Baines Flashcards
Methods for lateral movement
Fused human and mouse GFP
Heterokaryons
Fused by polyethylene glycol or Sendai virus
Rapid mixing of dyes
Rotational movement methods
Loss of fluorescence polarisation
Eosin maleimide reacts with AE1
Suggests a fast rotating and slow rotating population
Slow maybe linked to cytoskeleton
Flip flop movement methods
Membrane impermeable enzymatic probes
Side selective membrane bleaching
Freeze fracture of synapse and paranodes
RBCs show random distribution of IMPa
Other cells have adhesomes where IMPs near synapse
Overcomes free diffusion
Paranodes show high concentrations of ion channels
Mitochondrial IMPS
Subjected to electric field before freezing
Normally random, but seen at one end
Lateral diffusion after FRAP
Outer leaflet labelled
Bleach a small area
Lipid diffuse into bleached area restoring fluorescence
Diffusion coefficient can be determined
Integrins recovery is slow as bound to ECM
Single particle tracking
Used to measure lateral diffusion
Colloidal gold attached to Integrins, becomes stuck on matrix
Gold particles are corralled by the cytoskeleton
Gold-DOPE
Diffusion indexes
X10,000 difference between ACH and bacteriorhodopsin
Aggregation into purple patches stops diffusion
Hydrophobicity scales
Kyle Doolittle
Hopp-woods- potential antigenic sites of proteins. Rich in charged residues, so gives a hydrophilic index. Window size 6
False negatives and positives
Does not show the 7 TMs of GPCRs
More accurate is to use a hidden Markov model which is a statistical representation
Synthesis of class I proteins
N terminal signal sequence binds to a SRP
This SRP then binds to an SRP receptor before the protein is fed through the translocon
When both termini are on the same side it’s type I
N terminal signal sequence is cleaved by a signal peptidase
Will have the N in the lumen. Positive charge in near C
Synthesis of Class II proteins
Hydrophobic transmembrane sequence
Due to charges which flank the TM
Positive charges always face towards the cytoplasm not lumen
So if there is a positive next to the C, then type I
Positive next to N is type 2.
When the charges are swapped, this converts 2 to 1
But without swapping, the default is a type 2 with N into cytoplasm
Signal anchor will always insert in type 2 unless charges are switched
Type 2 will have the N terminus facing inwards
Mutations in the signal sequence don’t make a difference
Default in N in
With a signal sequence, N will always be in cytoplasm
Definition of channel
Approach free limits of diffusion
Not saturable
Direction by charge and gradient
Open and closing of tube
Definition of carriers
Stereo selective
Below free diffusion
Outward and inward facing
50 families
Aquaporin
6 TM helices and 2 half, 30 tilt with NPA loop
Cytoplasmic termini
Pore in tetrameric centre
His180 Arg195 make size and repel H3O
Water enters oxygen last but is switched by Positive dipoles of helix
Hg binds to C189
In tobacco plants transports CO2 shown by acidification
Potassium channel
8 TM helices, two from each tetrameric
Wide end of cone to extra cellular
C=O of pore loops coordinate to ion
4 sites but two at a time
Mutual repulsion drives through channel, no large barriers
1000/1 selective, selectivity loop removes water shell
Large vestibule rehydrates the ions
Movement of helices thought to restrict pore
Voltage gated postassium channel
4 TM helical extensions
4 beta subunits
S4 helix has Arg which is pulled to membrane
Depolarisation causes pull to extra cellular which opens channel
The M2 proton channel
Acidification of endosome release virus from receptor and detach RNA
Inhibited by amantadine
Homotetramer
4 Trps block channel, coordinate with His
As pH increases, Trp points downwards instead of across and opens channel
Two possible mechanisms-
His passes protons to water
Proton hopping of water
NA/k ATPase
3na out, 2 k in
Needed to maintain conc gradient and action of secondary transporters
A, b, y subunits
Beta is a chaperone and type 2
Gamma regulated by PKA and PKC by adrenaline , type 1
Alpha has Asp phosphorylation. Binds cholesterol to activate only at membrane
3 domains: nucleotide, actuator, phosphorylation
Drugs to affect NA/K ATPase
Ouabain- glycosides which inhibits, mimics cholesterol. Blocks NA leaving.
Digoxin- increases cytoplasmic NA, less NCX activity which increase Ca conc to give stronger contraction
Secondary transporters
Sodium always flows out to in, so after first pumping it out the reentry can be paired with another transporter
Symporters Glucose (creates internal x30,000), nucleosides
Antiporters
Extrusion of acid (NHE) or calcium (NCX)
Action potential opens Ca in membrane and SR
Moves troponin allowing contraction
Ca pumped back to SR using ATP or exchanged for NA entry
Secondary active transporters in kidney
SLC, KCC4, NKCC2 (NaKcl)
KCC3 in the PT, NCC and KCC4 in cortex and NKCC2 in outer medulla
Transporters of SLC12A are electro chemically neutral and regulate ion balance. Diuretic targets
SLC21 transporters in kidney
NKCC2 brings in NA, 2Cl, K
ROMK pumps K back out
NA is pumped back into blood by exchange for K
Cl into blood by CLCK
NCC brings in NA and Cl
TRPV5 brings in Ca
NA/K exchanger
NCX1 brings NA from blood exchanging for Ca
Ca can also be returned to blood by the H/Ca exchanger (PMCA1b)
Actions of diuretics
Sodium has a hydration she’ll
Acts on NA transporters to prevent water reabsroption
Furosemide- NA/KCl
Thiazides- NaCl
Transporters in homeostasis
Glycolysis makes pyruvate and H before kerbs making 6H MCT removes lactate and H AE removes HCO3- NHE removes H for NA NA is exchanged for K by NKA
NHE1 and metastasis
Large ATP at leading edge
Extrusion of acid
NHE1 and lysosomes are redistributed to the pseudopod tip to extrude acid and protease
Focalised proteolysis of ECM and cell attachments
Deteriorating environment gives positive feedback to NHE1
Mesenchymal to ameboid transition, moves through gaps in ECM
NHE1 regulated by cofillin
NHE1 removes 2H+ from the asp122,cofillin,his133 complex
At a low Ph His binds to PIP2
NHE1 makes alkali so loses PIP2 interaction
Complex is activated for barbed end formation
PIP2 -> IP3 and DAG by PLC
Tertiary transporter
NA/K ATPase
NA flows back in with AA1
AA1 then exchanged for AA2
Could be used to remove high amounts of AA1 to create useful exchange
GLUT1 structure
12 TM Lys mod by n-Hydroxy-succinimide esters -> 90% inhibition in Cys mutations Neutral bilayer interaction Few hydrophilic show complexes? Large N and O and cleft bottom shows C=O binding to sugars
In bacteria, transport H too to overcome energy, by mutating GLUT1 can create Glu/H symporter
Eukaryotes lost ability as constant high glucose
Beta barrel proteins
Humans have a low H gradient but high NA
Gram negative have peri plasm and peptidoglycan
Beta barrels excreted in peri plasm as hydrophilic before outer mem
Only needs 7 AA to cross
Tilted from 20-45 degrees
Porins for transport e.g. Maltose
Surface loops are antigenic sites
Variations in Eyelet in antibiotic resistant bacteria
Present in short-lived to inactive state delcour 1997
Squalene hopeless cyclase
Each half is enzyme
Substrate in ER membrane
Long chain to sterol ring
Two non polar plateaux