Fluid Mosaic Model 1 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 2 Membrane proteins: – Integral (intrinsic) proteins – Peripheral (extrinsic) proteins Membrane fluidity 3 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? 4 Lipids and membrane fluidity 5 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 6 Microbial growth at Cold temperatures Molecular Adaptation to Psychrophily 7 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 8 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 9 Bacteria have lipids rich in saturated fatty acids Archaea have lipid monolayer rather than bilayer Microbial Growth at High Temperature 10 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 11 The inner and outer leaflets of the membrane have different compositions of lipids and proteins Membrane asymmetry 12 Sphingomyelin and phophatidyl choline are located on the outer leaflet Phosphatdidylserine is located in the inner leaflet Membrane Rafts 13 Sphingolipids colocalize with cholesterol in membrane microdomains called lipid rafts. Membrane Rafts Caveolae are invaginated lipid raft domains of the plasma membrane They have roles in cell signaling and membrane internalization 14 Membrane Rafts 15 Caveolin is a protein associated with the cytosolic leaflet of the plasma membrane in caveolae. Caveolin interacts with cholesterol and selfassociates as oligomers that may contribute to deforming the membrane to create the unique morphology of caveolae. 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 has proteins such as collagen and fibronectin) 16 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 deficiency. Child dies by age 2 17 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 18 Biomembrane Membrane fusion and biological processes 19 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 20 Membranes play key role in cell-cell recognition Carbohydrates are usually branched oligosaccharides with fewer than 15 sugar units Membrane carbohydrates 21 Oligosaccharides on external of membranes are different among species, or individuals, or cells Membrane functions 22 Cell communication and signalling Cell-cell adhesion and cellular attachment Cell identity and antigenicity Conductivity Membrane functions Form selectively permeable barriers Transport phenomena – Passive diffusion – Mediated transport Facilitated diffusion – Carrier proteins – Channel proteins – 23 Gated or non-gated channels Active transport Transport of Ions and Small Molecules Across Cell Membrane 24 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. 25 Membrane transport 26 Solving the Problem Mechanisms by which cells solve this problem include: 1. Active transport 2. Facilitated diffusion 27 Active Transport Active transport is the pumping of molecules or ions through a membrane against their concentration gradient. It requires 28 a transmembrane protein (a complex of them) called a transporter Energy. ATP (source) Active Transport 29 Active transport enzymes couple net solute movement across a membrane to ATP hydrolysis. Active Transport 30 An active transport pump may be a uniporter, or it may be an antiporter that catalyzes ATPdependent transport of 2 solutes in opposite directions. Active Transport . 31 ATP-dependent ion pumps are grouped into classes, based on transport mechanism, genetic & structural homology. Active Transport The energy of ATP may be used directly or indirectly There are two types of active transport Direct / Primary 32 Indirect/Secondary Active Transport 33 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 – – 34 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. 35 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). 36 Energetics of active transport 37 Active transport – Metabolic energy expenditure is required. – Solute moves against a gradient of electrochemical potential. Carrier mediated membrane transport Carriers exhibit saturation kinetics with respect to solute concentration. Carriers exhibit stereospecificity. – 38 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). – 39 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. 40 Energetics of carrier-mediated transport Diffusion Passive transport (facilitated diffusion) – – – – 41 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 42 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 43 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 44 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 45 Classes of carrier proteins Uniport Uniport (facilitated diffusion) carriers mediate transport of a single solute. Examples include GLUT1 and valinomycin. 46 Classes of carrier proteins Uniport 47 These carriers can undergo the conformational change associated with solute transfer either empty or with bound substrate. Thus they can mediate net solute transport. Classes of carrier proteins Uniport Valinomycin is a carrier for K+. Valinomycin reversibly binds a single K+ ion. 48 Classes of carrier proteins Uniport Valinomycin is highly selective for K+ over Na+. Why??? 49 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. 50 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. 51 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+ 52 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 53 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. 54 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 55 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 56 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. 57 Uniport A Symport A B Antiport A B Classes of carrier proteins Antiport 58 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 59 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. 60 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: 61 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 62 O- Direct Active Transport The Na+/K+ ATPase 63 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 64 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+ 65 ADP + Pi Outward Potassium Flux The Na+/K+ ATPase transporter – Gradients for Na+ and K+ needed for action potentials & synaptic potentials Inward 3 Na+ Sodium Flux Extracellular Cytosol Mg++ ATP 2 K+ 66 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? 67 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 68 The Na+/K+ ATPase transporter Inhibited by : – Cardiac glycosides – Metabolic inhibitors – Heavy Metals Inward 3 Na+ Sodium Flux Extracellular Cytosol Mg++ ATP 2 K+ 69 ADP + Pi Outward Potassium Flux Digitalis inhibits the Na+/K+ Pump 70 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 Foxglove (Digitalis purpurea) 71 William Withering conducted studies on Foxglove Conducted the first scientific study on its effects “Old woman of Shropshire” 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) 72 Increase in intracellular levels of Ca 2+ enhances the ability of the cardiac muscle to contract Digitalis inhibits the Na+/K+ Pump 73 Inhibititors of the Na+/K+ Pump 74 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 2 Na+ and prevents the change in conformation necessary for transport of ions Inhibitors of the Na+/K+ Pump 75 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 form both ends Open channel Exit of K+ from cells Toxic Direct Active Transport The H+/K+ ATPase Transporter 76 The H+/K+ ATPase transporter plays a part in maintaining the acidity of the stomach Mammalian stomach contains a 0.1M solution of HCl This stongly acidic medium kills many ingested pathogens and denatures many ingested proteins before they can be degraded by proteolytic enzymes (pectin) that function at acid pH Direct Active Transport The H+/K+ ATPase Transporter HCl is secreted into the stomach by specialized epithelial cells called parietal cells in the gastric lining These cells contain the H+/K+ ATPase in their apical membrane which faces the stomach lumen This generates a million fold H+ gradient pH in stomach lumen is 1.0 whereas pH in the cytosol is 7.0 77 Direct Active Transport The H+/K+ ATPase Transporter 78 The H+/K+ ATPase is a P class pump that is similar in structure and function to the Na+/K+ pump found in the plasma membrane The numerous mitochondria in the parietal cells produces enough ATP needed for use by the H+/K+ pump The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 79 If parietal cells simply exported H+ ions in exchange for K+ ions , the loss of protons would lead to arise in OH- ions in the cytosol This would lead to an increase in cytosolic pH The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 80 This is avoided by using Cl/HCO3- antiporters in the basolateral membrane This exports the excess OHions in the cytosol into the blood This antiporter is activated at high cytosolic pH The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 81 In a reaction catalysed by carbonic anhydrase the excess cytosolic OH- combines with CO2 that diffuses in from the blood This forms HCO3- The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 82 Catalysed by the basolateral anionic antiporter, this bicarbonate ion is exported across the basolateral membrane in exchange for Cl- ions The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 83 The Cl- ions then exit through Cl- channels in the apical membrane, entering the stomach lumen The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 84 To preserve the electronuetrality, each Cl- ion that moves into the stomach lumen through the apical membrane is accompanied by a K+ ion that moves outward through a separate K+ channel The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 85 In this way the excess K+ ions pumped inward by the H+/K+ ATPase are returned to the stomach lumen Thus maintaining the normal intracellular K+ concentrations The H+/K+ ATPase Transporter Acidification of stomach lumen by parietal cells 86 The net result is secretion of equal amounts of H+ and Clions (HCl) into the stomach lumen While the pH in the cytosol remains nuetral and excess HCO3- ions are transported into the blood. Direct Active Transport The Ca 2+ ATPase Transporter 87 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 88 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 89 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 90 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 Ca2+ ions are trapped inside 91 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 92 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 93 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 94 SERCA:Sarco Endo(plasmic) Reticulum Ca 2+ ATPase 95 SERCA:Sarco Endo(plasmic) Reticulum Ca 2+ ATPase 96 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 97 Direct Active Transport ABC Transporters 98 The ATP bound to its domain provides the energy to pump the ligand across the membrane ABC Transporters Mechanism 99 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 10 0 Substrate binding results in a conformational change in the ATP binding cassette that increases their affinity for ATP ATP binds to the ATPbinding cassettes, changing their conformations so that the two domains interact strongly with each other ABC Transporters Mechanism 10 1 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) 10 2 Direct Active Transport ABC Transporters 10 3 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 10 4 Effects of ABC Transporters ABC transporters can confer antibiotic resistance in pathogenic microbes such as: Pseudomonas aeruginosa Staphylococcus aureus Candida albicans Neisseria gonorrhoeae 10 5 Transepithelial transport 10 6 Absorption of nutrients from the intestinal lumen occurs by: import of molecules on the luminal side of the intestinal epithelial cells And their export on the blood-facing (serosal) side, a two stage process called transcellular transport An intestinal epithelial cell is polarized Transepithelial transport Movement of glucose and amino acids across Eipthelia 10 7 In the first stage of this process, a 2 Na+/1glucose symport located in the microvillar membrane imports glucose against its concentration gradient from the intestinal lumen Transepithelial transport Movement of glucose and amino acids across Eipthelia 10 8 This symporter couples the energetically unfavourable movement of 1 glucose molecule to the favourable inward transport of 2Na+ Transepithelial transport Movement of glucose and amino acids across Eipthelia 10 9 In the steady state all the Na+ ions transported from the intestinal lumen into the cell during the Na+/glucose symport are pumped out across the basolateral membrane Transepithelial transport Movement of glucose and amino acids across Epithelia 11 0 Thus the low intracellular Na+ concentration is maintained This is accomplished by the Na+/K+ ATPase which is found exclusively in the basolateral membrane of the small intestine Transepithelial transport Movement of glucose across Eipthelia 11 1 The coordinated operation of these two transport proteins allows uphill movement of glucose from the intestine into the cell The first stage is ultimately powered by ATP hydrolysis by the Na+/K+ ATPase Transepithelial transport Movement of glucose across Eipthelia 11 2 In the second stage, glucose concentrated inside the intestinal cells by symporters are exported down their concentrated gradients into the blood via uniport proteins in the basolateral membrane. In the case of glucose this is mediated by GLUT2 Transepithelial transport Movement of glucose across epithelia 11 3 The net result of this twostage process is movement of Na+, glucose, and amino acids from the intestinal lumen across the intestinal epithelium into the extracellular medium that surrounds the basolateral surface of the intestinal epithelial cells Transepithelial transport Movement of glucose across Eipthelia 11 4 Tight junctions between the epithelial cells prevent these molecule from diffusing back into the intestinal lumen Eventually they move into the blood Transepithelial transport Movement of glucose across Epithelia 11 5 The increased osmotic pressure created by transcellular transport of salt, glucose and amino acids across the intestinal epithelium draws water from the intestinal lumen into the extracellular medium that surrounds the basolateral surface. Transepithelial transport Movement of glucose across Epithelia 11 6 Simple rehydration therapy depends on the osmotic gradient created by absorption of glucose and Na+ Treatment of chlolera and other intestinal problems (gastroenteritis). Giving patients a solution of sugar and salt helps in rehydration. Similar sugar/ salt solutions are the basis of popular sport drinks used by athletes (gatorade). This is used to get sugar as well as water into the body quickly and efficiently Faciltated Diffusion of Ions 11 7 Facilitated diffusion takes place through transmembrane proteins Water filled channel through which the ions can pass down its concentration gradient Transmembrane channels that permit facilitated diffusion can be opened or closed. They are said to be gated Facilitated Diffusion of molecules Small hydrophilic molecules such as sugars can pass through cell membranes by facilitated diffusion Examples: Maltoporin: allow disaccharide and maltose and few related molecules to diffuse into the cell of E.coli 11 8 Facilitated Diffusion of molecules Examples: Transmembrane proteins of red blood cells permit diffusion of glucose from blood into the cell NB. In facilitated diffusion the channels are selective. The structure of the protein only admits certain types of molecules 11 9 Ion Channels Ionophores: compounds that shuttle ions across membrane Valinomycin disrupts the (K+) channel Monensin disrupts (Na+) channel 12 0 Ion channels 12 1 Bacterial K+ channel Neuronal Na+ channel Nicotinic Acetylcholine receptor ion channel Ion channels Differences between Ion channels and transporters 1. The rate of flux through channels are much greater 2. Ion channels are not saturable 3. They are gated –opened or closed in response to some cellular event 12 2 Ion channels Types of Gated Channels 12 3 Ligand gated Mechanically- gated Voltage- gated Light -gated Voltage gated ion channel In cells such as neurons and muscle cells some channel open and close in response to changes in the charge (measured in volts) across the plasma membrane 12 4 Voltage gated ion channel Example: Impulse passes down neuron Reduction in voltage opens Na+ channels in adjacent portion of the membrane This allows the influx of Na+ into the neuron and hence the continuation of the nerve impulse 12 5 Ion Channels K+ Ion channel 12 6 The K+ ion channel is a tetramer of identical subunits The four subunits come together to form a pore that runs through the centre of the structure The pore starts with a diameter of 10 A and constricts to a smaller cavity (8 A) Ion Channels K+ Ion channel 12 7 Both the openings to the outside and central cavity of the pore are filled with water K+ can fit into the pore without losing its shell of bound water molecule Ion Channels K+ Ion channel 12 8 Approximately twothirds of the way through the channel the pore constricts The K+ ions will have to shed its bound water molecules Ion Channels K+ Ion channel 12 9 For K+ to relinquish their water molecule other polar interactions must replace those with water The restricted part of the core is built from residues contributed by two transmembrane alpha helices Ion Channels K+ Ion channel 13 0 A five amino acid stretch within this region functions as the selectivity filter that that determines the preference of K+ ions over other ions Ion Channels K+ Ion channel 13 1 K+ ion channels are 100-fold more permeable to K+ than to Na+ The key point is that the free energy cost of dehydrating Na+ ions are considerable. Ion Channels K+ Ion channel 13 2 The channel repays the cost of dehydrating K+ by providing compensating interactions with the carbonyl oxygen atoms lining the selectivity filter Ion Channels K+ Ion channel 13 3 These oxygen atoms are positioned so that they do not interact favourably with Na+ because it is too small The Na+ ions are rejected because they must stay hydrated to pass through the channel Ion Channels K+ Ion channel 13 4 Four K+ binding sites crucial for rapid flow are present in the constricted area of the K+ channel The ion can move within the 4 sites of the selectivity channel because they have similar ion affinities K+ binding sites on the selectivity pore of K+ channel Ion Channels K+ Ion channel 13 5 As each subsequent K+ moves into the selectivity filter, its positive charge will repel the K+ ion at the nearest site K+ binding sites on the selectivity pore of K+ channel Ion Channels K+ Ion channel 13 6 This causes it to shift to a site further up the channel and in turn push upward any K+ ion already bound to a site further up K+ binding sites on the selectivity pore of K+ channel Ion Channels K+ Ion channel 13 7 Insert photo K+ binding sites on the selectivity pore of K+ channel Ion Channels Na+ Ion channel (Voltage gated) 13 8 Typically more selective for Na+ over other monovalent or divalent ions The Na+ channel’s selection of Na+ over K+ ions depends on ionic radius It is sufficiently restricted so that small ions like Na+ and Li+ can pass through but larger ions are restricted Ion Channels Na+ Ion channel (Voltage gated) Na+ are activated by a reduction in the transmembrane potential -60 mV resting +30 mV activated 13 9 Ion Channels Na+ Ion channel (Voltage gated) 14 0 Insert photos Ion Channels Na+ Ion channel (Voltage gated) 14 1 The depolarization induced by the opening of Na+ channels causes voltage gated K+ channels to open, and the resulting efflux of K+ repolarises the membrane locally Changes in transmembrane potential produce subtle conformational changes in channel protein This is explained by the ball and chain model Na+ Ion channel (Voltage gated) 14 2 Ball and chain model for channel inactivation Na+ Ion channel (Voltage gated) 14 3 The first 20 residues of the K+ channel forms a cytoplasmic unit ( the ball) This is attached to a flexible segment of the polypeptide (the chain) Na+ Ion channel (Voltage gated) 14 4 When the channel is closed the ball rotates freely in aqueous soln When the channel is open the ball quickly finds a complementary site in the open pore and occludes to it Na+ Ion channel (Voltage gated) 14 5 The channel opens for only a brief interval before it undergoes inactivation by occlusion Shortening the chain speeds in activation Lengthening the chain slows inactivation Physiological consequences of defective ion channels Defect in Na+ channel results in : 14 6 Hyperkalemic (periodic) paralysis Paramyotonia congenita (muscles are stiff) Physiological consequences of defective ion channels 14 7 Many naturally occuring toxins act on ion channels Tetrodotoxin (produced by the puffer fish) and saxitoxin (produced by marine dinoflagellates Gonyaulax, which causes red tides) act by binding to the voltage gated Na+ channels of neurons and prevent normal action potentials Physiological consequences of defective ion channels 14 8 Eating shellfish that have been fed on Gonyaulax can be fatal Shellfish concentrate the saxitoxin in their muscles which becomes highly poisonous to organisms higher up on the food chain Physiological consequences of defective ion channels The venom of the Black mamba snake contains Dendrotoxin which interferes with the voltage gated K+ channels. Tobocurarine (active componet of curare) and two other toxins from snake venom Cobrotoxin & Bungarotoxin Blocks AcH receptors or prevent opening of its ion channel. By blocking signals from nerve to muscles these toxins can cause paralysis and possibly death 14 9 Ligand gated channels External ligands Bind to a site on the extracellular side of the channel Examples: Acetylcholine (ACh). Gamma amino butyric acid (GABA) 15 0 Binding of GABA at certain synapses ( designated GABAA) in the CNS admits Cl- ions into the cell and inhibits the creation of a nerve impulse Ligand gated channels Internal ligands Internal ligands bind to a site on the channel protein exposed to the cytosol Examples Cyclic AMP (cAMP) and cyclic GMP (cGMP) 15 1 cGMP (3`,5`-cyclic monophosphate) cAMP (cyclic adenosine phosphate) ATP is needed to open the channel that allows chloride (Cl-) and bicarbonate (HNO3-) ions out of the cell The acetylcholine receptor Ligand gated channel 15 2 Acetylcholine released by motor nuerons diffuses to the plasma membrane Binds to AcH receptor Conformational change in receptor Causes the ion channel to open Ca 2+ ,Na+ and K+ passes through with ease Depolarization of plasma membrane (contraction) Movement is unsaturable The acetylcholine receptor Ligand gated channel 15 3 Signaling in the nervous system is accomplished by a network of neurons, that carry an electrical impulse (action potential) from one end of the cell through an elongated cytoplasmic extension (the axon). The electrical signal triggers the release of neurotransmitter molecules at a synapse carrying signal to the next cell in the circuit The acetylcholine receptor Ligand gated channel 15 4 Three types of voltage-gated ion channels are essential to this signaling mechanism These are the voltage gated Na+ channels The voltage gated K+ channels The voltage gated Ca2+ channels The acetylcholine receptor Ligand gated channel Along the length of the axon are voltage gated Na+ channels that are closed when the membrane is at rest (60mV) They open briefly when the membrane is depolorised locally in responce to acteylcholine (or other nuerotransmitters) 15 5 The acetylcholine receptor Ligand gated channel 15 6 The depolorization caused by the opening of the Na+ channels causes the voltage gated K+ channels to open and the resulting efflux of K+ ions repolarizes the membrane locally The acetylcholine receptor Ligand gated channel 15 7 A brief pulse of depolarization traverses the axon as local depolarization triggers the brief opening of Na+ channels then K+ channels The acetylcholine receptor Ligand gated channel 15 8 After each opening of the Na+ channel a brief refractory period follows, during which the channel cannot open again. This results in a unidirectional wave of depolorization from the nerve cell body to the end of the axon The acetylcholine receptor Ligand gated channel 15 9 At the distal tip of the axon are voltage gated Ca2+ channels. They open and the Ca2+ enters from the extracellular space The rise in cytoplasmic Ca2+ triggers the release if acetylcholine into the synaptic cleft The acetylcholine receptor Ligand gated channel 16 0 Acetylcholine diffuses to the postsynaptic cleft where it binds to acetylcholine receptors and triggers depolorization The message is passed to the next cell in the circuit The acetylcholine receptor Ligand gated channel 16 1 Role of voltage gated and ligand gated ion channels in neural transmission 16 2 Initially the plasma membrane of the presynaptic neuron is polarized (inside negative) through the action of the electrogenic Na+ K+ ATPase, which pumps 3 Na+ out for every 2 K+ pumped into the neuron A stimulus to this neuron causes an action potential to move along the axon ( white arrow) away from the cell body. The opening of one voltage gated Na+ channel allows Na+ entry, and the resulting local depolorization causes the adjacent Na+ channel to open and so on. The directionality of movement of the action potential is ensured by the brief refractory period that follows the opening of each voltage gated Na+ channel Role of voltage gated and ligand gated ion channels in neural transmission 16 3 When the wave of depolarisation reaches the axon tip, voltagegated Ca 2+ channels open, allowing Ca 2+ entry into the presynaptic neuron The resulting increase in internal Ca 2+ triggers exocytic release of the neurotransmitter acetylcholine into the synaptic cleft Acetylcholine binds to a receptor on the postsynaptic neuron, causing its ligand gated ion channel to open Role of voltage gated and ligand gated ion channels in neural transmission 16 4 Extracellular Na+ and Ca 2+ enter through this channel depolarizing the postsynaptic cell. The electrical signal has now passed to the cell body of the post synaptic neuron and will move along the axon to a third neuron by the same sequence of events