Chapter 11 Notes
Chapter 11 Notes Biol 3302
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This 7 page Class Notes was uploaded by Rachael Couch on Wednesday February 10, 2016. The Class Notes belongs to Biol 3302 at University of Texas at Dallas taught by Burr; Srikanth in Spring 2016. Since its upload, it has received 16 views. For similar materials see Eukaryotic Molecular and Cell Biology in Biology at University of Texas at Dallas.
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Date Created: 02/10/16
Chapter 11: Transmembrane Transport of Ions and Small Molecules Pumps/transport Active transport – uses ATP to move ions against their concentration gradient Secondary active transport moves ions against concentration gradient but does not use ATP (antiporter) Antiporter moves 2 molecules in different directions; one goes down the gradient and the other goes against the gradient ATPpowered pumps Pclass pumps Structure o 2 alpha subunits and 2 beta subunits – tetramer Pclass is named because at least one of the alpha subunits gets phosphorylated Each alpha subunit has an ATPbinding site Alpha subunit 3 domains N domain = nucleotide binding domain o ATP binds this domain P domain o Aspartate is phosphorylated in this domain A domain = actuation domain o Phosphorylation causes a conformational change in the P site which changes the conformation of the A site which causes a conformational change in the transmembrane segment o Net effect: N “conveys” the conformational change (“transmits the message) from the P to the transmembrane segment Beta subunit help the alpha subunit fold correctly Not involved in ion movement but a mutation in the beta subunit could affect the movement of ions because it could cause the entire + protein to fold improperly and be completely nonfunctional H pump o Plasma membrane of plants, fungi, bacteria H /K pump o Apical plasma membrane of mammalian stomach o Pumps a cation out (K ) for every proton pumped in Na /K ATPase pump o Plasma membrane of higher eukaryotes o Net effect: 3 Na from cytosol to exterior and 2 K from exterior to cytosol Active transport both sodium and potassium are pumped against their concentration gradient + o Protein synthesis requires a high K concentration o Steps 1) Opening in the transmembrane segment faces the cytosol 2) Sodium binds first The pump has a higher affinity for sodium than potassium when not phosphorylated 3) ATP binds the N domain 4) Phosphate is transferred from ATP to aspartate in P domain ADP is released 5) Phosphorylation induces a conformational change that is transmitted by the A domain Opening is now facing the exterior 6) Potassium binds the opening and sodium leaves In phosphorylated form, the pump has a higher affinity for potassium than sodium 7) Potassium binding causes dephosphorylation 8) Dephosphorylation induces a conformational change (transmitted by A domain) 9) Potassium is released into the cell Pump is now back to where it started – ATP can bind and start the process over o Inhibition When inhibited by drugs (ouabain/digoxen), the cytosolic potassium concentration decreases and the cytosolic sodium concentration increases + 2+ This causes the Na /Ca antiporter to function less efficiently (pumps sodium in and calcium out of cells) Increased Ca concentration in the cytosol which causes the 2+ muscle to contract strongly Ca ATPase pump o Structure 10 membrane spanning helices in each alpha unit AA in 4 of these helices form 2 high affinity calcium binding sites One site is formed from negatively charged oxygen atoms from the carboxyl groups of glutamate and aspartate as well as from water The other site is made from main and side chains oxygen atoms o 2 locations Plasma membrane of all eukaryotic cells Sarcoplasmic reticulum (SR) membrane in muscle cells SR – special kind of smooth ER in muscle o Free calcium concentrations Very small in the cytosol Higher in contracting cells than resting but still low Relatively high in the sarcoplasmic reticulum (stores calcium) o Net effect 2 calcium ions move against their concentration gradient o Steps (same process as Na /K ATPase pump) 2+ Starts with opening facing the cytosol, ATP binds, Ca binds, induces phosphorylation conformational change, calcium released outside dephosphorylation conformational change 2+ o Calmodulin – regulates all Ca pumps Allosterically activates ATPase pump in the plasma membrane when bound to calcium Catalytic alpha subunit is similar to the alpha subunit of the muscle SR 2+ Ca pump Necessary because excess calcium in the cell causes excessive contractions must be tightly regulated Process 2+ Rise in Ca concentration (from the SR lumen) causes calcium to bind to Calmodulin Calmodulin (with calcium bound) then binds to the calcium pump in the plasma membrane and activates it Pump then pumps out calcium to return to normal resting state (low conc. calcium) Vclass proton pumps Function to maintain low pH Structure o 2 domains: Hydrophilic cytosolic domain (V ) a1d transmembrane domain (V₀) Binding and hydrolysis of ATP by the subunit in V1 provides the energy for pumping of protons through the proton conducting channel No phosphorylation Locations o Vacuolar membranes in plants, yeast, other fungi o Endosomal and lysosomal membranes in animal cells o Plasma membrane of osteoclasts and some kidney tubule cells Storage of metabolites and acidity in plant vacuoles o Higher concentration of metabolites in the vacuoles than in the cytoplasm but metabolites are still moved in against their concentration gradient o Lumen of the plant vacuole is acidic (pH = 36) cytosol 7.4 o In the vacuoles – 1 Vclass ATP powered proton pumps (sodium, calcium, and sucrose), 1 pyrophosphatepowered pump, 3 antiporters and 2 ion channel proteins o Vacuoles have a high concentration of protons and sucrose o Steps 1) Protons are pumped into the vacuole (against their gradient) by H pumping proteins that are powered by ATP hydrolysis and pyrophosphate hydrolysis 2) Protons are pumped down their concentration gradient from the inside of the vacuole to the cytosol Pumping the protons out does NOT change the pH 3) The energy gained from moving the protons down their concentration gradient is used to move sucrose, sodium, and calcium against their concentration gradient 4) The movement of positively charged molecules (sodium, calcium, and sucrose) into the vacuole causes chloride and nitrate to be pumped in via ion channel proteins This acidifies the lumen – not the buildup of protons Lysosomes o Uses 1 ATPpowered Vclass pump and a chloride ion channel o 1) ATP is used to pump protons from the cytosol into the lysosome This creates a potential difference/electrical potential (of 20mV) between the inside (+) and outside () = chemical energy Pumping the protons in does NOT change the pH, it only creates a potential difference Still neutral pH o 2) The electric potential causes chloride to come into the lumen of the lysosome This acidifies the lumen and removes the electric potential Fclass proton pumps F 1 0ATP Synthase o In the inner mitochondrial membrane in eukaryotes and plasma membrane of prokaryotic cells o Makes by using a proton gradient Moving protons down their concentration gradient moves a rotor which drives the catalytic portion of the pump production of ATP Other location thylakoid membrane of chloroplast ABC superfamily 2 transmembrane domains o Each transmembrane domain has 10 membrane spanning helices which form the channel for the metabolite to move from one side of the membrane to another 2 cytosolic ATPbinding domains Involved in the transport of metabolites (amino acids, vitamins, sugars, peptides) Location o Bacterial plasma membranes (amino acids, sugar, and peptide transporters) o Mammalian plasma membranes (transporters of phospholipids, small lipophilic drugs, cholesterol, other small molecules) E.coli BtuCD protein – a vitamin B12 permease o AKA permease because it helps the vitamin permeate from the outside o Brings vitamin B12 from the outside to the inside of the cell o The quantity of permeases in the cell membrane is (regulated) inducible by 1. Concentration of the nutrient in the medium 2. Metabolic needs of the cell Won’t bring in the nutrient unless deprived o Vitamin B12 flows through a porin protein to the periplasmic space Porin proteins allow small molecules to flow through the outer membrane o A soluble Vitamin B12 binding protein binds to the vitamin and directs it to the transmembrane domains of the permease o Permease then carries the vitamin across the plasma membrane Mammalian ABC transporters o All flippases o Same structure as prokaryotes o Discovered from multi drug resistance (MDR) Drug resistance comes from the ability to transport the drugs out of the cell into a different area to be degraded Can transport drugs because they are similar in structure to endogenous toxins o 50 different mammalian transporter proteins are known o Normal function of these proteins is to transport various natural and metabolic toxins into the bile, or kidney or lumen of the intestine o In general, defect in ABC protein disease o 4 different human ABC proteins Protein Tissue expression Function Other ABCB1 (MDR1) Adrenal, kidney, brain Exports lipophilic drugs 170,000 MW ABCB4 (MDR2) Liver Exports phosphatidyl choline into bile ABCB11 Liver Exports bile salts into bile CFTR Exocrine tissue Transports Cl ions Defect cystic fibrosis Experiment studying ABCB4 (MDR2) o Secretory vesicles that contain ABCB4 protein are isolated o The flippase faces the cytoplasm, with the APase domains on the cytosolic side o 1) Fluorescent phospholipids (PC) are added Position themselves in the membrane bilayer, mostly in the cytosolic leaflet o 2) ATP is added to half of the vesicles (those without ATP serve as a control) With ATP – All the fluorescent phospholipids move to the inside of the vesicle Without ATP – all the phospholipids remain on the outside High fluorescence is seen in both cases An see fluorescence from both leaflets o 3) All vesicles are quenched Quenching only works on the cytosolic (outside) leaflet In the group without ATP, more fluorescence is lost because most of the labelled phospholipids were on the outside and thus were quenched o 4) Add detergent (breaks open membrane) to all and quench Causes the bilayers to form micelles which exposes all of the phospholipids – only tails on the inside Both groups go to 0 fluorescence o Graph/diagram o Conclusion Contains flippase activity If not, there would not be a difference between ATP and no ATP added From this, it is inferred that all the ABC proteins have flippase activity because they are all structurally similar o Same experiment with “insideout” secretory vesicles (vesicles outside the cell) Cytosolic leaflet faces inside, exoplasmic leaflet outside ABCB4 protein still faces the cytoplasmic leaflet with the ATPase domains now on the inside of the vesicle PC integrates mostly into the exoplasmic leaflet ATP is added to some – only moves the few on the inside to the outside Those w/o ATP – mostly stay on outside – a few remain on inside Outside (exoplasmic leaflet) is quenched With ATP loses ALL fluorescence W/o ATP lose almost all (low or nondetectable levels) of fluorescence Maintenance of cytosolic PH Involves several antiporters Na HCO /Cl3antiporter increases cytosolic pH + 3 o Imports one Na ion and a HCO ion in exchange for the export of one Cl ion o The HCO ion 3issociates into CO and OH 2 o The OH combines with the H to form water o This raises the overall pH of the cytosol Na /H antiporter also increases cytosolic pH + + o Couples entry of one Na ion with the export of one H ion At high pH(>7.5) anion antiporter Cl/HCO comes i3to play o This protein exports one HCO ion f3r import of every Cl ion Cytosolic pH is maintained at 7.4
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