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Biomembrane Structure and Function Dr Sherline Brown Learning Objectives Describe the structural relationships of the components of the membrane and general functional roles served by each of them Describe the processes by which small solutes, ions and macromolecules cross biomembranes Describe various membrane transport pumps including their energy source, and functional significance Biomembrane structure Cell (plasma membrane): defines cell boundaries Internal membranes define a variety of cell organelles -Nucleus -Mitchondria -Endoplasmic reticulum -Golgi apparatus -Lysosomes -Peroxisomes -Chloroplasts Biomembranes Surrounds cell Separates cell from environment – Allows cellular specialization Separate some of the cellular organelles – Allows specialization within the cell Continuity of membranes between adjoining cells (tight junctions) can separate two extracellular compartments – Important in organ function Features of a biomembrane Sheet like structures Consists mainly of lipids and proteins Lipids have hydrophilic and hydrophobic moieties Specific proteins mediate specific functions of the membrane (pumps, channels, receptors etc) Features of a biomembrane Membranes are non-covalent entities Membranes are asymmetric Membranes are fluid structures Electrically polarized (plays a role in transport) Composition of a biomembrane Phospholipid bilayer (basic structure) Various membrane protein (depending on membrane function) Glycolipids and glycoproteins (proteins and lipids attached to carbohydrate) Cholesterol (in animal cells) Membrane lipids There are 5 general types of membrane lipids Glycerophospholipids :hydrophobic region consists of fatty acids joined to glycerol Glycolipids & sulfolipids :fatty acids esterified to glycerol, lack phosphate Membrane lipids Tetraether lipids : (archae) 2 long alkyl chains linked to gylcerol at both ends Sphingolipids: single fatty acid joined to fatty amine Sterols /cholesterol: 4 fused hydrocarbon rings Membrane Lipids Phospholipids -Major lipid component of most biomembranes -Amphipathic (hydrophobic and hydrophilic) -Examples phosphatidyl-choline P-serine P-ethanolamine P-inositol Phospholipid bilayer Membrane lipids Galactolipids and Sulfolipids Galactolipids Two galactose residues are joined to diacylglycerol Predominate in plant cells Located in thylokoid membranes of chloroplast Make up 70-80% of the total lipids of vascular plants They are the most abundant membrane lipids in the biosphere Membrane lipids Galactolipids and Sulfolipids Sulfolipids Found in plant membranes Sulfonated glucose residue is joined to diacylglycerol Membrane lipids Sphingolipids They are derivatives of the lipid sphingosine Sphingosine has a long hydrocarbon tail, and a polar domain that includes an amino group . Membrane lipids Sphingolipids They differ from phospholipids and galactolipids in that they contain no glycerol. Membrane lipids Sphingolipids Sphingosine may be reversibly phosphorylated to produce the signal molecule sphingosine-1phosphate. Other derivatives of sphingosine are commonly found as constituents of biological membranes Membrane lipids Sphingolipids The amino group of sphingosine can form an amide bond with a fatty acid carboxyl, to yield a ceramide. Membrane lipids Sphingolipids There are three main types of sphingolipids 1. Sphingomyelin has a phosphocholine or phosphoethanolamine head group. 2. Sphingomyelins are common constituents of plasma membranes of animal cells Membrane lipids Sphingolipids Membrane lipids Sphingolipids 2. Glycosphingolipids (outer face of plasma membrane) Two types: Cerebrosides (single sugar linked to ceramide) Globosides (2 or more sugars linked to ceramide) They are nuetral glycolipids Membrane lipids Sphingolipids Glycosphingolipids Membrane lipids Sphingolipids 3. Gangliosides is a ceramide with a polar head group that is a complex oligosaccharide, including the acidic sugar derivative sialic acid Ganglosides are negatively charged Functions of Sphingolipids Protection of the cell surfaces against environmental factors Cell Signaling Cell recognition Determination of human blood groups (glycosphingolipids) Membrane lipid Cholesterol -Steriod; lipid soluble -Found in both leaflets of bilayer -Amphipathic -Found in animal cells -Membrane fluidity buffer -Synthesized in membranes of ER Membrane proteins Two types of membrane protein A. Integral protein (interacts extensively with hydrocarbon chains of membrane lipids) B. Peripheral membrane proteins (bound to membranes primarily by electrostatic and hydrogen bond interactions) Membrane proteins Integral Proteins Penetrate bilayer or span membrane Can only be removed by disrupting bilayer Membrane proteins Integral Proteins Types Transmembrane proteins – Single-pass or Multiple-pass Covalently tethered integral proteins Membrane proteins Integral Proteins Many are glycoproteins Covalently-linked via asparagine, serine, or threonine to sugars – Synthesized in rough ER – Function: enzymatic, receptors, transport, communication, adhesion Membrane proteins Integral Proteins Membrane proteins Types I and II (1 transmembrane helix) Type III (multiple transmembrane helices in a single polypeptide) Type IV (transmembrane helices in many polypeptides forms channels Membrane proteins Type V (held by covalent bonds to the lipids) Type VI (transmembrane helices and lipid anchors) Membrane proteins Peripheral (extrinsic) proteins – Do not penetrate bilayer – Not covalently linked to other membrane components Form ionic links to membrane structures • Can be dissociated from membranes • Dissociation does not disrupt membrane integrity – Located both extracellular and intracellular sides of membrane Synthesis • Cytoplasmic (inner) side – cytoplasm • Extracellular (outer) side – made in ER and exocytosed Membrane proteins Lateral mobility Biomembranes are a two-dimensional “mosaic” of lipids and proteins Most membrane lipids and protein can move through the membrane plane within limits Fluid Mosaic Model Membranes are two-dimensional solutions of oriented lipids and globular proteins The lipid bi-layer acts as solvent for integral membrane proteins and a permeability barrier Membrane lipids: supporting structure – Phospholipids – Glycolipids – Cholesterol Fluid mosaic model Membrane proteins: – Integral (intrinsic) proteins – Peripheral (extrinsic) proteins Membrane fluidity Many membrane processes depend on membrane fluidity -transport -signal transduction Membrane fluidity is dependent on the properties of the fatty acid chain Transition temperature is dependent on the length of the fatty acid chains and on their degree of unsaturation Membrane fluidity Movement of hydrophobic tails Depends on temperature and lipid composition How does lipid composition affect fluidity? Lipids and membrane fluidity Interactions between hydrophobic tails decrease fluidity (movement): – Shorter tails have fewer interactions – Unsaturated fatty acids are kinked and decrease interactions Lipids and membrane fluidity Cholesterol “buffers” fluidity: Prevents interactions Restricts tail movement Microbial growth at Cold temperatures Molecular Adaptation to Psychrophily The cytoplasmic membranes of psychrophiles have a higher content of unsaturated fatty acids. This helps to maintain a semifluid state of the membrane at low temperatures Microbial growth at Cold temperatures Molecular Adaptation to Psychrophily Lipids of some psychrophiles contain polyunsaturated fatty acids or other long chained hydrocarbons with multiple bonds These fatty acids remain more flexible at lower temperatures than saturated or monounsaturated fatty acids Microbial Growth at High Temperature Molecular Adaptations to Thermophily – Modifications in cytoplasmic membranes to ensure heat stability Bacteria have lipids rich in saturated fatty acids Archaea have lipid monolayer rather than bilayer Microbial Growth at High Temperature Archaea have lipid monolayer rather than bilayer Lipid monolayers are quite resistant to peeling apart When the lipid layers peel apart they cause cell lysis Membrane asymmetry The inner and outer leaflets of the membrane have different compositions of lipids and proteins Membrane asymmetry Sphingomyelin and phophatidyl choline are located on the outer leaflet Phosphatdidylserine is located in the inner leaflet Biomembrane Cell to cell interactions and adhesions • Integrins are transmembrane proteins of the plasma membrane • They act to attach cells to each other • They carry message between the extracellular matrix and the cytoplasm (extracellular matrix Biomembrane Cell to cell interactions and adhesions • Integrins regulate many processes - platelet aggregation at the site of a wound - tissue repair -activity of immune cells -invasion of tissue by a tumor Mutation can result in leukocyte adhesion Biomembrane Cell to cell interactions and adhesions • Other plasma membrane proteins involved in surface adhesions: • Cadherins • Immunoglobin-like proteins • Selectins: essential part of the blood clotting process Biomembrane Membrane fusion and biological processes Integral proteins (fusion proteins) facilitate this event Membrane continuity is maintained Entry into host cell by viruses Fusion of sperm and egg Release of neurotoxins by exocitosis Membrane carbohydrates Membranes play key role in cell-cell recognition Carbohydrates are usually branched oligosaccharides with fewer than 15 sugar units Membrane carbohydrates Oligosaccharides on external of membranes are different among species, or individuals, or cells Membrane functions Cell communication and signalling Cell-cell adhesion and cellular attachment Cell identity and antigenicity Conductivity Transport of Ions and Small Molecules Across Cell Membrane Membrane transport All cells require the molecules and ions they need from ECF (extracellular fluid). There are two problems to be considered Relative concentrations -diffusion -active transport 2. Lipid bilayers are impermeable to most essential molecules and ions 1. Membrane transport Solving the Problem Mechanisms by which cells solve this problem include: 1. Active transport 2. Facilitated diffusion Active Transport Active transport is the pumping of molecules or ions through a membrane against their concentration gradient. It requires a transmembrane protein (a complex of them) called a transporter Energy. ATP (source) Active Transport Active transport enzymes couple net solute movement across a membrane to ATP hydrolysis. Active Transport The energy of ATP may be used directly or indirectly There are two types of active transport Direct / Primary Indirect/Secondary Active Transport Primary /Direct – The transport system is an ATPase. – The energy for transport comes directly from ATP. – Some cation transport systems fall into this category. The NaK-pump is the prime example. Active Transport Secondary/Indirect – – The transport system utilizes the Na+ electrochemical gradient as an energy source to move a solute against its electrochemical gradient. Na+ is transported down its electrochemical gradient in the process. This is also referred to as a Na-coupled or gradient-coupled transport. Active Transport Indirect Active Transport. Transporters use energy already stored in the gradient of a directly pumped ion. Membrane Transport Transporters are of two general classes: carriers and channels. These are exemplified by two ionophores (ion carriers produced by microorganisms): valinomycin (a carrier) gramicidin (a channel). Energetics of active transport Active transport – Metabolic energy expenditure is required. – Solute moves against a gradient of electrochemical potential. – Assymetrical Km for carrier loading. Km is generally higher on that side of the membrane toward which active transport occurs. Carrier mediated membrane transport Carriers exhibit saturation kinetics with respect to solute concentration. Carriers exhibit stereospecificity. – Glucose carrier transports D-glucose but not Lglucose. Carrier mediated membrane transport Carriers are susceptible to inhibition. Carrier rates are susceptible to hormonal control (although channels may be as well). Influence of insulin on the glucose transporter Influence of aldosterone on the Na-K transporter (NaK-pump). – Kinetics of transport carriers Carriers exhibit Michaelis-Menten kinetics. The transport rate mediated by carriers is faster than in the absence of a catalyst, but slower than with channels. A carrier transports only one or few solute molecules per conformational cycle. Energetics of carrier-mediated transport Diffusion Passive transport (facilitated diffusion) – – – – No metabolic energy required. Solute moves down a gradient of electrochemical potential in combination with a carrier. Km is the same on the two sides of membrane. Example - glucose transport in most cells. Carrier proteins Proteins that act as carriers are too large to move across the membrane. They are transmembrane proteins, with fixed topology. Example: GLUT1 glucose carrier, found in plasma membranes of various cells, including erythrocytes. GLUT1 is a large integral protein, predicted via hydropathy plots to have 12 transmembrane ahelices. Carrier proteins conformation change conformation change Carrier-mediated solute transport Carrier proteins cycle between conformations in which a solute binding site is accessible on one side of the membrane or the other. Carrier proteins conformation change conformation change Carrier-mediated solute transport There may be an intermediate conformation in which a bound substrate is inaccessible to either aqueous phase. With carrier proteins, there is never an open channel all the way through the membrane Classes of carrier proteins Uniport A Symport A B Antiport A B Classes of carrier proteins Uniport Uniport (facilitated diffusion) carriers mediate transport of a single solute. Uniport A Symport A B Antiport A Examples include GLUT1 B Classes of carrier proteins Uniport These carriers can undergo the conformational change associated with solute transfer either empty or with bound substrate. Thus they can mediate net solute transport. Uniport A Symport A B Antiport A B Classes of carrier proteins Symport Symport (cotransport) carriers bind 2 dissimilar solutes (substrates) & transport them together across a membrane. Transport of the 2 solutes is obligatorily coupled. Uniport A Symport A B Antiport A B Classes of carrier proteins Symport An example is the plasma membrane glucoseNa+ symport. A gradient of one substrate, usually an ion, may drive uphill (against the gradient) transport of a cosubstrate. Uniport A Symport A B Antiport A B Classes of carrier proteins Symport Trans-epithelial transport: In the example shown, 3 carrier proteins accomplish absorption of glucose & Na+ in the small intestine. glucose Na+ glucose-Na+ symport apical end Na+ glucose ATP ADP + Pi basal end Na+ pump GLUT2 K+ intestinal epithelial cell Classes of carrier proteins Symport . The Na+ pump, at the basal end of the cell, keeps [Na+] lower in the cell than in fluid bathing the apical surface. Na+ glucose-Na+ symport glucose apical end Na+ glucose ATP ADP + Pi basal end Na+ pump GLUT2 K+ intestinal epithelial cell Classes of carrier proteins Symport . •The Na+ gradient drives uphill transport of glucose into the cell at the apical end, via glucose-Na+ symport. [Glucose] within the cell is thus higher than outside. Na+ glucose-Na+ symport glucose apical end Na+ glucose ATP ADP + Pi basal end Na+ pump GLUT2 K+ intestinal epithelial cell Classes of carrier proteins Symport . •Glucose flows passively out of the cell at the basal end, down its gradient, via GLUT2 (uniport related to GLUT1). Na+ glucose-Na+ symport glucose apical end Na+ glucose ATP ADP + Pi basal end Na+ pump GLUT2 K+ intestinal epithelial cell Classes of carrier proteins Antiport Antiport (exchange diffusion) carriers exchange one solute for another across a membrane. Uniport A Symport A B Antiport A B Classes of carrier proteins Antiport Example: ADP/ATP exchanger (adenine nucleotide translocase) which catalyzes 1:1 exchange of ADP for ATP across the inner mitochondrial membrane. Uniport A Symport A B Antiport A B Classes of carrier proteins Antiport Usually antiporters exhibit "ping pong" kinetics. One substrate is transported across a membrane and then another is carried back. Uniport A Symport A B Antiport A B Active Transport ATP dependent ion pumps are grouped into classes, based on transport mechanisms as well as genetic and structural homology. Examples include P-class pumps F-class pumps V-class pumps Active Transport There are four types of Direct Active transport 1. 2. 3. 4. The Na+/K+ ATPase The H+/K+ ATPase The Ca 2+ ATPases The ABC transporters P-Type transporters 1.Na+/K+ ATPase H+/K+ ATPase Ca 2+ ATPase They use the same basic mechanism: Conformational change in proteins as they are reversably phosphorylated by ATP All three pumps can be made to run backwards If the pumped ions are allowed to diffuse back through the membrane complex, ATP can be synthesized from ADP and inorganic phosphate P-Type transporters The reaction mechanism for a P-class ion pump involves transient covalent modification of the enzyme. O Enzyme-C OH ATP Pi ADP H2O O Enzyme- C O O P O- P-Class Pumps O- Direct Active Transport The Na+/K+ ATPase K+ is 20 X higher in cytosol than extracellular fluid Na+ in extracellular fliud is 10X greater than in cytosol Concentration gradient is maintained by active transport of both ions The Na+/K+ ATPase transporter does both jobs Direct Active Transport The Na+/K+ ATPase The Na+/K+ ATPase transporter uses energy from the hydrolysis of ATP to Actively transport 3 Na+ ions out of the cell For each 2 K+ ions pumped into the cell The Na+/K+ ATPase transporter – Na+/K+-ATPase, in plasma membranes of most animal cells, is an antiport pump. Inward 3 Na+ Sodium Flux Extracellular Cytosol Mg++ ATP 2 K+ ADP + Pi Outward Potassium Flux The Na+/K+ ATPase transporter Gradients for the Na+ and K+ is needed for action potentials and synaptic potentials Inward 3 Na+ Sodium Flux Extracellular Cytosol Mg++ ATP 2 K+ ADP + Pi Outward Potassium Flux Direct Active Transport The Na+/K+ ATPase Transporter What does this accomplish It helps to establish a net charge across the plasma membrane The accumulation of sodium ions outside of the cell draws water out of the cell and enables it to maintain osmotic balance. Why is this important? Direct Active Transport The Na+/K+ ATPase Transporter What does this accomplish The gradient of sodium ions is harnessed to provide the energy to run several types of indirect pumps Mechanism Na+/K+ pump • The mechanism is similar to that of the muscle calcium pump • In the E1 conformation the Na+/K+ ATPase has three high affinity Na+ binding site and two low affinity K+ sites accessible on the cytosolic side Mechanism Na+/K+ ATPase pump • In the E1 conformation the enzyme can bind ATP and the Na+ ions occupy its binding site on the enzyme • The phosphoryl group is the transferred to aspartate Mechanism Na+/K+ ATPase pump • The three bound Na+ ions become accessible to the exoplasmic face and simultaneously the affinity for the 3 Na+-binding sites become reduced • The Na+ ions then dissociate one at a time to the Mechanism Na+/K+ ATPase pump • The transition to the E2 conformation generates two high-affinity K+ sites accessible to the exoplasmic face, binding the K+ ions • Upon dephosphorylation the enzyme undergoes another conformational change and releases the The Na+/K+ ATPase transporter Inhibited by : – Cardiac glycosides – Metabolic inhibitors – Heavy Metals Inward 3 Na+ Sodium Flux Extracellular Cytosol Mg++ ATP 2 K+ ADP + Pi Outward Potassium Flux Digitalis inhibits the Na+/K+ Pump Digitalis is a mixture of cardiotonic steroids Digitoxigen and ouabain inhibitors cardiotonic steriods – strong effect on heart Increases the force of contraction of the heart Digitalis inhibits the Na+/K+ Pump Inhibit dephosphorylation of the phosphorylated form of ATPase on the extracellular face of the membrane Leads to higher Na+ in the cytosol Diminished Na+ gradient leads to slower exclusion of Ca 2+ by Na-Ca exchanger (antiporter) Increase in intracellular levels of Ca 2+ enhances the ability of the cardiac muscle to contract Digitalis inhibits the Na+/K+ Pump Inhibititors of the Na+/K+ Pump Oubain (Samali for arrow poison steriod derivative of ouabain) Binds to the form of the Na+K+ ATPase that is open to the extracellular side Locks in Na+ and prevents the change in conformation necessary for transport of ions Inhibitors of the Na+/K+ Pump Palytoxin (produced by coral found in Hawaii) Binds to Na+K+ ATPase and locks it in position so that the ion binding sites are permanently accessible from both ends Open channel Exit of K+ from cells Toxic The H+/K+ ATPase Transporter (H+, K+)-ATPase, involved in acid secretion in the stomach, is an antiport pump. The H+/K+ ATPase Transporter It catalyzes transport of H+ out of the gastric parietal cell (toward the stomach lumen) in exchange for K+ entering the cell. Direct Active Transport The Ca 2+ ATPase Transporter The Ca 2+ ATPase is located in the plasma membrane of all eukaryotic cells 1 ATP is used to pump 1 Ca 2+ out of the cell 20,000 fold conc gradient between Ca 2+ in the cytosol and that in the extracellular fluid (ECF) The Ca 2+ ATPase Transporter Ca 2+ -ATPase pump, in endoplasmic reticulum (ER) & plasma membranes catalyze transport of Ca 2+ away from the cytosol, either into the ER lumen or out of the cell. There is some evidence that H+ may be transported in the opposite direction. – Ca 2+ -ATPase pumps keep cytosolic Ca 2+ low (10-7M vs. 10-3 M in plasma), allowing Ca 2+ to serve as a signal. Direct Active Transport The Ca 2+ ATPase Transporter Resting skeletal muscle there is a higher conc of Ca 2+ ions in the endoplasmic reticulum than the cytosol Activation of muscle fibre allows Ca 2+ to pass into the cytosol, triggering contraction Direct Active Transport The Ca 2+ ATPase Transporter After contraction the Ca 2+ is pumped back into the sarcoplasmic reticulum This is done by another Ca 2+ ATPase pump Uses energy from each molecule of ATP to pump 2 Ca 2+ ions The Ca 2+ pump is called SERCA The Ca 2+ ATPase Transporter • The catalytic cycle begins with the enzyme in its unphosphorylated state with 2 calcium ions bound • In the E1 conformation the enzyme can bind ATP. Conformational change occurs and the The Ca 2+ ATPase Transporter • The phosphoryl group is then transferred from ATP to aspartate • Upon ADP release the enzyme changes its overall conformation (E2-P). This process is called eversion The Ca 2+ ATPase Transporter • In the E2-P conformation the calcium binding sites become disrupted and the calcium ions are released to the side of the membrane opposite to which they entered • E2-P is then hydrolysed releasing the inorganic phosphate The Ca 2+ ATPase Transporter • With the release of the phosphate the stabilization of the E2 form is lost and the enzyme everts back to the E1 conformation • The binding of two calcium ions from the cytosolic side completes the cycle SERCA:Sarco Endo(plasmic) Reticulum Ca 2+ ATPase SERCA:Sarco Endo(plasmic) Reticulum Ca 2+ ATPase Direct Active Transport ABC Transporters ABC (ATP-Binding-Cassette) transporters are transmembrane protein that Expose a ligand-binding domain at one surface and a ATP-binding domain at the other surface The ligand binding domain is restricted to a single type of molecule Direct Active Transport ABC Transporters The ATP bound to its domain provides the energy to pump the ligand across the membrane ABC Transporters Mechanism The catalytic cycle begins with the transporter being free of both ATP and substrate The transporter can interconvert between closed and open forms Substrate enters the central cavity of the open form of the transporter from inside the cell ABC Transporters Mechanism Substrate binding results in a conformational change in the ATP binding cassette that increases their affinity for ATP ATP binds to the ATP-binding cassettes, changing their conformations so that the two domains interact strongly with each other ABC Transporters Mechanism The strong interaction between the ATP-binding cassettes induces a change in the relation between the two domains releasing the substrate to the outside of the cell The hydrolysis of ATP and the release of ADP and inorganic phosphate resets the transporter for another cycle ABC Transporters (Mechanism) Direct Active Transport ABC Transporters The human genome contains 48 genes for ABC transporters. CFTR- the cystic fibrosis transmembrane conductance regulator TAP-the transporter associated with antigen processing ABC transporters that pump chemotherapeutic drugs out of cancer cells Physiological effects of defects in ABC Transporters Genetic diseases such as: Cystic fibrosis Tangier disease Retinal degeneration Anemia Liver failure Effects of ABC Transporters ABC transporters can confer antibiotic resistance in pathogenic microbes such as: Pseudomonas aeruginosa Staphylococcus aureus Candida albicans Neisseria gonorrhoeae