarity 5. Active Transport Primary Active Transport: Coupling Transport to ATP Hydrolysis Fig 4.46: Steps Explanation 1. 2. 3. 4. 5. In step 1, the ATP pump is in E1 conformation and allows Na+ to binds inside of the cell. This leads to bind three Na+ ions and an ATP. Protein gate closes so Na+ ions can no longer flow back into the cytosol. Hydrolysis of ATP causes the change in conformation from E1 to E2 and this results in binding site more accessible to the ions in the extracellular compartment. Once the three Na+ ions are released the protein picks up two K+ ions. Binding of Potassium to the protein and dephosphorylation causes the protein to snap back to its original conformation and allow potassium ions to diffuse into the cells. 5. Active Transport Primary Active Transport: Coupling Transport to ATP Hydrolysis Fig 4.46: • The Na+/K+ ATPase (sodium-potassium pump) maintains a gradient with a ratio of Na+:K+ pumped is 3:2 per ATP molecule that is hydrolyzed • This excess K+ inside the cells is balanced by negative charges of proteins and nucleic acids; whereas the Na+ excess outside the cell is balanced by Cl- ions • The ATPase is a P-type pump, in which phosphorylation causes changes in conformation and ion affinity that allow transport against gradients: – E1: conformation: Ion binding sites are accessible to the inside of the cell – E2: Ion binding sites are accessible to the outside of the cell • • The sodium–potassium pump is found only in animal cells EX: Digitalis drug (Digoxin) is used to treat certain heart conditions such as strengthen the heart’s contraction by inhibiting the Na+/K pump (obtain from dried leaves of Foxglove plant)-binds to allosteric site of ATPase (inhibits Na+/K+ pump) leads to a chain of events that increases Ca+ availability inside the muscle cells of the heart and leads to increase in cardiac concentration . https://www.youtube.com/watch?v=ljm1JSubTOc Foxglove plant The Human Perspective Defects in Ion Channels and Transporters as a Cause of Inherited Disease Defects in ion Channels Quick Review Quick Review Q) What is p pump? Driven by phosphorylation Q) What is NOT true about Digitalis? a) It strengthens the heart’s contraction by inhibiting the Na+/K+ pumps b) It is steroid and obtained from the foxglove plant and has been used for 200 years as the treatment for heart disease c) The use of Digitalis in turns increases the Ca+2 availability inside the muscle cells of the heart d) It is steroid and obtained from the foxglove plant and has been used for 200 years as the treatment for sickle cell anemia 5. Active Transport Primary vs Secondary Active Transport • In each of the previous cases, chemical energy, in the form of ATP hydrolysis, is used to transport ions or small molecules and is called primary active transport (Remember- this transport occurs by the EXPENSE OF ATP) • If the generated electrochemical gradient is utilized to drive the formation of a gradient for another solute (ion or molecule), then this would be called secondary active transport (Remember- this transport not use ATP- instead gradient generated by the EXPENSE OF ATP is used to move the substance) In Secondary active transport substance can be moved in the same direction or opposite direction https://d2gne97vdumgn3.cloudfront.net/api/file/TZV6mBX1TImdMRlUgcpa Secondary Active Transport: Substance move from lower conc. to the higher conc. because of the gradient generated by another solute 5. Active Transport Co-Transport: Coupling Transport to Existing Ion Gradients • Secondary active transport of glucose is an example of symport, two transported species moving in the same direction • Antiporters, or exchangers, move two transported species in opposite directions • For example, cells can maintain a proper cytoplasmic pH by coupling the inward movement of Na+ with the outward movement of H+ http://previews.123rf.com/images/extender01/extender011507/extender01150700003/42176187Symport-and-antiport-types-of-cell-membrane-transport-systems-Stock-Vector.jpg Fig 4.49: Secondary transporter: the Na+ gradient helps to transport glucose by a Na+/glucose cotransporter 5. Active Transport Secondary Active Transport (or Co-Transport): Coupling Transport to Existing Ion Gradients • Potential energy stored in ionic gradients is utilized to perform work, including the transport of other solutes. • The movement of glucose across the apical plasma membrane of the epithelial cells, against a concentration gradient, occurs by cotransport with sodium ions – Na+ concentration is kept low by a Na+/K+ATPase pump. – Diffusion of sodium ions down a concentration gradient drives the cotransport of glucose molecules into the cell against a concentration gradient (moves 2 Na ions and one glucose) • Once inside, the glucose molecules diffuse through the cell and are moved across the basal membrane by facilitated diffusion Fig 4.49: Secondary transporter: the Na+ gradient helps to transport glucose by a Na+/glucose cotransporter Membrane Potentials • Irritability: response to external stimulation; all organisms have it! • The basis of this is the propagation of nerve impulses • Potential differences exist when charges are separated, and membrane potentials have been measured in all types of cells Fig 4.51: The structure of a nerve cell  Neurons are specialized cells for information transmission using changes in membrane potentials  Dendrites receive incoming information; the cell body contains the nucleus and metabolic center of the cell; the axon is a long extension for conducting outgoing impulses  Most neurons are wrapped by a lipid-rich myelin-sheath  The place where no myelin sheath is called Node of Ranvier and that is the site where action potential is generated- Why?  (Uninsulated and rich in ion channels) Membrane Potentials The Resting Potential • The resting potential is the membrane potential of a nerve or muscle cell, subject to changes when activated. • K+ gradients maintained by the Na+/K+ATPase are responsible for the resting potential. • The Nernst equation is used to calculate the voltage equivalent of the concentration gradients for specific ions. • In Fig 4.52 A- when both electrodes are on the outside of the cells- no potential difference is measured. • In Fig 4.52 B- When one electrode penetrates the PM the potential difference drops to -70 mV and approaches the equilibrium potential for potassium ions Fig 4.52: Measuring a membrane’s resting potential Membrane Potentials The Action Potential • When cells are stimulated, Na+ channels open, causing membrane depolarization • When cells are stimulated, voltagegated Na+ channels open, triggering the action potential • Na+ channels are inactivated immediately following an AP, producing a short refractory period when the membrane cannot be stimulated It is a period in which a nerve cell is unable to fire an action potential. • • Excitable membranes exhibit all-ornone behavior Fig 4.53: Formation of an action potential refractory period Membrane Potentials Explanation: The Action Potential LEAP: Less negative Excitation (Depolarization) Action Potential Time 1, left box: The membrane in this region of the nerve cell exhibits the resting potential, in which only the K + leak channels are open and the membrane voltage is approximately −70 mV. Time 2, middle box: The depolarization phase: The membrane has depolarized beyond the threshold value, opening the voltage‐regulated sodium gates, leading to an influx of Na + ions. The increased Na + permeability causes the membrane voltage to temporarily reverse itself, reaching a value of approximately +40 mV. Time 3, right box: the repolarization phase: Within a tiny fraction of a second, the sodium gates are inactivated and the potassium gates open, allowing potassium ions to diffuse across the membrane and establish an even more negative potential at that location (−80 mV) than that of the resting potential. Propagation of Action Potentials as an Impulse Saltatory conduction: Propagation of an impulse by forming an action potential only at the nodes of Ranvier Speed Is of the Essence: Speed of neural impulse depends on axon diameter (the greater the diameter the lower the resistance and more rapid action potential) and whether the axon is myelinated. Nearly all of the Na+ ion channels of a myelinated neuron reside in the unwrapped gaps, or nodes of Ranvier, between adjacent Schwann cells or oligodendrocytes that make up the myelin sheath. The nodes of Ranvier are the only sites where action potentials can be generated, jumping from node to node. Fig 4.55: Saltatory conduction Jumping of impulse from node to node is called saltatory conduction. (impulse travel in myelinated axon is 20x more faster than the unmyelinated axon). Multiple sclerosis (MS) is a disease associated with deterioration of the myelin sheath that surrounds axons in various parts of the nervous system- start in young adulthood and patient has difficulty in walking, weakness in hands and vision problems : Propagation of an impulse by forming an action potential only at the nodes of Ranvier Let’s Label the Diagram (Check Test) Depolarization Action potential Repolarization Refractory Period Nodes of Ranvier Myelin Propagation of Action Potentials as an Impulse APs produce local membrane currents depolarizing adjacent membrane regions of the membrane that propagate as a nerve impulse. The large depolarization from an action potential creates a difference in charge along the inner and outer surfaces of the plasma membrane. Once triggered, a succession of action potentials passes down the entire length of the neuron without any loss of intensity, arriving at its target cell with the same strength it had at its point of origin. Q) Do you think stronger stimuli will produce a bigger impulse? All impulses traveling along a neuron exhibit the same strength, so stronger stimuli cannot produce bigger impulses, however the strength of stimuli can make a difference can active more nerve cells (scalding, hot, water versus warm water) Fig 4.54: Propagation of an impulse results from the local flow of ions unidirectionally Neurotransmission: Jumping the Synaptic Cleft Presynaptic neurons communicate with postsynaptic neurons at a specialized junction, called the synapse, across the synaptic cleft (a narrow gap of about 20 to 50nm) . Chemicals (neurotransmitters) released from the presynaptic cleft diffuse to receptors on the postsynaptic cell. Bound transmitter can depolarize (excite) or hyperpolarize (inhibit) the postsynaptic cell. Q) How neurotransmitter maintains in our body? Transmitter action is terminated by reuptake or enzymatic breakdown. Fig 4.56: The neuromuscular junction Neurotransmission: Jumping the Synaptic Cleft Neurotransmission: Jumping the Synaptic Cleft Sequence of events during synaptic transmission with acetylcholine as the neurotransmitter Fig 4.57: • Depolarization of pre-synaptic cell causes Ca2+ channels in membrane to open, Ca2+ stimulates fusion of vesicles with membrane. • Calcium ions diffuse from extracellular fluid into the terminal knobs of the neuron and elevated calcium triggers the fusion of synaptic vesicles to the plasma membrane. • Neurotransmitter bind to the receptor in the postsynaptic plasma membrane • Neurotransmitter binding to ion channel receptors can either stimulate or inhibit action potential (cation-selective channels –more positive membrane potential, anion-selective channels –more negative membrane potential) Action of Drugs on Synapses • A neurotransmitter (NT) can be eliminated by two ways: enzyme that destroy NT in the synaptic cleft and NT reuptake process • Milder inhibitors of acetylcholinesterase (enzyme that hydrolyze acetylcholine) are used to treat the symptoms of Alzheimer’s disease, which is characterized by the loss of acetylcholine-releasing neurons. • Many drugs act by inhibiting the transporters that sweep neurotransmitters out of the synaptic cleft such as antidepressants. • Inhibiting the reuptake of serotonin, mood disorders can be treated. • Cocaine, interferes with the reuptake of the dopamine in the synaptic cleft of the limbic system and produces a short-lived feeling of euphoria and a strong desire to repeat the activity. Q1) Do neurotransmitters have always stimulatory effects? Q2) Can Neurotransmitter may induce different effect (Stimulatory and inhibitory effect) by binding to postsynaptic receptors? Q3) Do neurotransmitter always stay in the body? What happened to them? Summary of Chapter 4 - Quick Check or Can you answer? • What is Plasma membrane (trilaminar structure) and its function • Plasma membrane (PM) is dynamic in nature; the ratio of lipid to protein varies in type of organism and cells (e.g inner mitochondrial versus myelin sheath) • Membrane lipids classification -Phospohoglycerides (head and tail) -Sphingolipids (derivatives of sphingosine, an amino alcohol with a long hydrocarbon chain) –classification like ceramide (R), Sphingomyelin (Ceramide+Phosphatidylcholine), Cereboside (Galactose), Ganglioside (sugar and sialic acid) Main examples glycolipid and ABO blood group) -Cholesterol • Liposome – what are they and how they work- use to deliver drugs or DNA within the body • Are the membrane lipids same on both sides of the membrane- Asymmetry of the membrane lipids by experiment Summary of Chapter 4 - Quick Check or Can you answer? • Membrane proteins have different affinities and interaction with the lipid bilayer -Integral (receptors, channels, agents for electron transport)- can identify transmembrane segments by using a hydropathy plot - Peripheral (Mechanical support for membranes, Enzymes and factors that transmit signals) -Lipid-anchored (adhesion molecules and enzymes) • Membrane fluidity and how it maintain with the temperature change by remodelling enzymes such as desaturases, phospholipases and acyltransferases) • Phospholipids move within the membrane by enzymes (Flippases) • Diffusion of membrane proteins after cell fusion (experiment mouse and human cell fusion) • -membrane and lipid mobility in the PM (experiment) Summary of Chapter 4 - Quick Check or Can you answer?  What is the lipid composition of membranes?  How many kinds of membrane lipids are there? Can you identify the differences between them? Where are they found and their importance in a living system?  Why Cholesterol is an important molecule? How it plays a role in membrane fluidity?  What is liposome, how it forms and its importance in therapeutics?  How do you know that plasma membrane is dynamic in nature? Explain with cell fusion experiment.  Can proteins move? If yes, how and in which directions?  Can lipids move in the membrane? How do you know? Summary of Chapter 4 - Quick Check or Can you answer?  Give an example of heterogeneous proteins in a plasma membrane.  Can you identify the ways by which solute cross cell membranes?  What is the relationship between partition coefficient and membrane permeability?  What are different kinds of ion channels? Which one is important for therapeutic point of view?     Differentiate between Active and passive transport Primary and Secondary active transport Symport and Antiport  What is membrane potential? How it generates?  What is Node of Ranvier and Saltatory conduction?  What is neurotransmitter? How they acts and action of drugs on synapses