Carriers and Pumps Flashcards
(45 cards)
transporter energetics
passive transport:
-diffusion
-channel
-uniporter
active transport:
-symporter- secondary
-antiporter- secondary
-pump- primary
transporter energetics pt. 2
-different in terms of energetics
-facilitated forms of diffusion in the form of proteins that can function as channels or uniporters, they allow substances to go from region of high [] to low []
-all called channels because when it opens allow access from both sides at the same time
channels vs transporters
-when open, channels allow access from both sides at the same time, whereas transporters you bind something like a sugar on one side and close up so the sugar is momentarily occluded within the transporter- no access to either side then opens on opposite side for the sugar to come off (at no time is the membrane accessible from both sides)
what type of transporter allows ions to go down their [] gradient but designed to capture energy and do some osmotic work?
-symporter- substance goes from region of low to high [] (up gradient) and because both substances are going in the same direction (outside to inside of cell)
-antiporter- ion going downhill has energy captured to move something else in opp direction and build up gradient
-pump- generates gradients- always move something from low to high []
-symporters and antiporters depend on the gradient being generated in the first place and pump is active transporter that sets up gradient to be used by symporters and antiporters to move large numbers of substances
ion and solute pumps
-all Exs. of primary active transport, directly link to source of energy, and transport is always uphill
-they do energy coupling- need to capture energy from one source and convert it to another source, which is the gradient –> because energy coupling is strict and complicated to happen they tend to have high specificities
-proton pump is almost always going to transport only protons
-ion channels may have selectivity for certain ions but may still transport other ions
-pumps have low transport rates compared to channels
energy sources
-pumps can harness light- found in halobacteria, surface of membrane is purple because it’s almost crystalline array of rhodopsin- carrying proteins arranged along the surface
–> bacteria capture light and light is used to drive proton or Cl pumps
-mitochondrial membrane in bacteria- source of energy is redox potential, component of ETC
-obscure transporters that can couple to different organic rxns- decarboxylation or breaking a pyrophosphate bond- 2 phosphates broken down to phosphate –> generally found in bacteria, fungi, and plants
-ATP is most common source of energy- transport ATPases use energy of ATP hydrolysis to pump ions or other solutes –> found in all types of organisms
ion gradients drive other energy-requiring processes
-primary pump on all of our cells uses quarter of all the energy the cell produces
-gradients it develops (Na and K gradients) itself form of energy that can do its own work
-ion gradients can do ATP synthesis (form of chemical energy), drive antiporters and symporters, move solutes, nutrients, metabolites across the membrane (chemiosmotic work)
-principle ways cells regulate volume is by osmotically- moving ions across membrane and water follows passively
-transporters do a lot of cell homeostasis- regulate pH, remove toxic compounds, and drugs
-ion gradient can drive flagellar rotation (form of mechanical work)
-function of channels- when they open, K and Na ions run down gradients using gradients produced by pumps- energy used by pumps is involved in signalling across cells in multi-cellular organisms
transport ATPases
-energy from ATP hydrolysis = delta G ATP
-delta G ATP resides for an ion pump in 2 components- [] gradient of ion being transported and membrane potential because ion is charged and separation of charge will develop a voltage difference across the membrane
-these 2 components ([] gradient and voltage gradient) are interchangeable
example of transport ATPases
-pump is electroneutral by moving for each cycle a positive ion in one direction and a positive ion in the opposite direction- net result is zero
-change in V is 0 and all the energy from ATP is used to build up [] gradients of protons and K across the membrane
-Ex. of this is the H/K ATPase in stomach lining- stomach is acidic because it’s the first place where digestion takes place with pH of 1-2 compared to neutral pH of surrounding cells- huge proton gradient and generated by this pump- for every ATP, it moves one proton in the stomach lumen and one K back –> electroneutral
-Ex. vacuole proton pump is the opposite Ex.- lysosome is also acidic but vacuolar pump for every ATP it transports 2 protons across into the lumen of the lysosome –> because this is uncompensated, you can quickly build up positive charge- VATPase will stall its energy of ATP hydrolysis- all of it is residing in the voltage gradient
–> to see pH gradient across the lysosome, you’d need to open a different kind of channel like the Cl transport –> Cl ions move in and protons being pumped in are compensated by Cl ions moving through different transporter and the two together work to make lysosome acidic
-work done by ATP is 2 components: [] gradient and charge –> these 2 are interchangeable
classes of transport ATPases
-F-type- F0F1-ATPase- found in the inner membrane of mitochondria and chloroplasts and bacterial membranes –> complex with multiple subunits and mostly transport ions rarely Na ions
-V-type- V0V1-ATPase- lysosomal proton pump- similar structure but doesn’t work in direction of synthesis, only uses ATP and generates proton gradients –> found in vacuoles of plants and fungi, lysosomes of animal cells and all sorts of intermembrane compartments like endosomes and synaptic vesicles
-P-type- P stands for phosphor enzyme intermediate- bunch of ion-cation pumps and some can transport lipids, peptides but most classical ones transport positively charged ions like Na, K, Ca, and so on –> different structure from F and V-type with single large subunit with sometimes an accessory subunit and found in all membranes of eukaryotic cells and also found in prokaryotes
-ABC- ATP binding cassette- CFTR, MDR- number of subunits is variable- transports solutes, drugs, entire proteins
F1F0 ATP synthases
-F1 has ATPase activity and F0 could be inhibited by oligomyosin
-lollipop-like structures- F1F0 ATP synthase- sits next to ETC (redox-driven proton pump) and protons then come back into matrix from intermembrane space through ATP synthase to make ATP –> fully reversible in vitro- you can give it ATP and put it in vesicle it will generate proton gradient and if you give it to proton gradient, ADP and phosphate, it will make ATP
–> in bacteria this works since not enough O2 to drive ETC to end and they’ll just make ATP from glycolysis, use it to work ATP synthase as ATPase like hydrolysis, and generate proton gradients
–> in the mitochondria and chloroplasts- only designed for ATP synthesis and they stop themselves from going the other way in vivo
-mitochondria have protein that gets activated when cell is anaerobic and it moves from cytoplasm into mitochondria and inserts itself in the pump- bocks it physically from going the wrong way
F1 and F0 sectors
-at no time is there a pore through membrane open on both sides
-F1 sector- sticks into matrix of mitochondria or cytoplasm of bacteria- makes ATP
–> if it falls off the membrane it’s water soluble because it’s cytoplasmic
-consists of ring of alpha (ATP binding) and beta (catalytic sites) subunits alternating with gamma subunit in the center
-F0 sector- embedded in membrane- conducts protons
-consists of C ring- each one of these has negative charge that binds proton and adjacent subunit called A subunit has positive charge that can interact with negative charge –> A subunit consists of 2 hemichannels not connected to one another
–> proton enters half channel and binds to negatively charged carboxyl group and because the other groups are proton bound they shuffle around until last one comes in and at this point proton is shed to exit to the opp side of membrane
-2 sectors connected by one subunit that extends across
proton conduction through ATP synthase
-individual subunits each has a hairpin- 2 helices
-pKa- probability of proton coming off of a binding and that defines high vs low affinity
binding change mechanism
-hexameric part has 3 catalytic subunits and 3 catalytic sites- each one of them is able to form or break down ATP reversibly but they have different affinities at any time
-tight site- ATP is bound and formed but requires energy to be kicked off
-intermediate affinity- bound loosely
-low affinity- open
–> somehow energy put into system that opens tight site and allows ATP to come off then in intermediate site it becomes a tight site where ATP can be reversibly formed and broken down
–> open site becomes intermediate affinity site
-binding change mechanism- 3 catalytic subunits goign through alternating catalysis and driven by sequential change in binding affinity
-central subunit has different contacts- gives one site special properties then rotating and contacting another site
–> rotating within hexamer as it rotates causes changes in binding affinity of each of the sites
as C subunit ring turns in the lipid bilayer, it causes torque or strain on central subunit which at intervals releases and ratchets from one active site to another
ratcheting motion of gamme (central) subunit and as it contacts each of the catalytic subunits, their affinity for ATP changes and ATP is either synthesized or released
ATP synthase
-attached throguh his tags to a nickel coated glass slide to orient hexamer to it then predicted that if you add ATP you would see it rotate
-you can add a really long actin filament through crosslink and you can see actin filament rotate when ATP gets added and pauses at 120 degree angle for each of the catalytic sites
V1V0 ATPase
-common evolutionary origin with F1F0 ATPase
-found not only within the cells but also on the PM of specialized cells like ones that secrete proteins
Ex. macrophages engulfs bacterium through acidic region to kill bacterium and in kidney there’s intercalated cells that secrete protons into urinary filtrate and osteoplasts that break bone with tight seals around bone and secrete protons to help proteases do work
-1st function- acidification- important for how receptor binds ligand as endocytose and signal for ligand to fall off is pH, as it gets acidic, there’s a conformational change and ligand falls off
-proton gradient- important for taking up solutes via proton transport Ex. synaptic vesicle- neurotransmitter has to be []ed in vesicles so when vesicle fuses there’s a release of this neurotransmitter- []ed using proton gradient- protons will go out b/c that’s direction of gradient and neurotransmitter will go in through transporters
F- and V-type pumps are similar
-V-type has more subunits
-F-ATPase is well-behaved- take 2 parts apart, the F part will just be an ATPase and bottom part will be a proton channel
-V-ATPase if taken apart the top part is dead and part in membrane is dead- doesn’t transport protons
–> V-ATPase is regulated by assembly and disassembly
Ex. in yeast cells with no glucose around you want to conserve ATP- V-ATPase falls apart and part in membrane isn’t open and V1 part in cytoplasm is specifically turned off –> only when these 2 sectors come together do things start working
disorders of V-ATPases
-certain subunits expressed in specific tissues mutated
-2 isoforms B1 and A4- if your kidney can’t secrete protons, you have metabolic acidosis and hearing deficit in the inner cells of ear you need to regulate pH
-osteopetrosis- if you have mutation in V-ATPase, bones become very dense and lethal
what mutation in which transporter phenocopy of V-ATPse mutation in osteopetrosis
Cl transporter
P-Type transporters functions
-transport all sorts of different ions- Ca, copper, Na, K
-main pump = Na pump, Na/K ATPase found in the PM of every cell in the body
–> pumps Na out and K in- Na/K ATPase
-H/K ATPase- gastric proton pump
-Ca/H ATPase- golgi, ER, plasma membrane- keep cytoplasmic Ca very low- sets it up for signalling –> Ca rushes out to cytoplasm through Ca channel and binds to different effectors
-copper/? ATPase- cofactor in many enzymes- Cu pumped into compartments and loaded onto specific enzymes- remove Cu from body that’s in excess
Reaction cycles of SR Ca-ATPase
-Ca binds on cytoplasmic side- Ca ATPase is called E1, conformation that binds Ca with high affinity –> binds ATP to form Ca-ATP complex and ATP is hydrolyzed and phosphate transferred to protein by binding to aspartic acid residue through covalent intermediate (aspartyl phosphate reaction intermediate) and ADP can come off –> high energy phosphate enzyme intermediate
-during course of conformation change from E1 high energy to low energy phosphate, Ca site is open and changes to face opposite side and sub micromolar affinity is millimolar affinity and Ca comes off
–> protons come along as counterion and bind to the same site and go in the opp direction –> cycle is finished, completed by phosphate bond being hydrolyzed
–> phosphate comes off and protons are released to the cytoplasm and ready for another cycle
-K equilibrium for the hydrolysis of ATP and phosphate enzyme intermediate is close to one –> energy is captured by protein and used to change affinity of Ca binding site from sub micromolar to millimolar
-binds substrate on one side of the membrane with high energy, changes its conformation so ion faces opp side of membrane and binding site is ripped apart and affinity falls drastically so ion can come off
-sometimes pumps can be electrogenic or electroneutral
all P-type pumps look alike
part of the membrane, ATP binding parts, sometimes 2nd subunit
Ca ATPase
-ions are binding in the middle of the membrane whereas on the cytoplasmic side, which is quite far –> this is where ATP is being bound and hydrolyzed- has to have energy coupling from a part of the pump that’s quite distant to this membrane part to change the affinity –> happens through huge motion with helices going up and down
