Lecture 4: Transport I PDF

1 Introductory Physiology Lectures – S1A Foundations for understanding Systems & Integrated Physiology 1 Overview and Scientific Process Think! How the body 2-3 Systems, tissues, cells (genes)...

1 Introductory Physiology Lectures – S1A Foundations for understanding Systems & Integrated Physiology 1 Overview and Scientific Process Think! How the body 2-3 Systems, tissues, cells (genes), body water is organised Basis for 4 Transport processes (across cell membranes) Understanding System Function - Bio-electric potentials (and ion distribution) Just 1 of 11 8 Nerve Physiology systems 2 4: Transport across cell membranes I How do solutes and H2O cross cell membranes? What is a solute? - anything dissolved in water e.g. Na+, glucose, vitamins, drugs Why is it important that solutes cross membranes? - absorb oxygen for respiration - food through gut - maintain membrane potential Essential to understand - change membrane potential how all tissues function 3 4: Transport across cell membranes I Learning Objectives Describe how solutes or water can cross a lipid bilayer Permeability and electrochemical gradient Passive versus active transport - different carrier proteins Cells have a Resting Membrane Potential (RMP) of -70 mV Mechanisms of osmotic fragility of red blood cells And some self-directed learning for the weekend! 4 Nutrients in Waste Out Circulation movement in the blood Blood Transport crossing cell membrane(s) Cells 5 Circulation and Transport Glucose from intestine to blood to muscle Intestine Interstitial space Blood Glucose must: leave intestine and enter blood (crossing 4 lipid membranes and interstitial space) circulate in blood stream to desired location leave blood and enter muscle cell (crossing 3 Target Cell more lipid membranes and interstitial space) 6 Challenge of transport inside cells Multiple intracellular membranes Molecules/ions/gases must cross internal membranes e.g. Mitochondria, nuclear membrane, sarcoplasmic reticulum 7 4: Transport across cell membranes I Learning Objectives Describe how solutes or water can cross a lipid bilayer Permeability and electrochemical gradient Passive versus active transport - different carrier proteins Cells have a Resting Membrane Potential (RMP) of -70 mV Mechanisms of osmotic fragility of red blood cells And some self-directed learning for the weekend! 8 Cell Membranes: a selectively-permeable barrier Two factors determine if a substance crosses a membrane: Permeability or will the membrane let it through ? Electro-Chemical Gradient or is there a “force” on the substance to cross the membrane ? 9 If gradient is outwards, there is a force to leave a cell Chemical Gradient Substances move from high Flux Arrow: widest part indicates conc. to low conc. by diffusion highest concentration If gradient is inwards, there is a force to enter a cell 10 -ve Negative ions forced -ve -70 mV outwards -ve -ve Electrical Gradient Inside of cells is negative Flux Arrow: widest part indicates relative to outside highest concentration Resting membrane potential -70 mV +ve +ve Positive ions attracted -70 mV inwards +ve +ve 11 Cell Membranes: a selectively-permeable barrier Which substances will the membrane allow through? 12 An ion of molecule will only move across a membrane spontaneously if it is moving down (or with) its electrochemical gradient and the membrane is permeable to that ion or molecule 13 Gases & hydrophobic substances will cross membrane spontaneously in the direction of the electrochemical gradient Passive diffusion O2 or CO2 or NO It doesn’t require metabolic energy Steroid Hormones It doesn’t require & Ethanol carrier proteins C2H5OH 14 Ions and hydrophilic substances cannot spontaneously cross the membrane even if there is a electrochemical gradient… K+ or Na+ or Cl- (charged) Glucose or Amino Acids or Water (polar) Proteins & DNA (-ve charged) 15 Ions and hydrophilic substances cannot spontaneously cross the membrane even if there is a electrochemical gradient… K+ or Na+ or Cl- (charged) … CARRIER PROTEINS are present in the Glucose or Amino membrane which make Acids or Water the membrane permeable (polar) Proteins & DNA (-ve charged) Secretion of proteins is a special case: see PL3005 16 Fick’s Law of Diffusion for Biologists dn = dtPDc Rate of diffusion = permeability coefficient x electrochemical gradient Thus, if the membrane is permeable (or is made permeable by a carrier protein) and there is an electrochemical gradient, a substance will cross the cell membrane spontaneously Q. What is the rate of diffusion if P = 0, or c = 0? 17 Are nerve impulses sent by diffusion Diffusion times of ions and molecules to acheive 99% equilibrium Distance (µm) Time (s) 0.1 0.000 000 5 Nerve synapse 1 0.000 5 10 0.05 Typical Cell 100 5 1000 (1 mm) 500 (8 min) 1000000 (1 m) 500 000 000 (15 y) Longest Nerve 18 Are nerve impulses sent by diffusion Diffusion times of ions and molecules to acheive 99% equilibrium Distance (µm) Time (s) Processes in normal cells may occur by diffusion, but processes reliant on diffusion can only occur over small distances 10 0.05 Typical Cell Transmission of nerve impulses does NOT occur (directly) by diffusion (Mechanism explained later in the module) 1000000 (1 m) 500 000 000 (15 y) Longest Nerve 19 Part I – Summary Spontaneous movement of ions or molecules across a lipid bilayer Membranes act as selectively permeable barriers Membrane must be permeable, or made permeable with a carrier protein An electrochemical gradient across the membrane Solutes move by diffusion – rate calculated by Fick’s law Diffusion is rapid over short distance… … slow over long distance 20 4: Transport across cell membranes I Learning Objectives Describe how solutes or water can cross a lipid bilayer Permeability and electrochemical gradient Passive versus active transport - different carrier proteins Cells have a Resting Membrane Potential (RMP) of -70 mV Mechanisms of osmotic fragility of red blood cells And some self-directed learning for the weekend! 21 Passive versus Active transport and the role of the different carrier proteins Regulated CHANNELS Non-regulated PASSIVE Uniporter TRANSPORTERS Symporter Antiporter ACTIVE PUMPS ATP-Pump 22 Regulated CHANNELS Non-regulated PASSIVE Channels form “pores” in membrane with specificity for a single substance (e.g. Na+ only or H2O only) Flux of ions (or flow of H2O) is passive - dictated by elec/chem gradient Regulated (gated) or non-regulated (constitutive - always open) 23 PASSIVE Uniporter TRANSPORTERS Symporter Antiporter ACTIVE 24 PASSIVE Uniporter TRANSPORTERS Uniporters bind and move larger molecules (e.g. glucose) down concentration gradient across the membrane without creating a pore that is completely open at any one time Regulated by insertion/removal of the uniporter from cell membrane (e.g. GluT1 - glucose transporter in kidney) 25 PASSIVE Uniporter TRANSPORTERS Uniporters bind and move larger molecules (e.g. glucose) down concentration gradient across the membrane without creating a pore that is completely open at any one time Regulated by insertion/removal of the uniporter from cell membrane (e.g. GluT1 - glucose transporter in kidney)  Q - why glu transporter rather than glu channel? 26 K+ ion K+ A - Charge, Shape and Size 27 K+ ion K+  Glucose channel would allow other ions & H2O 28 ie - loss of selectively permeable nature of cell membrane K+ ion K+ 29 passive versus active transport and the role of the different carrier proteins Regulated CHANNELS Non-regulated PASSIVE Uniporter TRANSPORTERS Symporter Antiporter ACTIVE PUMPS ATP-Pump 30 Active transport Ions or molecules can move up/against concentration gradient, but this requires additional energy - spontaneous diffusion won’t occur TRANSPORTERS Symporter Antiporter ACTIVE PUMPS ATP-Pump 31 Active transport Primary (1º) active transport by ion pumps - ATP dependent Example: Na+ ions are actively extruded (movement against electrochemical gradient) from the cell by the Na+ pump - uses ATP Essential to maintain cell volume, nerve impulses and muscle contraction (later in the module) PUMPS ATP-Pump 1˚ ACTIVE 32 Active transport - examples Secondary (2º) active transport by symporters and antiporters - Uses energy stored in conc. grad. established by 1˚ active transport Example Na+ ions (high concentration in the gut) can provide energy to move glucose against its concentration gradient into the body (details in next lecture) TRANSPORTERS Symporter 2˚ ACTIVE Antiporter 33 passive versus active transport summary Regulated CHANNELS Non-regulated PASSIVE Uniporter TRANSPORTERS Symporter 2˚ ACTIVE Antiporter PUMPS ATP-Pump 1˚ ACTIVE 34 4: Transport across cell membranes I Learning Objectives Describe how solutes or water can cross a lipid bilayer Permeability and electrochemical gradient Passive versus active transport - different carrier proteins Cells have a Resting Membrane Potential (RMP) of -70 mV Mechanisms of osmotic fragility of red blood cells And some self-directed learning for the weekend!  Resting membrane potential (RMP) is –70 mV 35 Inside is negative relative to outside milliVolts -70 0 +70 ICF ECF  Why is RMP –70 mV? 36 An imbalance of charge across the membrane +ve ions are roughly the same concentration on either side (though different ions) -ve ions and molecules much higher concentration inside than outside Na+ 150 mM why don't some of the ions cross the membrane to neutralise the charge: K+ ICF 150 mM +ve charges enter Prot17- and/or Org P- 130 mM ATP4- ECF -ve charges leave  Why is RMP –70 mV? 37 Two factors explain this: Fick's law and Na/K Pump Fick’s law dn = PC dt Na+ and K+ Prot17- Na+/K+ pump  Why is RMP –70 mV? 38 Two factors explain this: Fick's law and Na/K Pump Fick’s law dn = PC dt Na+ DC for –ve ions is high, but P = 0 Therefore: K+ ATP4- and proteins17- cannot leave Prot17- to “neutralise” the negative charge "Trapped Anions"  Why is RMP –70 mV? 39 Two factors explain this: Fick's law and Na/K Pump Fick’s law dn = PC dt Na+ DC for Na+ ions is high, but P = 0 Therefore: K+ Na+ cannot enter resting cells Prot17- to “neutralise” the negative charge But some channels will open from time to time, so could RMP eventually be neutralised?  Why is RMP –70 mV? 40 Two factors explain this: Fick's law and Na/K Pump Na+/K+ pump uses ATP to: 3 Na+ ATP Na+/K+ Pump 3 Na out of cell + Pump 2 K+ and simultaneously ADP Prot17- + Pi Pump 2 K+ into cell The pump is “electrogenic” with an overall export of +ve charge Constant use of ATP to export +ve ions helps maintains RMP at -70 mV 41 4: Transport across cell membranes I Learning Objectives Describe how solutes or water can cross a lipid bilayer Permeability and electrochemical gradient Passive versus active transport - different carrier proteins Cells have a Resting Membrane Potential (RMP) of -70 mV Mechanism of water movement & osmotic fragility of red blood cells And some self-directed learning for the weekend! 42 How does H2O cross the membrane? … and why is it important Water moves by a process known as osmosis if the cell membrane is permeable Permeability Water channels (aquaporins) Osmotic gradient Water moves from a region of low osmolarity to a region of high osmolarity 43 What is Osmolarity… … how is it measured for molecules? Osmolarity is the amount of solute dissolved in 1 litre* of water e.g. 1 mole of glucose in 1 litre of water = 1 Osmolar (1 Osm) If we can measure osmolarity on either side of a membrane we can predict which way the water will flow * Strictly speaking 1 Kg but the difference is small with dilute biological fluids 44 What is Osmolarity… … how is it measured for salts? Osmolarity is the amount of solute dissolved in 1 litre* of water e.g. 1 mole of NaCl in 1 litre of water = 2 Osmolar (2 Osm) NaCl dissolves in water to create two osmotically active ions If we can measure osmolarity on either side of a membrane we can predict which way the water will flow * Strictly speaking 1 Kg but the difference is small with dilute biological fluids 45 What is Osmolarity… … how is it measured for biological (complex) fluids? Plasma contains Na+ 150 mM K+ 5 mM Ca2+ 2 mM Other solutes ~130 mM How do we measure osmolarity - determine concentration of all the individual components? Or is there a simple way… 46 Osmolarity is measured by … Depression of Freezing Point Freezing point of a solution is affected by total concentration of osmotically active particles One mole of solute depresses the freezing point of water by 1.86˚C 1 M (1 Osm) glucose will freeze at -1.86 ˚C 0.5 M (1 Osm) NaCl will freeze at -1.86 ˚C plasma freezes at approx -0.52 ˚C Osmolarity of plasma = -0.52 x 1.0 Osm = 0.28 Osm = 280 mOsm -1.86 Knowledge of solute composition is not required to measure osmolarity  ICF and ECF solute compostions differ… 47 … but there is no net movement of water between ICF and ECF - why? ECF 14 L ICF 28 L Example Plasma Muscle Cell Solutes (mM) Na+ 150 (high) 10 (low) K+ 5 (low) 150 (high) Ca2+ 2 (high) 0.0004 (low) Other ~130 (variable) ~130 (variable) No net movement because osmolarity is the same in both Let’s explore this idea! Osmolarity (mOsm) ~280 ~280 48 Osmotic fragility of Red Blood Cells ICF = 280 mOsm Plasma RBC membrane contains aquaporins 280 What happens in plasma (280mOsM)? mOsm 280 mOsm Is there an osmotic gradient? No (the solutions are isosmotic) Is there net osmosis? No, therefore RBC maintains its normal size & shape (and function!) 49 Osmotic fragility of Red Blood Cells ICF = 280 mOsm 140mM NaCl RBC membrane contains aquaporins 280 What happens in 140 mM NaCl mOsm 280 mOsm Is there an osmotic gradient? No (the solutions are isosmotic) Is there net osmosis? No, therefore RBC maintains its normal size & shape (and function!) 50 Osmotic fragility of Red Blood Cells ICF = 280 mOsm Pure water RBC membrane contains aquaporins 280 What happens in pure water mOsm 0 mOsm Is there an osmotic gradient? Yes (Osmolarity greater inside) Is there net osmosis? Yes, water rushes into cell and it explodes (and loses function)! 51 Osmotic fragility of Red Blood Cells ICF = 280 mOsm Sea water RBC membrane contains aquaporins 280 What happens in sea water mOsm 1000 mOsm Is there an osmotic gradient? Yes (Osmolarity greater outside) Is there net osmosis? Yes, water rushes out of cell and it implodes (and loses function)! 52 What’s really happening… Water moves by osmosis if the cell membrane is permeable Osmosis is the same as diffusion Water moves spontaneously down its concentration gradient! Water has a concentration? … confused, don’t be… 53 What is the concentration of H2O? In grammes per litre Concentration of water is defined as 1000g per litre In moles per litre (molarity) Mr of H2O = 18  1000 g H2O is 1000/18 = 55.5 moles 55.5 moles per 1 litre = 55.5M 54 How can the concentration of H2O be changed? Join the two compartments with a membrane permeable only to H 2O Compartment on left = [H2O]high Compartment on right = [H2O]high Water will flow down its conc. grad. until equilibrium H2O with no H2O with no glucose [H2O] high [H2O] high glucose H 2O Volume of solution = 1 litre = 1 litre Amount of H2O = 55.5 moles = 55.5 moles Concentration = 55.5 M = 55.5 M Osmolarity = 0 mOsm = 0 mOsm 55 How can the concentration of H2O be changed? Join the two compartments with a membrane permeable only to H 2O Compartment on left = [H2O]high Compartment on right = [H2O]low Water will flow down its conc. grad. until equilibrium … which is the same as saying water moves from an area of low osmolarity to a region of high osmolarity H2O with no H2O with 100mM glucose [H2O] high [H2O] low glucose H 2O e.g. 1 ml H2O Volume of solution = 1 litre = 1 litre (~ 0.1 mole) Amount of H2O = 55.5 moles = 55.4 moles Concentration = 55.5 M = 55.4 M Osmolarity = 0 mOsm = 100 mOsm 56 Understanding Physiology saves lives Infusion of fluids is essential during operations, etc. What happens if you infuse 2 litres of water instead of saline? Some drugs can only be administered in a drip formula e.g. cancer chemotherapy What happens if you make them up incorrectly (or are responsible for the person who does)? Haemolysis 57 4: Transport across cell membranes I Learning Objectives Describe how solutes or water can cross a lipid bilayer Permeability and electrochemical gradient Passive versus active transport - different carrier proteins Cells have a Resting Membrane Potential (RMP) of -70 mV Mechanisms of osmotic fragility of red blood cells And two slides of reading … 58 TONICITY AND OSMOLARITY - I See your textbook (Fox) for details but briefly, solutions that have the same osmolarity as plasma (such as 0.9% NaCl and 5% glucose) are said to be isosmotic to plasma. Solutions with a lower osmolarity than plasma are hypoosmotic; solutions with a higher osmolarity than plasma are hyperosmotic. However, you may be more familiar with the terms isotonic, hyoptonic and hypertonic. These terms are far more physiologically (and thus clinically) relevent – why? The terms isosmotic, hypoosmotic and hyperosmotic just refer to differences in the osmolarity of the solutions (fine for chemistry), but they do not take into account the permeability of the cell membranes (essential for physiology). The terms isotonic, hypotonic and hypertonic do take into account the permeability of the cell membranes, and that’s why they are routinely used by physiologists and clinicians. When red blood cells (with an ICF osmolarity of 280 mOsm) are in a solution of 280 mOsm glucose, the glucose solution is referred to as being isosmotic (the same osmolarity) to the plasma of the cells. However, it is also isotonic with the plasma of the cell because the glucose cannot cross the cell membrane; there are no glucose transport membranes in RBC membrane. Using the same logic, when red blood cells are in a solution of 140 mOsm glucose, the glucose solution is referred to as being hypotonic. In contrast, when RBC are in a solution of 560 mOsm glucose, the glucose solution is referred to as being hypertonic. In the glucose examples, RBC in a hypertonic solution (eg 560 mOsm glucose) will shrivel up (see lecture 4) whereas RBC in a hyoptonic solution (eg 140 mOsm glucose) will start to swell and may burst (see lecture 4) 59 TONICITY AND OSMOLARITY - II Why bother with the two terms – does it ever make a difference, when on the face of it, for many solutions, osmolarity and isotonicity mean the same thing? The simple answer is yes it makes a difference, and potentially a very big difference. The classic example to explain the difference is to observe what happens when RBC are placed in a solution of 280 mM Urea; the urea solution is referred to as being isosmotic (the same osmolarity, 280 mOsm) as the plasma of the cell, but is NOT isotonic because urea can cross the RBC membrane (via urea transporters) down its concentration gradient. If RBC are placed in a 280 mM urea solution, the urea will cross the RBC membrane by diffusion, moving down its concentration gradient, until it reaches an equal concentration on either side of the membrane (140 mM either side). If you think about it, this means that the total osmolarity inside is now 420 mOsm (280 + 140) and the osmolarity outside is 140 mOsm. Water will move into the cell and it will explode (see lecture 4). Clearly, such a solution may have started as isosmotic, but it is not isotonic as the solute (urea) can penetrate the cell membrane. Simply put: If a solution is isosmotic to the ICF of the cell, and the solute CANNOT cross the cell membrane, the solution is also isotonic. If a solution is isosmotic to the ICF of the cell, but the solute CAN cross the cell membrane, the solution is NOT isotonic. Rare Disease - Clinical Relevance Just for information, there is a rare disease in which urea transporters are absent from RBC and such cells are resistant to lysis, even in 2 M urea (http://www.ncbi.nlm.nih.gov/pubmed/18028269). 60 Lecture 4: Summary – Transport I Learning Objectives Describe how solutes or water can cross a lipid bilayer Permeability and electrochemical gradient Passive versus active transport - different carrier proteins Cells have a Resting Membrane Potential (RMP) of -70 mV Mechanisms of osmotic fragility of red blood cells Self-Directed Learning Read Tonicty and Osmolarity