Biological Membranes Flashcards
What are the three types of secondary transporters?
Uniporters, Symporters and Antiporters
What are Uniporters?
Like channels, a single substrate is transported each cycle
What are Symporters?
Both substrates go in the same direction. E.g leuT, GltPh, VcINDY
What are antiporters?
Substrates move in opposite directions.
What are antiporters and symporters known as?
Co-transporters
Describe Symporter alternating access mechanism?
Substrate binding site is alternated from one side of the membrane to the other.
1. Molecules bind in open facing state, substrate then binds
2. Once loaded, it transitions through the membrane to release substrates into the cytoplasm via its inward facing state.
3. Once empty, it moves back through the membrane, returning to outward-facing state.
What does alternating access maintain?
Tight coupling as it can only move through different conformational states when it is fully loaded or empty
What an example of the antiporter transport mechanism?
Ping-pong mechanism.
1. Substrate from cytoplasm binds to inward facing binding site.
2. Induces conformational change, flipping the transporter to its outer-facing state.
3. Substrate is released extracellularly.
4. This allows other substrates to bind. Once bound, this induces the inward facing state.
5. Transporter releases contents into cytoplasm. Allows substrate binding once again
Why are antiporters needed?
- Maintenance of Ionic Asymmetry
- Organelle Acidification
- pH Homeostasis
- Extrusion of toxic compounds
What are passive mechanisms of Adaptations to bacterial acid stress?
- Natural buffering capabilites of amino acids.
- Proteins
- Polyamines
What are the active mechanisms of adaptations to bacterial acid stress?
Physiological, Metabolic and Proton-consuming
What do physiological adaptations consist of?
- Modifications of the lipid bilayer.
How does modification of the lipid bilayer resist acid stress?
Enzyme produced that modifies unsaturated fatty acids into cyclopropane. Effects stability of the membrane.
Thickening of the membrane also does this, reducing ability of protons entering.
What is produced that assists modification of lipid bilayer against acid stress?
PolyP and Cadaverine - inhibitors of the outer membrane porins.
They reduce the amount of protons entering the cell.
What do proteins in the periplasm do to withstand acid stress?
Conformationally change. Chaperones can keep the proteins attached to them until the pH returns to 7, which then triggers them to refold.
How do metabolic conditions resist acid stress?
Protons are pumped out of the periplasm via a set of enzymes.
How does active H+ consuming resist acid stress?
Multiple codons in E.coli that encode enzymes and transporters, modify substrates to bind a proton to the substrate.
Pumps out proton as well as the substrate.
Which antiporters are involved in bacterial acid stress?
AdiC, GadC, CadB and PotE
What sort of mechanism is AdiC proposed to deploy?
Ping-Pong, binding of arginine and agmatine.
Steps involved in AdiC function?
Binding of arginine in outward facing state = isomerisation of the protein inwards. Leads to inward facing state conformation and release of substrate.
Reverse happens with agmatine
What happens when the outside is acidified with AdiC function?
Activity is increased
What prevents rebinding of arginine in AdiC?
The Outward facing state having a 5 fold reduced affinity for Agm. Changes in conformation reduce affinity.
Which transporters have been shown to have involvement in drug resistance?
MATES - multidrug and toxin extrusion transporters
Which was the first MATE to be discovered?
NorM
What makes NorM able to transport lipid soluble drugs?
The transport unit has a lipid bilayer accessible vestibule in it.
What is the mechanism of NorM hypothesized to be?
Allosteric Antiport mechanism. They have cations bound within them.
What is the universal lipid carrier?
C55P, attached to for flipping across the membrane
What do antiporters have involvement in to do withh bacterial structure?
Peptidoglycan synthesis
What is the process of peptidoglycan synthesis?
C55P flips precursors of peptidoglycan which makes NAM, leading to lipid 2 formation.
MurJ then flips Lipid 2 from inner to outer leaflet, where it is used by enzymes and added to peptidoglycyan.
C55P is then flipped and recycled to be used again.
What does MurJ have the same fold as?
MATES
What is different about MurJ?
Has hydrophobic groove and 2 other binding sites.
Chloride Ion in MurTA, not protons.
Sodium in MurEc.
Sodium ion site in binding sites of these is similar to PfMATE
What is the mechanism of MurJ transport?
- Tube in membrane, lipid B enters into the core this way. Tail fits into groove.
- Sodium binding induces conformational changes, and allows the head group of Lipid II to exit.
- Protein enters the outward facing transitional state.
- Chlorine rebinds and returns to the inward facing state.
What is MurJ?
Lipid 2 Flippase
MurJTa is ___ independent but ___ dependent.
Na+, Cl-
What disrupts lipids in a bilayer?
Scrambalase
Experiment showing lipid transport mechanisms?
- Tag lipids with fluorescence marker and add a reducing agent.
- In scrambling lipid, the action of protein flipping moves lipids from one side to the other = contact with fluorescence on both sides.
What does addition of Dithionite and Triton X 100 mean in lipid transport experiments?
Quenching of fluorescence, all lipids present will be exposed.
What is Dithionite?
Reducing agent used in lipid experiments
What does Dithionite cause in lipid experiments?
Reduction of lipids, quenching of fluoresence = reduction.
What are the 3 types of secondary transporter mechanisms?
- Rocker Switch - Moving Barrier
- Rocking Bundle - Moving Barrier
- Elevator - Fixed barrier
How do the mechanisms of secondary transport vary?
In position of the barrier, position of the binding site and differences in the type of barrier.
What is the aim of secondary transporter?
Bind to a substrate in one conformation and switch to a different composition to expose the binding site from one side of the membrane to the other.
What are examples of transporters in the synaptic cleft?
EAATs, GLUTs and NSS
What is the largest transporter family?
Major Facilitative Superfamily (MFS)
What is the general architecture of MFS transporters?
12 transmembrane helices, 2 symmetrical related bundles of 6.
Which transmembrane helices of MFS interact closely with the substrate and form gating helices?
1,4,7,10
Which transmembranes of MFS are involved in interdomain contacts?
2,5,8,11
Which transmembranes of MFS are involved in structure support?
3,6,9,12
What is GLUT’s mechanism of alternating access?
- OFS favoured in absence of substrate. Cytoplasmic salt bridge formed.
- Substrate binds and TM7b moves, coordinating the sugar.
- Conserved asparagine coordinates the bound sugar. Tyrosine occludes it.
Inward occlusion from TM10 - TM7 and TM10 no longer in contact, TM10 breaks. Salt bridge is lost, inward form opens.
- Substrate is released, OFS favoured once again.
What are GLUTs adapted to?
No large environmental changes, glucose-rich extracellular medium
How can we show GLUTp is proton driven?
We can analyse transporters in cells, vesicles from cells or from reconstituted liposomes
What is ascorbate added for in pH experiments?
To generate a pH gradient
What is CCCP and what does it do?
Proton Ionophore, dissappaites proton gradients
Which residues of GlcPSEs structure are essiential for protein coupling?
D22 and R102
What happens when D22 of GlcPSE is protonated?
It cannot interact with R102
What is required of GlcpSE for binding to substrates?
Protonation to enter inward facing state
What are DAT and SERT essential for?
Resetting the neural system and allowing it to fire again
What is the structure of LeuT?
12 transmembrane helices.
2 structurally repeated inverted loops
Conserved motifs
What happens when you break helices and loops form?
Backbone carbonyl bonds form, which form interactions with the ligands and sodium ions
How do we know LeuT is sodium driven?
By purifying it, reconstituting it into liposomes and doing transport assays with radiolabelled substrates.
What gives an indication that LeuT is sodium driven?
Sodium ions form intimate relationships in the binding site
Which mechanism of alternating access does LeuT deploy?
Rocking bundle
How does LeuT’s rocking bundle mechanism work?
Protein is in OFS, and binding of sodium ions helps to form the substrate binding state.
Once bound, the gates close, allowing passage across the membrane.
EAATS
Excitatory amino acid transporters
What are EAAT malfunctions associated with?
Strokes, ALS, schizophrenia and depressiom
What is GltPH an example of?
EAAT
What is the structure of GltPH like?
8 transmembrane helices, 2 reentrant loops
What play a crucial role in GltPh?
Hairpin Loops HP1 and HP2
How does the gated mechanism of GltPh?
- Gate is open, the substrate binds
- Binding causes closure
- HP1 opens, allowing substrate to move to cytoplasm
What can Primary Transporters import?
Metals/Cofactors/Nutrients/Virulence determinants
What can primary transporters Efflux?
Drugs, Antimicrobials, byproducts and Toxins
What are examples of energy sources for Primary active transporters?
Primary sources such as ATP Hydrolysis, Redoz and Light
What are examples of energy sources from Secondary active transporters?
Voltage, Sodium gradients and H+ Gradients
What is required for Primary Transporters alternating access mechanism?
A minimum of 2 Conformational changes
What are Primary transporters an example of?
Active transport, they work against gradients, meaning they are usually coupled with energy sources
What are the 4 types of primary transporter?
- P-type = Na+/K+ ATPase, Ca2+
- F-type = Mitochondrial/Chloroplast ATP synthase
- V-type = Vacuolar ATPase
- ABC = ATP binding cassette transporters
Examples of ABC transporters?
P-glycoprotein and Maltose uptake system
What are P type ATPases?
Family of Cation transporters
How do P type ATPases function?
Through reversible phosphorylation by ATP at a conservved Asparagine residue
What are the functions of Na/K+ ATPases?
- Establishment of an electrochemical cation gradient for other transporters
- Maintenance of cytosolic Ca2+
- Acidification of gastric juices
- Pump heavy toxic metals out of cells
What is the relevance of Na+/K+ ATPase in the Synaptic cleft?
It generates and maintains sodium gradients which are used for Neurotransmitter release.
Imbalance of Na+ and K+ across the membrane is essential for Action potential generation and powering of Primary transporters.
Main importance of Na+/k+ atpase?
Establishes electrochemical potential
What is Na/K+ ATPase formed of?
Primary alpha subunit composed of 10 TM helices, and 3 cytosolic domains
What are Na/K+ ATPases 3 cytosolic domains?
- Actuator - A
- Phosphorylation - P
- Nucleotide Binding - N
What can be added to minimal conformation Na/K+ ATPase?
Auxillary subunits
What are examples of auxiliary subunits that can be added to Na+/K+ ATPase?
Beta and Gamma subunits
What is Beta auxiliary subunit important for?
Trafficking of the alpha subunit to the plasma membrane
What is the gamma auxiliary subunit important for?
Tissue specific activity, fine tunes activity mainly through Na+ and K+ affinity
Which two enzymatic states compose Na+/K+ ATPase’s catalytic cycle?
E1 and E2
What does the Enzymatic state E1 mean?
High affinity state for Na+
What does the Enzymatic state E2 mean?
Low affinity state for Na+, high for K+
How does conformational change arise in Na+/K+ ATPase?
Through Hydrolysis/Phosphorylation of the P by ATP
What does phosphorylation/Hydrolysis of Na+/K+ ATPase lead to?
K+ binding due to release of Na+.
How does Na+/K+ ATPase return to its original conformation?
Through P domain being dephosphorylated, causes a conformational change meaning lower K+ affinity and higher sodium affinity
How does each Na/K ATPase domain assist conformational changes?
P domain = site that gets phosphorylated
N domain = Binds ATP which donates phosphate
A domain = Dephosphorylates P domain
How does alternating access occur in Na+/K+ ATPase?
- 3 Na+ bind - triggers phosphorylation
- Binding causes E1 –> E1-P = occluded state
- ATP is processed to ADP, phosphate released and H+ binds
- Formation of E2P state = reduced Na+ affinity
- 3Na+ fall off, 2K+ ions bind, forms occluded E2 state
6.ATP binds to N domain, reenters E1 state and K+ released
Examples of diseases caused by Na+/K+ ATPase related mutations
Hypertension and Familial Hemiplegic Migraines
Which section of Na/K ATPase is mutated for disease to occur/
Disease occurs if any part of the pump is mutated
How can Na/K ATPase be targeted for therapeutic measures?
Digitoxin is structurally related to Cardiac Glycoside
Can be administered to decrease Na+ gradients which block Na+/Ca2+ activity
Increased Ca2+ as a result leads to stronger contractions
What do ABC transporters use to induce conformational changes?
Binding and Hydrolysis of ATP
What does binding and hydrolysis of ATP in ABC transporters lead to?
Transduction of the transporter into the TM domain, allowing it to transition from IFS to OFS
What do ABC importers have that exporters do not?
A substrate-binding protein to present to the TM domain
What do substrate binding protein presence mean for ABC importers?
Allows unidirectionality
What are ABC importers important for?
Nutrient uptake, Toxin export, Antimicrobial resistance, biofuel export and lipid flipping
How are ABC transporters related to Tuberculosis?
Shown to be involved in their virulence mechanisms, recycling mechanism of Trehalose, utilises ABC transporter to traffic Trehalose back into the cell for further cell wall construction
What is Maltose uptake ABC transporter composed of?
MalE - substrate-binding protein
MalF and MalG = TM domains (each 6 helices)
Mal K= fuel source, nucleotide-binding domain, binds and hydrolyses
How does MalK act as a fuel source?
- Binds to 2 ATPs
- Conformationally linking MalK lobes together, causing transduction into the membrane
- Hydrolysis= separation and phosphate release
How is MalK conformational change linked the TM domain?
Via EAA loops, which are coupling helixes.
Each TM has one.
How do the EAA loops work?
They attach TM regions to the two lobes of MalK
What happens when the EAA loops are brought together following substrate binding?
OFS is formed and the cytoplasmic side is blocked off.
What transporter is upregulated in every resistant cancer cell?
P-glycoprotein ABCB1
