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Chemical Potential and Molecular Transport Across a Permeability Barrier Solute Transport in Plants 1. Physical & Chemical Principles Governing Transport Chemical Potential Passive & Active Transport 2. Molecular Mechanisms Governing Transport Ion Transport Across Membranes Nernst Equation Membrane Transport Proteins Channels Carriers Pumps Active Transport Primary & Secondary Aquaporins ABC Binding Cassettes Development of a Diffusion Potential and a Charge Separation Chemical Potential Equation Nonionizing Solute Membrane preferentially permeable to K+ uj = uj* + RTlnCj uj = chemical potential of a solute (Joules mol-1) uj* = chemical potential of a solute under u us Charge imbalance across membrane K+ diffuses faster Æ Charge separation Æ diffusion potential Chemical Potential Nonionizing Solute like Sucrose μ~i = μ* + RT ln C i Chemical Potential Inside the Cell μ~ o = μ* + RT ln C o Chemical Potential Outside the Cell s s s s s Δμ~ = μ~i − μ~ o s s s i C Δμ~ = RT ln s s Co s s Chemical Potential Difference Between inside and outside of cell Chemical Potential Difference is a Concentration Difference standard conditions RTlnCj = concentration component (activity) Chemical Potential Nonionizing Solute like Sucrose C i Chemical Potential Difference is ~ Δμ = RT ln s a Concentration Difference s Co s C i 1.0 ~ s = = 10 Higher concentration inside cell Positive Δμ s ~ o 0 . 1 Positive Δμ Movement Out of Cell C s s Higher concentration outside cell Negative Δμ~ C i 0.1 s s = = 0.1 Negative Δμ~ Movement Into Cell o s 1 . 0 C s Chemical Potential Ionizing Solute like Potassium (K+) Chemical Potential of an Ionizing Solute u = uj* + RTlnCj + zjFE uj = chemical potential of a solute (Joules mol-1) Δμ~ uj* = chemical potential of a solute under standard conditions Δμ~ RTlnCj = concentration component (activity) zjFE = electrical potential component Zj = electrostatic charge of an ion (e.g. +1 for a monovalent cation) F = Faraday’s constant (charge on 1 mole of protons) E = electric potential of a solution (relative to ground) Nerst Equation & Electrochemical Potential 0.0 μ *j + ( RT ln C oj + z j FE o ) = μ *j + ( RT ln C ij + z j FE i ) Difference in Electrical Potential at Equilibrium At equilibrium, Electrochemical potential must be equal on both sides of a membrane Rearranging the equation or ΔE j = E i − E o = Ei − Eo = o RT ⎛⎜ C j ⎞⎟ ln i z j F ⎜⎝ C j ⎟⎠ o RT ⎛⎜ C j ⎞⎟ ln i z j F ⎜⎝ C j ⎟⎠ = μ~i + − μ~ o+ K+ K K+ [ ] + ZFE ) − (RT ln[K ] + ZFE ) [K ] + F (E − E ) = ( RT ln [K ] = ( RT ln K Δμ~ K+ o RT ⎛⎜ C j ⎞⎟ ln z j F ⎜⎝ C ij ⎟⎠ Application of Nernst Potential For a univalent cation at 25 C ⎛ Co ⎞ ΔE j = 59mV log⎜ ji ⎟ ⎜C ⎟ ⎝ j⎠ Distinguishing between active and passive transport. If Co is 10x > Ci Æ Voltage drop = 59mV + i + o i o + i i o + o Chemical Potential = Concentration + Difference Difference Electrical Potential Difference Nerst Equation Difference in Electrical Potential at Equilibrium Nernst equation 1. Applies to ions 2. At equilibrium, concentration difference is balanced by electrical potential difference 3. ΔE j Voltage Drop is balanced by the Concentration difference ΔE j = E i − E o = = Chemical Potential Difference K ΔE j = E i − E o = c oj ⎞ 2.3RT ⎛ ⎜⎜ log i ⎟⎟ zjF ⎝ cj ⎠ o RT ⎛⎜ C j ⎞⎟ ln z j F ⎜⎝ C ij ⎟⎠ Application of Nernst Potential For a univalent cation at 25 C ⎛ Co ⎞ ΔE j = 59mV log⎜ ji ⎟ ⎜C ⎟ ⎝ j⎠ Distinguishing between active and passive transport. If Co is 10> Ci, Voltage drop = 59mV Conclusions about ion movement based on Nernst Potentials in Pea Root Cells Relevance of Proton Pumping in Plasma Membrane (Dashed lines fi passive transport Solid lines fi active transport) 1. K+ can accumulate passively in cytosol & vacuole. 2. Na+1 Ca+2 and are actively transported out of cytosol or into vacuole. 3. All anions are actively transported into cytosol from external environment. 4. Anions like NO3- and H2PO4- are transported from vacuole to cytosol. 5. Protons transported out of cytosol to external environment 1. ATPase pumps protons out of cell, causing generation of a membrane potential. 2. Pumping protons out creates more negative potential in cytosol: 1. Driving force for passive diffusion of K+ into cell. 2. Responsible for proton cotransport of anions into cytosol 3. In soil, H+ displaces other nutrient cations. Lyotropic series Al+3>H+>Ca+2>Mg+2>K+1=NH+4>Na+1 Membrane Transport Proteins 1. Channels 2. Carriers 3. Pumps & Primary active transport 4. Secondary active transport Symport Antiport 5. Aquaporins 6. ABC Binding Cassettes Membrane Transport Proteins - Channels 1. Transmembrane protein with specific size and charge 2. Transport is always passive. 3. Usually limited to ions & water. 4. Channels variation regulated by 1. Hormones 2. Voltage 3. Light 5. Rapid ≈ 108 molecules/second 6. K+ Channels may be 1. Inward rectifying 1. E.g. in stomatal opening 2. Outward rectifying 1. E.g. in stomatal closing Membrane Transport Proteins - Carriers 1. Proteins but pores don’t cross membrane 2. Carriers bind transported molecules. 3. Highly specific. 4. Rate: 100-1000 molecules/second (106 times slower than channel transport) 5. Saturation kinetics. 6. Can be passive = facilitated diffusion or active transport. Membrane Transport Proteins - Primary Active Transport 1. Require source of energy 1. ATP…called ATPases 2. Oxidation-reduction 3. Light (bacteriorhodopsin) 2. 1o Active Transport Membrane Proteins = Pumps 3. Transport: 1. Protons 2. Ions 3. Large Organic Molecules 4. Can be electrogenic 1. Leads to charge imbalance 5. Can be electroneutral 1. No change in electrical potential Proton Transport in a plant cell Membrane Transport Proteins Channels Carriers Pumps Solute Concentration Accumulated Protons Kinetic Analyses of Transport Distinguishing between simple diffusion and Carrier Transport. Carrier transport Æ saturation kinetics Membrane Potential Relies on ATP Membrane potential of a pea cell Solute transport properties can change over a range of concentrations 1. Cyanide poisons respiration. ATP synthesis blocked by CN- Solute transport Can be both carrier-mediated or through channels Water Diffusion Across Cell Membrane Sucrose transport in soybean cells Channel? mediated Diffusion across cell membrane Diffusion through aquaporin Carrier-mediated Roles of aquaporins 1. Water diffusion 2. Uncharged solute movement 3. CO2 ? Plasma Membrane H+ - ATPase 1. P-type ATPase fi enzyme is phosphorylated as part of catalytic cycle. Vacuolar H+ - ATPase 1. Electrogenic proton pumps transport protons from cytosol to vacuole. 2. 10 membrane-spanning domains 3. Regulated by pH, temperature, [ATP] 2. V-type ATPase fi enzyme is like F-ATPase (ATPsynthase) in mitochondria. 4. Inhibited by orthovanadate (a phosphate analog) 3. Rotation of subunits drives proton transport across the membrane. 5. Enzyme has an autoinhibitory domain 4. Inhibited by bafilomycin and high [nitrate] 1. Influenced by light, hormones, pathogen attack 5. Functions of Vacuolar H+ - ATPase 2. H+ - ATPase activation by 1. Dephosphorylation of residues This will 1. Drive anion uptake into vacuoles due to electrical component 2. Drive cation and sugar uptake by secondary transport (antiport). 6. Lemons are sour because of hyperacidification Membrane Transport Proteins Secondary Active Transport 1. Acidifies vacuolar sap to pH 5.5. 1. Symport 1. Anions 2. Sugars 3. Amino Acids 2. Antiport 1. Sodium ATP Binding Cassette Transporters Transport at the Tonoplast ABC transporters Æ use the energy of ATP hydrolysis directly form a phosphorylated intermediate during catalysis Æ pump organic molecules … large anionic molecules Æ Glutathione (Glu-Cys-Gly) conjugates with molecules being transported Conjugates going into vacuole Herbicides …detoxificaiton Heavy metals chelated to polypeptides…detoxification Anthocyanins…pigment accumulation Conjugates going into vacuole anti-fungal terpenes across the plasma membrane ATP Binding Cassette Transporters Transport at the Plasma Membrane End Solute Movement Transport at the Plasma Membrane