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13 The Plasma Membrane Chapt. 13 Student learning outcomes: • Diagram structure, explain function of lipid bilayer plasma membrane: types of phospholipids, proteins • Explain mechanisms for transport of small molecules: • Passive transport • Active transport • Describe uptake of larger molecules: • Phagocytosis • Receptor-mediated endocytosis Bilayer structure of the plasma membrane Cells are surrounded by plasma membrane: • separates cell from environment • selective barrier, mediates interactions with environment. Fundamental structure is phospholipid bilayer: • Proteins embedded in bilayer carry out functions: Mammalian red blood cells (erythrocytes) good model • no nuclei or internal membranes, • easy to isolate pure plasma membranes. Fig. 13.1 . Polar head groups: dark lines because bind electron-dense metal stains. Hydrophobic fatty acid chains in center are lightly stained Fig 13.2 Lipid components of plasma membrane Bilayers are viscous fluids, not solid Asymmetric plasma membrane phospholipids: (Table 1) • Outer leaflet — phosphatidylcholine (37%), sphingomyelin. • Inner leaflet — phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol. Glycolipids — only in outer leaflet, carbohydrate on surface. Cholesterol — lot • 23% epithelial cell • Affects fluidity Fig. 13.2: note negative charge on PI, PS Figure 13.4 Visualization of lipid rafts in plasma membrane Cholesterol and sphingolipids (sphingomyelin and glycolipids) cluster in small patches (lipid rafts) • Rafts highly-ordered versus phospholipid bilayer. • Sphingolipids - different melting temperatures than phospholipids derived from glycerol. • Visualize lipid rafts with fluorescent probe Laurdan, sensitive to rigidity of phospholipid bilayer (false color: red = ordered). Figs. 13.3,4: Fluid mosaic model of plasma membrane Plasma membrane ~ 50% lipid, 50% protein by weight. • Proteins larger than lipids, → ~1 protein per 50–100 lipids Fluid mosaic model of membrane structure Integral proteins (often transmembrane) Peripheral proteins Distinguish by ease of disruption of protein from membraneIntegral need detergents Some integral anchored behave as peripheral *Fig. 13.5: membrane proteins Solubilization of integral membrane proteins by detergents Integral membrane proteins insert into lipid bilayer; • Dissociate with reagents that disrupt hydrophobic interactions – detergents Detergents - amphipathic (both hydrophobic and hydrophilic): solubilize integral, transmembrane proteins ex. Octyl glucoside, sodium dodecyl sulfate (SDS) Fig. 13.6: detergents solubilize integral membrane proteins Integral membrane proteins of red blood cells Transmembrane proteins: • • • • Membrane-spanning s α helices (~ 20 to 25 hydrophobic aa) Inserted into ER membrane during synthesis (chapt. 10) Carbohydrate groups added in ER and Golgi Most are glycoproteins: oligosaccharides on cell surface Ex. Red blood cell: Glycophorin unknown function (131 aa; 16 chains carbohydrates) Band 3 anion transporter for HCO3 – and Cl– ions (929 aa; dimer). Fig. 13.8: red blood cell transmembrane Bacterial outer membranes Porins: Transmembrane proteins in outer membrane of bacteria such as E. coli. (Gram-negative) • Porins cross membrane as β barrels. • Very permeable to ions, small polar molecules • Porins also in outer membrane of mitochondria and chloroplasts Fig. 13.10: bacterial porins Proteins anchored in plasma membrane by lipids and glycolipids Outer leaflet has proteins anchored by glycosylphosphatidylinositol (GPI) anchors on C terminus. • Proteins glycosylated in ER, Golgi, exposed on cell surface • In lipid rafts Inner leaflet has proteins anchored by covalently attached lipids. • From free ribosomes, modified by myristic acid, prenyl, palmitic acid. • Often roles in signal transmission (ex. Src and Ras) • May behave as peripheral proteins Fig. 13.11: membrane-anchored proteins See also Figs. 10.17, 8.33, 8.36 Mobility of membrane proteins Lateral movement of proteins and lipids in membrane: • First demonstrated in 1970. • Fused human and mouse cells in culture • Analyzed membrane proteins using fluorescent antibodies Some membrane proteins have restricted movement: • • • • bound to cytoskeleton, other membrane proteins proteins on adjacent cells, extracellular matrix. Fig. 13.12: A polarized intestinal epithelial cell EX. Polarized epithelial cell plasma membranes divided into apical and basolateral domains. Small intestine: • apical surface covered by microvilli – increase surface area for absorption. • basolateral surface mediates transfer of nutrients to blood Tight junctions restrict movement of proteins between domains (tighter than adherens junctions) (Fig. 14.26, 27 Fig. 12.16) Fig. 13.13 Structure of the Plasma Membrane Lipid composition can affect protein movement. • GPI-anchored proteins cluster in lipid rafts. • Proteins may move in and out of rafts, facilitating processes such as cell movement, cell signalling. Figs. 13.3, 13.11 The glycocalyx Glycocalyx: coat of carbohydrates of glycolipids, glycoproteins on outer face of plasma membrane: • Protects cell from ionic and mechanical stress • Barrier to invading microorganisms • Cell-cell recognition (details Ch. 14) Figs. 13.14 Intestinal epithelium Binding of selectins to oligosaccharides Glycocalyx oligosaccharides participate in cell-cell interactions. Ex. White blood cells (leukocytes) adhere to endothelial cells lining blood vessels • leave circulatory system • mediate inflammatory responses. • adhesion involves transmembrane proteins - selectins • Selectins bind sugars Figs. 13.15 selectins bind glycosylated transmembrane proteins Membrane Transport: Permeability of phospholipid bilayers 2. Selective permeability of plasma membrane: Maintains internal composition of cell Passive diffusion: • Molecules dissolve in phospholipid bilayer, diffuse across • Direction of transport from high concentration to low • Small, relatively hydrophobic molecules passively diffuse Figs. 13.15 permeability See also Fig. 2.27 Transport of Small Molecules Facilitated diffusion: • Concentration gradients control direction of movement (from high to low). • Transmembrane proteins take polar and charged molecules across membrane • Carrier proteins bind molecules on one side; undergo conformational change to allow molecule to pass through membrane to other side. • Channel proteins form open pores through membrane; allow free diffusion of molecule of appropriate size and charge. Fig. 2.28 Structure of glucose transporter *Carrier proteins Facilitated diffusion: sugars, amino acids, nucleosides. • Glucose transporter has 12 α-helical transmembrane segments (typical carrier protein). Figs. 13.17: purple = polar residues in lipid bilayer; bind glucose See Fig. 2.28 also Model for facilitated diffusion of glucose Glucose transporter alternates between two conformational states: • Once glucose is taken up, it is rapidly metabolized • Intracellular glucose concentration remains low; • Glucose continues to be transported into cell. [Glucose can be transported in opposite direction] Fig. 13.18 carriers See Fig. 2.28 also Channel Protein: Structure of an aquaporin Channel proteins • Open pores in membrane, molecules pass freely • Porins, Gap junctions (also Ch. 14) Aquaporins: • H2O molecules cross membrane more rapidly than diffusion through phospholipid bilayer; • Impermeable to charged ions Fig. 13.19 aquaporin Red = water molecules See Fig. 2.28 also Transport Small Molecules Ion channels: Well-studied in nerve and muscle cells; Fig. 13.20 • Open, close for transmission of electric signals. • Transport extremely rapid: >million ions per second. • Ion channels very selective; different channel proteins allow passage of Na+, K+, Ca2+, and Cl– • Most have “gates” (open if specific stimuli). • Ligand-gated channels open in response to binding of neurotransmitters or other signaling molecules. • Voltage-gated channels open in response to changes in electric potential across membrane. study ion channels: the patch clamp technique Role of ion channels in transmitting electric impulses elucidated using giant squid axons (Hodgkin and Huxley,1952). Electrodes inserted in axon measured changes in membrane potential, from opening, closing of Na+ and K+ channels Patch clamp technique: • Study of activity of individual ion channels • Micropipette isolates small patch of membrane, allows analysis of ion flow through single channel Fig. 13.20 Patch Clamp; (Neher and Sakmann,1976) Transport of Small Molecules Ion pumps • Use energy from ATP hydrolysis to actively transport ions across plasma membrane to maintain concentration gradients (Na+/K+ pump) • Ionic composition of cytoplasm very different from extracellular fluids. Ion gradients, resting membrane potential of giant squid axon Ions are electrically charged → transport results in electric gradient across membrane. Resting squid axons have electric potential ~ -60 mV: inside of cell is negative with respect to outside • Potential arises from ion pumps (Na+/K+ pump) & open membrane channels • Resting membrane has open K+ channels, so flow of K+ (out) makes largest contribution to resting potential. Fig. 13.22* squid axon resting potential Membrane potential and ion channels during action potential As nerve impulses (action potentials) travel along axons, membrane depolarizes: • Membrane potential changes from –60 mV to +30 mV in < 1 msec • Rapid sequential opening, closing of voltage-gated Na+ and K+ channels propagates impulse along axon. Fig. 13.23 action potential Fig 13.24 Signaling by neurotransmitter release at synapse • Depolarization of adjacent regions of plasma membrane (voltage-gated Na+/ K+ channels) allows action potentials to travel length of nerve cell. • At nerve end, chemical neurotransmitters release into synapse, bind receptors on another cell to open ligand-gated ion channels ex. Acetylcholine release binds channel protein on muscle cell: (Na+ enters; opens voltage-gated Ca2+ channel, activates myosin binding to actin, contraction (Fig. 12.28) Fig. 13.24* neurotransmitter Model of the nicotinic acetylcholine receptor Ligand-gated ion channel Ex. Nicotinic acetylcholine receptor: • 5 subunits in cylinder • Closed, pore is blocked by side chains of hydrophobic amino acids. • Acetylcholine binding induces conformational change, hydrophobic side chains shift out of channel, opens a pore for positive ions (negative aa line channel) Fig. 13.25 nicotinic acetylcholine receptor: Nicotine keeps channel open; Neurotoxins block receptor (curare) Ion selectivity of Na+ channels Voltage-gated Na+ and K+ channels very selective. • Na+ (0.95 Å) is smaller than K+ (1.33 Å), • Na+ channel pore is too narrow for K+ or larger ions Fig. 13.26 Selectivity of Na+ channel Selectivity of K+ channels Voltage-gated K+ channels • Part of channel pore lined with carbonyl oxygen (C=O) atoms from polypeptide backbone; displace water to which K+ is bound, and K+ goes through. • Na+ too small to interact, remains bound to water. Fig. 13.27 Selectivity of K+ channel (3-D structure of by X-ray crystallography). Transport of Small Molecules Voltage-gated Na+, K+, and Ca2+ channels: Family of related proteins 1 protein each K+ channel 4 subunits a-helix 4 has positive aa, senses voltage Fig. 13.28 Transport of Small Molecules ** Active transport • Molecules transported against concentration gradients. • Energy provided by coupled reaction (ATP hydrolysis) Ion pumps examples: • Na+-K+ pump (Na+-K+ ATPase) uses energy from ATP hydrolysis to transport 3 Na+ and 2 K+ against electrochemical gradient Fig. 13.29 a subunit blue; b green; g (red) regulatory; Fig 13.30 Model for operation of the Na+-K+ pump Na+-K+ pump uses ATP-driven conformational change: • 3 Na+ transported out of cell and 2 K+ transported into cell for every ATP used Na+-K+ pump uses 25% of ATP in many animal cells. gradients necessary: • for propagation of electric signals in nerve and muscle cells • active transport of molecules • maintain osmotic balance and cell volume. Fig. 13.30 Ion gradients across plasma membrane of typical mammalian cell Differences in ion concentrations: • balance high concentrations of organic molecules inside cells, • equalize osmotic pressure, prevent net influx of H2O Fig. 13.31 mammalian cell ionic composition Structure of the Ca 2+ pump Ca2+ pump structurally related to Na+-K+ pump, also powered by ATP hydrolysis • Ca2+ transported out of cell or into ER lumen, so intracellular Ca2+ concentrations are extremely low (0.1 mM) • Transient, localized increases in intracellular Ca2+ important in cell signaling (muscle contraction) Fig. 13.32 3 cytosolic subunits; 3 transmembrane subunits Transport of Small Molecules H+ Ion pumps in bacteria, yeasts, and plants actively transport H+ out of cell • H+ pumped out of cells lining stomach, → acid gastric fluids. • Structurally distinct pumps actively transport H+ into lysosomes and endosomes. Fig. 2.29 H+ pump in bacteria ATP synthases of mitochondria and chloroplasts also H+ pumps: • pumps operate in reverse, movement of ions down electrochemical gradient drives ATP synthesis. Structure of an ABC transporter ABC transporters: highly conserved ATP-binding domains or ATP-binding cassettes. • • • • More than 100 members of family; prokaryotic, eukaryotic ATP hydrolysis transports molecules in one direction. Bacteria transport nutrients in: ions, sugars, amino acids. Eukaryotes transport toxic substances out of cell: Ex. products of mdr (multidrug resistance) genes. – Often expressed high levels in cancer cells – Can remove variety of chemotherapy drugs. Fig. 13.33 ABC transporters cystic fibrosis transmembrane conductance regulator (CFTR) Cystic fibrosis (CFTR protein): • Defective Cl– transport in epithelial cells → very thick, sticky mucus, obstructs respiratory passages • Protein CFTR (cystic fibrosis transmembrane conductance regulator) in ABC transporter family • Autosomal recessive disease: • Mutant protein not fold properly • Gene therapy targets Fig 13.34 Active transport of glucose Active transport of glucose can be driven by Na+ gradient (or H+ gradient in prokaryotes). • Glucose transporters in apical domain of intestine epithelials transport 2 Na+ and 1 glucose into cell. • Flow of Na+ down electrochemical gradient provides energy for transport , accumulation of high intracellular glucose concentrations. [Cell will need to pay ATP to pump out those Na+ (Na+/K+ pump)] Fig. 13.34 use of ion gradient to drive accumulation of nutrient Two kinds of Glucose transport by intestinal epithelial cells Apical domain uses active uptake of glucose by cotransport with Na+ (symport) Basolateral domain: glucose is transferred to underlying tissue by facilitated diffusion (uniport) System is driven by Na+-K+ pump, also found in basolateral domain. Fig. 13.35 uptake glucose by intestinal epithelial, transfer to blood Examples of antiport Antiport : 2 molecules transported opposite directions. • Ca2+ is exported from cells by Ca2+ pump, Na+-Ca2+ antiporter that transports Na+ into cell and Ca2+ out. • Na+-H+ exchange protein helps regulate intracellular pH. Fig. 13.36 antiport Phagocytosis 3. Endocytosis: cells take up macromolecules, fluids, large particles such as bacteria. • Material is surrounded by plasma membrane, which buds off inside cell to form vesicle with ingested material • Pinocytosis (cell drinking): property of all eukaryotic cells. • Phagocytosis (cell eating) occurs in specialized cell types: – Phagosome vesicle formed from pseudopodia after bind particle – Phagolysosome – after fuse with lysosome Fig. 13.37 endocytosis Examples of phagocytic cells Amoebas use phagocytosis to capture food particles such as bacteria (role of Actin) Multicellular animals use phagocytosis as defense against invading microorganisms, to eliminate aged or damaged cells. Macrophages and neutrophils (white blood cells) are “professional phagocytes.” Fig. 13.38 Phagocytosis: A, amoeba eating protist;B, macrophage and red blood cells Endocytosis * Receptor-mediated endocytosis: mechanism for selective uptake of specific macromolecules. • Cell surface receptors in regions (clathrin-coated pits). • Pits bud as clathrin-coated vesicles, fuse with early endosome • Dynamin, GTP help Fig. 13.39 Receptor-mediated endocytosis; see also Fig. 10.37 for clathrin and lysosomal proteins Endocytosis Receptor-mediated endocytosis first elucidated in patients with familial hypercholesterolemia • they have defective LDL receptor (LDLR gene): • not bind LDL, or not internalize LDL after bound • Cholesterol transported through bloodstream mostly in form of low-density lipoprotein, or LDL particle • 1500 cholesteryl esters • 500 cholesterols • 800 phospholipids • 1 protein (apoprotein B100) Fig. 13.40 LDL particle The LDL receptor Mutant LDL receptors that do not bind in coated pits have altered internalization signal in cytoplasmic tail of receptor. Internalization signal is 6 aa, including tyr (-> cys in mutant). [Similar internalization signals are found in other receptors taken up via clathrin-coated pits] Fig. 13.41 Formation of clathrin-coated pits • Internalization signals bind adaptor proteins, which bind clathrin. • Clathrin assembles into basketlike structure, forms invaginated pits. • Dynamin-GTP releases coated vesicles into cell • Clathrin-coated pits occupy to 2% of cell surface; lifetime 1 to 2 minutes • After internalization, clathrin-coated vesicles shed coats, fuse with early endosomes • Molecules sorted, recycled to plasma membrane, or remain in early endosomes, mature to late endosomes and lysosomes Figs. 13.42,44 Endocytosis Early endosomes acidic pH (6.0 to 6.2), (membrane H+ pump) • Dissociates many ligands from receptors. • Receptors return to plasma membrane via transport vesicles. • Ligands (LDL) remain, degraded to release cholesterol • Late endosomes more acidic (pH 5.5 to 6.0); • mature into lysosomes (pH 5) : endocytosed materials are degraded by acid hydrolases. Fig. 13.44 Endocytosis In nerve cells, after synaptic vesicles release neurotransmitters, are recovered in clathrin-coated vesicles which fuse with early endosomes. • Synaptic vesicles regenerated by budding from endosomes. Fig. 13.45 Review questions 7. Curare binds nicotinic acetylcholine receptors and prevents them from opening; how would it affect contraction of muscles? 9. How can glucose be transported against its concentration gradient without the direct expenditure of ATP in intestinal epithelial cells? 10. How does the mdr gene confer drug resistance upon cancer cells? 12. What have studies on cells from children with familial hypercholesterolemia told us about the mechanisms of receptor-mediated endocytosis?