M and R Flashcards
What are the general functions of membranes?
Selective permeable barrier enclosed environment Communication recognition of signaling molecules signal generation
What are some of the specific functions a membrane can have ?
Interaction with adjacent cells absorption or secretion changing shape for transport synapses electrical signal conduction
Describe the composition of a plasma membrane
40% lipid
60% Protein
1-10% carbohydrates
How are plasma membranes stabilised?
H bonds with h2o
Give an example of a phospholipid
Phosphatidylcholine
Give an important property of phospholipid molecules
Amphipathic
How are phospholipid molecules named?
By their head
How can chain length of phospholipid molecules vary ? What are the most common lengths?
C14-C24
Most common C16 and C18
Describe the structure of a phospholipid
Glycerol backbone, 2 FAs with phosphate group and head
What is a plasmalogen? Give an example of one
Lipid not based on glycerol eg sphingomyelin
What is a glycolipid?
Lipid containing sugar
What are the two types of glycolipids?
Cerebroside - contain a sugar monomer
Ganglioside- contain a sugar oligosaccharide
What movements are lipid able to do in a bilayer?
Flip flop
Rotation
Lateral drift
Flexion
A double bond in a phospholipid has what effect?
Introduces a kink reducing packaging and therefore increasing fluidity
How can a protein move in a bilayer?
Rotation. lateral drift and conformational change
What type of movement can a lipid do but not a protein in a plasma membrane?
Flip flop - requires too much energy for the hydrophobic and hydrophilic moieties to swap.
Describe an integral protein
Hydrophobic interactions with membrane - has at least one transmembrane domain
Describe a peripheral protein
Held in place by electrostatic interactions or H bonds - no transmembrane domain
What evidence is there for proteins ?
Freeze fracture
Fractionalization and SDS page
Specificity of function
Why do proteins have reduced movement than lipids?
Tethering to cytoskeleton
Aggregates
Lipid mediated effects - areas of low cholesterol
Basolateral junctions
Where would you find the cytoskeleton?
Cytostolic face of membrane
Describe spherocytosis anemia
Spectrin depleted by 40-50% leading to spherical shaped RBCs
Describe eliptocytosis
Defect in spectrin meaning they are unable to form tetrameres leading to fragile ellipoid cells
What is topology?
Mechanisim of inserting proteins meaning proteins have very specific orientation in the membrane- membranes are therefore asymmterical.
What proportion of a membrane is cholesterol?
45%
How does cholesterol stabilise the membrane ?
Through H bonds
CHOLESTEROL ABOLISHES THE ENDOTHERMIC PHASE TRANSITION OF THE LIPID BILAYER
The sterol ring decreases packaging and increased fluidity
The long tail reduces chain motion decreasing fluidity
What types of molecules are able to easily pass through the bilayer?
Hydrophobic - oxygen, carbon dioxide, nitrogen and benzene
Small uncharged polar molecules - water, urea, and glycerol
What molecule cannot pass through the bilayer?
Ions
Large uncharged polar molecules
What are the uses of proteins in the bilayer?
Maintain ionic composition
Maintain cell volume
Maintain pH
Concentration of metabolites and anabolites
Remove waste products
Generation of ionic gradients for function
What does diffusion depend on?
Gradient and permeability
What three methods of transport are there when using a protein?
Uniport
Antiport
Symport
What is the problem with transport of molecules using proteins ?
They are saturatable
Give the extracellular concentration of Na ions
145mM
Give the extracellular concentration of Cl ions
123mM
Give the extracellular concentration of Ca ions
1.5mM
Give the extracellular concentration of K ions
4mM
Give the intracellular concentration of Na ions
12mM
Give the intracellular concentration of Cl ions
4.2mM
Give the intracellular concentration of Ca ions
10-7M
Give the intracellular concentration of K ions
155mM
Describe and give the properties of the Na+ pump
3 sodium out
2 K in
Uses one ATP
Provides the concentration gradient and therefore energy for lots of secondary active transports
Describe the NCX transporter
sodium calcium exchanger
1 calcium out for 3 sodium in
Low affinity high capacity
Electrogenic
Describe the PMCA
Plasma membrane Ca ATPase
One calcium out for one H in
Brings in H to increase electrochemical efficiency
High affinity low capacity
Describe the SERCA
Sarco(endo)plasmic reticulum Ca ATPase
One calcium out for one H in
located on ER - creates store of Ca
Describe the NHE
one H out for One sodium in
extrudes acid using NA gradient - role in pH conc
Describe the AE
Anion exchanger
Bicarbonate out and Cl in
Extrudes base and electrochemically neutral
How do proteins help control cell volume?
Movement of osmotically active ions or organic osmolytes - h20 follows
How do proteins help control cell pH?
Using Na gradient
Extrude / influx acid or base
What is the membrane potential ?
Electrical potential different across a membrane controlled by movement of ions
-20–90mV
What is the equilibrium potential ?
The potential difference across a membrane that is selectively permeable to only that ion- electrical chemical gradient equals concentration gradient leading to no net movement
How do you calculate the equilibrium potential for an ion?
Use of the Nernst Equation
E= 61/Z log 10 (conc out/ conc in)
How is the resting membrane potential brought about?
Membrane selectively permeable to K+
K+ down concentration gradient and in down chemical gradient but anions cannot follow making cell negative compared to outside.
Na pump has minor role but is mainly used to created gradients
What types of gating are there?
Ligand - Fast ( intrinsic ion channel) slow ( GPCR)
Mechanical
Voltage
Give four properties of an AP
All or nothing
Only occur if reach threshold
Distinct signals
Propagated without loss of amplitude
Define depolarisation
Cell interior less negative
Define hyperpolarisation
Cell interior more negative
During the AP which ions conductance changes rapidly and which one slowly
Na - quickly
K- Slowly
What type of feedback is involved in an AP?
Postive
Describe the channel and ion channels leading to depolarization and then repolarisation?
Na channels open leading to Na entering the cell which increases the number of Na channels open and therefore the influx of Na into the cell. This depolarises the cell.
Depolarisation opens K channels leading to influx of K into the cell and causes inactivation of Na channels stopping the Na influx resulting in repolarisation.
What happens during the absolute refractory period ?
All Na channels are inactivated- excitatory of cell is at 0
What happens during the relative refractory period?
Na channels are recovering- excitability returns to normal as number inactivated decreases
Describe accommodation
Constant sub threshold stimulus leads to more and more Na channels in the inactivated state meaning a larger stimulus for AP to be generated.
Eventually AP cannot be generated
Describe the structure of a Na channel ?
1 subunit = 4 domains = 1 functional protein
6 transmembrane domains - S4 channel voltage sensor- stimulated by change in transmembrane voltage
Describe the structure of a K channel?
1 subunit = 1 domain = 1/4 functional protein
Need 4 subunits
What is the length constant?
Distance it takes for initial signal to fall by 37% of original amplitude
What affects the length constant?
Diameter - larger = faster
Resistance - higher = faster
Capacitance - lower = faster
What is the effect on transmission of myelination? Why?
Quicker at larger axon diameter due to saltatory conductance
What effect does myelination have on resistance and capacitance ?
Increased resistance
Decreased capacitance
Where is high density of ion channels found in a myelinated neurone?
At nodes of ranvier
Describe the consequences of MS
Demyelination of neurones leads to a decreased length constant
Initially - total blockage of signal
Later - slower conductance as channels spread along the axon
How do you calculate unmyelinated neurone speed?
Proportional to square root of diameter
What is the maximal speed of an unmyelinated neurone?
20ms-1
How do you calculate the speed of a myelinated neurone?
Proportional to diameter
What is the maximal speed of an myelinated neurone?
120ms-1
At what point does speed in myelinated neurone equal that of unmyelinated?
1mm
Describe neurotransmitter release?
Ca entry though Ca channels bind to synaptotagmin
Vesicles brought close to membrane
Snare complex make a fusion pore
Transmitter released through this pore
What heads can you find on a phospholipid? What property do they all have ?
Choline, amine, amino acid, sugar
All polar
Describe the formation of the bilayer?
Amphipathic molecules form bilayer spontaneously in h20 driven by VdWs between hydrophobic tails
Structure is then stabilised by non covalent forces ; electrostatic and H bonds between hydrophilic moeites and interactions between hydrophillic moeites and h20
How are peripheral protiens removed?
pH or ionic strength
How are integral proteins removed?
Agents that compete for non polar interactions with bilayer
How is the orientation of proteins in the membrane determined?
Protein synthesis determines this with addition of highly hydrophobic stop transfer sequence. When protein is being translated and fed into ER lumen. The stop signal will remain in the ER once it has been translated - rest of the protein is translated in cytoplasm and protein will then span the membrane.
Give the properties of the stop transfer sequence
18-20 AA
Hydrophobic smaller uncharged AAs
What is a hydropathy plot? What are the axis?
y= hydropathy index
x= aa number
Shows how hydrophobic/ hydrophilic an AA sequence is
Can work out the number of TMDs a protein has
Define facilitated diffusion
Permeability of membrane to a substance is determined by specific proteins in the bilayer - modes include carrier molecules and channel proteins
What substance inhibits the Na pump?
Oubain
What happens to the NCX in ischaemia?
Decreased ATP so no Na pump leading to an increase in Na conc in the cell so the NCX reverses drawing Ca into cell.
What control is there on the NHE?
Activated by growth factors and inhibited by amiloride
Describe bicarbonate reabsorption in the proximal tube?
Na pump removes Na- NHE can therefore pump Na from the lumen into cells along gradient in exchange for H ions - H into lumen and picks up HCO3- bringing into cell.
Used to retain base for buffers
What are aquaporins?
Allow h2o to move increased easily through membrane - inclusion in membrane in kidney epithelial cells stimulated by ADH increasing water reabsorption
Describe loop diuretics ?
Block Na uptake in thick ascending limb of loop of henle
Describe amiloride
Potassium sparing diuretic acts on both ENAC and proximal NHE tubules to prevent Na reuptake
Describe the effects of excess aldosterone
Up regulate ENAC and NHE to increase Na uptake - contribute to hypertension
Treated by spironolactone
Describe glucose uptake
SGLUT1 - Na and glucose mover through faciliated diffusion- using conc gradient from Na pump
GLUT transport moves glucose from cell to blood stream
GLUT 1&3 found throughout the body - maintains basal level
GLUT 2 found in hepatocytes and pancreatic b cells
GLUT 4 found in striated muscle and adipose cells - insulin stimulates up regulation
How is it maintained that glucose never moves back into the lumen of the vessels from cells ?
Glucose is quickly converted into glucose-6-phosphate
What type of molecules move via passive transport?
Non polar molecules
How does rate change with changing the concentration gradient in passive transport?
Rate increases linearly with increase conc gradient
Describe the difference between active transport and other types of transport
Requires energy from ATP hydrolysis/ electron transport/ light
Against unfavorable conc and or electrical gradient
Describe the mitachondrial uniporter
High conc to buffer when potientally harmful- one ca in for breakdown of one ATP
Explain the issue with transporters in cystic fibrosis
Transport of Na of cell by Na pump allows NKCC2 channel but faulty CFTR protein leads to accumulation of Cl- in cell meaning water moves into cell via osmosis and viscous mucus in lumen
Explain the issue with transporters in diarrhoea
CFTR over activated by phosphorylation PKA- Cl- excessively transported into lumen water then follows.
In a 70kg male how much water is there? What is the breakdown of this ?
42L
Intracellular 28L
Extracellular 14L - interstitial 9.4L, plasma 4.6L
What would happen if there was a sudden change in Na levels ?
increase would lead to peripheral oedema or decrease would lead to shrinkage of interstitial space and blood volume –> organ misfunction
How is resting membrane potiental expressed?
Potiental inside the cell relative to outside
How can you measure a membrane potential ?
Using a very fine micropipette that can penetrate the cell and is filled with conducting solution KCl
What is the resting membrane potential of nerve cells?
-50–75mV
What is the resting membrane potential of smooth muscle cells?
-50mV
What is the resting membrane potential of skeletal / cardiac muscle ?
-80–90mV
Why is the resting membrane potential not equal to Ek of potassium?
Other ion channels are open
What does changing the permeability of K have on the membrane?
Changes Ek therefore changes resting potential.
What ions cause depolarisation of a membrane ?
Na and Ca
What ions cause hyperpolarisation of a membrane?
Cl and K
Describe fast synaptic transmission
Receptor is also an ion channel
Depolarising transmitter open channels with a positive reversal potential leading to excitation in cells causing excitatory post synaptic potential
Hyperpolarising transmitter open channels with -ve reversal potentials leading to an inhibitory post synaptic potential.
Describe slow synaptic transmission
Signal leads to channel being opened via a GTP binding protein.
Define an action potential
Change in voltage across a membrane. they are dependent on ionic gradient and relative permeability of the membrane. Generated by an increased permeability to Na in the membrane leading to movement towards Ena
What happens to other channels during an action potential?
open or close brought on by a conformational change
Why does the cell go into a state of hyperpolarisation after depolarisation in an AP?
Voltage gated k channels open therefore increased conductance of K
Describe the actions of procaine and its usage
Topical anaesthetic for medicine and dental surgery- vasoconstrictor and increases quality of anaesthetic.
Binds and blocks Na channels meaning AP cannot rise
Blocks conduction small myelinated axon, non myelinated axon and large myelinated axon so SENSORY BEFORE MOTOR
2 pathways
hydrophobic - pass through membrane becoming charged
hydrophilic- use dependent
Describe propagation of an action potential
Change in membrane potential at one part of the membrane affects adjacent sections of axon
local current causes spread of current
conduction velocity determined by how far axon current can spread
AP initiated at site of depolarisation
How does high membrane resistance lead to high conduction velocity?
Number of channels open - lower resistance increased channels open and more loss of local current across membrane limiting spread of current
How does low membrane capacitance lead to high conduction velocity?
Capacitance is the ability to store charge - high capacitance more current to charge and cause a decrease in spread of local current
Why does large axon diameter lead to high conduction velocity?
Decreased cytoplasmic resistance
Describe the composition of myelin
40% h2o
Dry mass
70-85% lipid and 15-30% protein
Which neurones is it easier to stimulate using electrodes and why?
Myelinated neurones as decreased capacitance
When does myelination start during development and when does it finish?
4th month of development and continues into 1st year of life
How long does regeneration in PNS take ?
1-3mm
Where are L type calcuim channels found?
Lungs, muscle and neurones
What chemical blocks L type calcuim channels?
Dihydropyridines
What types of calcuim channels are found in the heart?
R and T
Describe the structure of calcium channels ?
Very simillar to Na channels
Extra proteins enabling correct regulation including glycosylation and phosphorylation sites
What are the blockers of nACHr?
Competitive and non competitive
Describe and give an example of a competitive blocker on nACHr?
Tubocurarine
Fits where ACH does - over come by increasing Ach conc
same amount of Ach will cause less depolarisation so may not reach threshold
Describe and give an example of a non competitive blocker on nACHr?
DEPOLARISING BLOCKER eg succinycholine
maintains depolarisation inhibits Achesterase
Na channels are inactivated and receptors desensitised.
sodium channels cannot activate adjacent ion channels as they are inactivated leading to MEPP - mini end plate potentials being activated.
What are MEPP?
mini end plate potentials - small random spontaneous release of vesicles causing a small amount of neurotransmitter being released
Describe myasthenia gravis and the treatment
nAchR receptors activated by target antibodies causing weakness and a reduction in end plate potentials amplitude
Treat with AchEsterase
What is ca needed for?
Control of fertilisation, proliferation, secretion, neurotransmitter, metabolism, contraction, memory , apoptosis and necrosis.
What are the advantages and disadvantages of maintaining the large gradient between Ca conc in and outside a cell?
Advantage - changes in conc occur rapidly with little Ca movement
Disadvantage - ca leads to loss of regulation and death
How is the large gradient in Ca set up?
Relative impermeability of the plasma membrane
Ability to expel Ca across the membrane
Ca buffers
Intracellular stores ( rapidly releasable and not)
Explain the activation of CaATPase?
Increased Ca conc
Ca binds to calmodulin
Ca- calmodulin complex binds to ATPase and ATPase removes Ca
What effect does buffers have on the ability of ca to diffuse?
Decrease as diffusion dependent on conc of binding molecule and level of sat
How is release from intracellular storage of calcium mediated?
By GPCRs activating Gaq which binds to phospholipid PIP2 releasing IP3 which binds to SERCA triggering release
CICR- Ca binds to ryanodine receptors on side of S/ER triggering release of increased Ca
In what organ is CICR very important and why?
Cardiac myocytes for coordinated powerful contraction- early depolarisation also allows NCX to reverse leading to increased Ca into the cell
Where is the non rapidly releasable store of Ca in the cell?
Mitachondria
When does uptake into non rapidly releasable ca stores occur?
When Ca conc high and in normal conditions to create microdomains
What is the function of Ca uptake to the mitachondria?
Ca buffering
regulate pattern and extent of signalling
stimulation of mitachondrial metabolism ( increased Ca increased Met increased ATP )
Role in apoptopic cell death
Describe ca store refilling
Termination of signal
Ca is recycled using store operated channels based on depleted signal - specific proteins interact following depletion to activate channels (STIM and ORAI)
What chemicals may be used during chemical signals?
Hormones, neurotransmitters and local chemical mediators
Define a ligand
Any small molecule that binds specifically to a receptor site
define a agonist
a ligand that produces activation of a receptor
define an antagonist
a ligand that combines with a receptor site without causing activation
define a partial agonist
agonist that stimulates a receptor but are unable to elicit maximal cell response
define a receptor
a molecule that specifically recognises a ligand or family of ligands and in response to binding brings about regulation of a cellular process.
UNBOUND THEY ARE FUNCTIONALLY SILENT
define an acceptor
operate in absence of a ligand - binding of a ligand has little or no effect
How can signaling between cells be done?
Secreted molecules or siganlling via plasma membrane bound molecules using adhesion molecules
Define paracrine
local chemical mediator secreted into interstitial space and bind with adjacent cells causing a whole tissue response
Define endocrine
hormone released into blood stream and arrives at distal site to cause a response
Where are hydrophilic molecules receptors?
On the cell surface
Where are hydrophobic molecules receptors ?
Intracellular - nucleus or cytoplasm
Give some simillarities between receptors and enzymes
specific sites
binding governed by shape of binding cleft
binding is reversible
specificity of binding confers specificity to regulation of process involved
binding induces a conformational change and change is activity of molecule
no chemical modification of ligand in binding
Give the differences between receptors and enzymes
Affinity - receptors higher affinity
ligand binded to receptor site is not modified chemically whereas substrate bound in an enzyme if modified in chemical reaction catalysed by active site
How are receptors classified?
Specific physiological signalling molecule recognised
Affinity to a series of antagonists
Explain the process of signal transduction by membrane bound ion channel
Membrane bound ion channels- binding leads to conformational change and opening of gated ion channels permitting flow of ions eg nAchR, GABA, gylcine and glutamate
Describe the structure of a membrane bound ion channel
Classic -Pentameric structure - 4 TMDs- 1 lining pore, 2 bind ligand and each has charge attracting ions
SEE DIAGRAM
Non classic - eg IP3
Explain the process of signal transduction by membrane bound receptor with integral enzyme activity
Ligand binds to extra cellular domain of receptor activity causing conformational change activating intrinsic enzyme activity contained within the structure of the receptor
eg growthe factor receptors for insulin, EGF and PDGF linked to tyrosine
Explain tyrosine kinase linked receptors
Binding of hormone to binding sites activates protein kinase activity in cytoplasmic domain which autophosphorylates tyrosine residues on cytoplasmic domain of receptor. This is recognized by transducing proteins or directly by enzymes containing phosphotyrosine recognition sites. The enzyme is then activated by phosphorylation or allosterically.
Explain the process of transduction via membrane bound receptors which couple to effectors via GTP binding regulatory proteins to enzymes or channels
Receptor bindinf results in a conformational change which activates a GTP/GDP exchange in GTP binging regulatory proteins
eg mAchR and all adreonceptors
Describe the structure of a GPCR
7 TMDs
N cytoplasmic C in cell part of G protien coupling domain
LOOK AT SHEET
Describes signal transduction via intracelllular receptors
Binds to monomeric receptors in cytoplasm or nucleus
Stabilized in resting state by HSP or chaperone proteins - activated receptor dissociates from chaperone protein in nucleus where it binds to control regions in DNA defined by specific sequences leading to regulation of gene expression
Describe by the structure of intracellular receptors
C region- binding domain
Middle - DNA binding domain
N region - nothing
How is amplification achieved?
Cascade
What in lay mans terms is receptor mediated endocytosis?
Membrane internalisation
What cells can perform phagocytosis?
neutrophils and macrophages
Explain the process of phagocytosis?
Particles bind to receptor in plasma membrane, cell extends pseudopod to permit further interactions and membrane evagination and particle internalisation via membrane zippering mechanism.
Internalised phagosome then fuses with a lysosome forming a phagolysosome and particle is degenerated
Define pinocytosis
invagination of plasma membrane to form lipid vesicle permits uptake of impermeable extracellular solutes and retrieval of PM.
What are the two types of pinocyosis
Fluid form and receptor mediated endocytosis
Define receptor mediated endocytosis
Selective internalisation of molecules into cell by binding of molecules to specific cell surface receptor
Describe the uptake of cholesterol by receptor mediated endocytosis
LDLs bind to receptors for ApoB on clathrin pits which form spontaneously these are invaginated and pinch of the PM to form coated vesicles. The vesicles are uncoated by an ATP dependent process and then fuse with larger smoother vesicle endosome. In the endosome the receptor and LDL dissociate. The transmembrane receptors is sequestered off to a domain within endosome membrane budding off as a vesicle to be recycled. LDL is then degraded
Describe an LDL particle
Originates in the liver
core of cholesterol molecules esterified to FAs surrounded by a lipid monolayer containing phospholipids cholesterol and ApoB.
Which cells express an ApoB receptor
Those requiring cholesterol
Why does the receptor and ligand dissociate in the endosome?
Endosome ph 5.5-6 maintained via ATP dependent proton pump
Describe the structure of of clathrin coated pit
Minimum stucture - triskeleton ( 3 legged) contain clathrin and 2 light chains
Triskeletons associate to form basket structure making hexagon and pentagon structures
Describe the uncoating of a clathrin pit
Carried out by an ATP dependent uncoating protein which binds and stabilises the freed coated proteins
How is the clathrin pit attached to the PM and what is its function?
By a number of integral membrane adapter proteins which form associations both with clathrin and receptors.
Locate receptors over the clathrin pit
Describe the defects that may be present in the receptors in a patient with hypercholesterolaemia?
Receptor deficiency- prevent expression of LDL receptor
Non functional receptor
Receptor binding normal - no internalisation due to deletion of C terminus meaning no interaction with a clathrin pit
Describe iron uptake
2 Fe3+ ions bind to apotransferrin forming transferrin which binds to receptors at neutral pH and is internalised. At acidic pH iron is released by apotransferrin remains bound to the receptor- complex is sorted in CURL for recycling back to PM.
Ligand and receptor are recycled
Describe uptake of occupied insulin receptors
Only over clathrin pits when bound as binding induces a conformational change so now recognised by clathrin pits. In CURL insulin remains bound to receptor and complex is degraded by lysosome
Ligand and receptor are both degraded
Why is it beneficial that both the ligand and the receptor are degraded in insulin receptor uptake ?
Allows for the reduction in number of insulin receptors on membrane desensitising the cell to continues high levels
Explain the process of transcytosis
Ligands remain bound to receptors and are transported accross the cell
Give and explain an example of transcytosis
Maternal immunoglobulins to foetus via placenta
Transfer of immunoglobulin A from circulation to bile to liver - during this receptor cleaved resulting in release of immunoglobulin with bound secretory component from receptor
What two pathogens take advantage of receptor mediated endocytosis?
Chlorea and diptheria
How do pathogens take advantage of receptor mediated endocytosis?
Binding to cells by fotuitous association with cell receptors and entering cells via pits - unfolding hydrophobic domains in membrane fusion proteins at lower pH. They they insert the membrane fusion proteins into endosome membrane leading to this fusing with the membrane and release of the genomic RNA into cell cytoplasm. They then use the host machinery to replicate RNA and caspid proteins to bind new viruses at cell membrane.
Why must signal transduction occur?
Most signals cannot enter cells to cause a response so must activate a protein on the cell surface and use this to transduce into the cell.
How do G proteins alter activity of effectors?
Via activation of guanine nucleotide binding proteins
What are G proteins responsible for?
Muscle contraction, stimulus secretion coupling, catabolic and metabolic processes , light/smell and taste perception
Describe the structure of G proteins
Same generic heteromeric structure made up of 3 subunits ( alpha, beta and gamma)
Beta and gamma bind each other so tightly they can be classed as one unit
Describe the method of activation and termination of activation of G proteins
1) Basal state G protiein present at inner face of PM predominately in heteromeric form
2) Alpha subunit binds guanine nucleotide of GTP to hydrolyse to GDP
3) Activated receptor has high affinty for G protein and protein -protein interactions.
GDP released from alpha subunit and replaced by GTP
4) Binding of GTP reduced affinity of aplha subunit for receptor and beta-gamma subunit. Therefore these are released to interact with effectors
5) Effector interactions is terminated by intrinsic GTPase activity of alpha subunit turning GTP back to GDP leading to increased affinity again.
Why can a G protein be classed as an on off switch?
On - receptor facilitated GTP/GDP exchange
Off- determined by time taken for GTP hydrolysis ( timer function)
How does cholera interact with a G protein?
Deactivation of Gs protein mediated signalling inrreversibly- Gsa subunit uncouples
How does pertussis interact with a G protein?
Prevents Gi protein activation by GPCRs- irreversibly
What does cholera and pertussis interaction with G proteins have incommon?
ADP- ribosylate specific G proteins
What are the signalling cascade three components ?
Receptor
GPCRs
Effector molecule (ion channel or enzyme )
What is the consequence of activation of Gs ?
Increased adenylyl cyclase leading to lipolysis and glycogenolysis
What is the consequence of activation of Gq ?
Increased phospholipase C leading to Smooth muscle contraction
What is the consequence of activation of Gi?
Decreased levels of adenylyl cyclase and stimulation of k channels leading to slowing of cardiac pacemaker
What is the consequence of activation of Gt?
Stimulation of cyclic GMP phosphodiesterase leading to visual excitiation
What does cyclic GMP phosphodiesterase do ?
hydrolyses cyclic GMP to 5’ GMP
What does phospholipase C do?
PIP3 –> InsP3 and DAG
Mutations to GPCRS cause what?
disease through loss or gain of function
What is retinitis pigmentosa?
Loss of function mutation to rhodpsin
What is nephrogenic diabetes insipidus?
Loss of function to V2 vasopressin receptors
What is familial male precocous puberty?
Gain of function mutation to LH receptor
What is the function of adenylyl cyclase ?
Hydrolyses cellular ATP to generate cyclic AMP which interacts with specific protien kinases to phosphorylate a variety of other proteins within a cell
What is the effect of increased adenylyl cyclase?
Glycogenolysis, gluconeogenesis, lipolysis
Relaxation of variety of types of smooth muscle, positive inotrophy and chronotrophy
What is the function of phospholipase C?
Membrane phospholipid PIP to IP3 which interacts with specific intracellular receptor on ER to allow Ca ions into cytoplasm
Describe the deactivation pathways of GPCRs?
While activated receptor susceptible to variety of kinases that phosphorylate the receptor and prevent it activating further G proteins - desensitisation phenomenon
Lifetime of alpha GTP may be limited by cellular factors that interact GTPase
Basal states are favoured
Enzymatic cascades activated downstream - act to oppse GPCRs effect
Explain the regulation of chonotrophy of the heart?
Rate of AP generation can be affected by Ach release to M2 receptors which increase potassium channel opening leading to hyperpolarisation slowing intrinsic firing rate resulting in negative chronotropic effect.
Explain the regualtion of inotrophy of the heart?
Circulating adrenaline and sympathetic innervation B1 receptors activated causes increase open probability of voltage operated calcuim channels.
Gs also indirect effect by increasing cAMP –> PKA –> phosphorylation and activation of VOCC leading to influx of Ca
Explain the regulation of arteriolar vasoconstriction
NA on alpha 1 receptors to stimulate phospholipase C and IP3 production via Gq- IP3 release on ER ca leading to contractile response
Explain the regulation of modulation of neurotransmitter release?
Presynaptic GPCRs can influence the release eg pre synaptic u-opoid receptors stimulated by endogenous opoids or analgesics to couple Ga1
Gby subunits liberated interact with VOCC to decrease Ca release
Where do drugs bind to ?
Receptors or proteins - GPCRs and enzymes most common
What concentration is important in determining drug action?
Concentration of drug molecule around receptor
When do drugs have the same concentration of drug molecules?
Drugs with equivalent molar conc NOT equivalent conc by weight
How many particles does 1M contain?
6x10^23
What is drug receptor binding governed by?
Association and dissociation rates - related to concentration of reactants and products
What must a substance have to be an agonist?
Both affinity and efficacy
What does an antagonist have in terms of affinity and efficacy?
ONLY affinity
What is affinity?
Likelihood of ligand binding to its target.
Reciprocally measured by dissociation constant
What is efficacy?
Likelihood of activity - governs receptor activation
What is Bmax?
Max binding capacity- information about receptor number
What is Kd?
Dissociation constant ( measure of affinity)- conc needed for 50% occupancy
What does a small Kd show?
Increased affinity
What is Kd known as if it is determined pharmalogically?
Ka
What does a conc response curve show?
Response in cells/ tissue
What does dose response curve show?
Response in whole body
What is E50?
Effective [ ] giving 50% maximal response - measure of potency
What is potency?
Combination of affinty, efficacy and number of receptors - tissue dependent factors
How good a drug is at generating response measured by EC50.
What is I50?
Inhibitory drugs - inhibitory concentration gicing 50% of maximum inhibition
With the same Emax does a drug have to have the same efficacy?
No could have different affinity
What is asthma?
Reversible airflow obstruction and bronchiospasm
What is the treatment goal of asthma?
Activate B2 adrenoceptors to relax airways but avoid B adrenoceptors else where in the body
Describe the properties of salbutamol and salmeterol
Salmeterol - lower Kd for B2 compared to B1 therefore increased affinity- selective efficacy
Route of adminstration limits B1 affects
Salmeterol - longer acting no selective efficacy prevents b1 side effects by different affinty
What is the problem of giving salbutamol in a drip to a patient with a heart condition?
No selectivity so causes increased heart rate by activation of B1 receptors and therefore reduces filling of coronary vessels leading to angina symptoms
When are spare receptors often seen and why?
receptors that show catalytic activity as amplification in signal transduction pathway and response limited by poster receptor event
What is the function of spare receptors?
Increase sensitivity allowing for response at decreased conc
changing receptor number affects what?
Changes agonist potency - affects maximal response that can be induced
With increased activity what happens to the number of receptors found on a cell membrane ?
Decrease
What are partial agonists?
Drugs that cannot produce maximal effect even when full receptor occupancy
Can a partial agonist be increased potent than full agonist?
Yes - depends on tissue and biological response
When can a partial agonist turn into a full agonist?
Increased receptor number - still show low efficacy but sufficient receptors
Give a use of partial agonist
Opoids
Morphine - full agonist
Buprenorphine - partial agonist - increased affinity but decreased efficacy provides adequate pain relief but decreases respiratory depression.
Addicts still get withdrawel symptoms
What are the 3 types of antagonists?
Reversible competative antagonists
Irrervsible competative antagonist
Non competative antagonist
Describe reversible competitive antagonists
Relies on dynamic equilibrium between ligands and receptors - increased conc increases inhibition
overcome by increasing agonist conc which causes a parralell shift to R of conc response curve
EG Naxolone for opoid respiratory depression
Describe irreverisble competitive antagonists
Slow or no dissociation
Causes a parrallel shift to R of agonist conc response curve and at an increase conc supress maximal response - spare receptors filled by antagonist so not enough free receptors to elicit maximal response
EG Phenoxybenzamine - non selective alpha 1 blocker used in hypertension episodes of phenochromocytoma
Describe non competative antagonists
Allosteric binding to receptor molecules - increase or decrease effect of binding of agonist
Describe desensitisation
Loss of functional response usually reversible due to down regulation of receptors and uncoupling of receptor-effector molecules eg during repeated drug application
Define tolerance
Diminishing effect of a drug due to prolonged repeat exposure - have to give increased amounts for same effect
Define supersensitivity
Enhanced response of binding of agonist
Define tachyphylaxis
Response to a drug gets smaller when a drug is given at high doses / repeatedly
Describe homologous desensitisation
Receptor decrease its response to signal when agonist present at high conc
Describe heterologous desensitisation
Prolonged stimulation to one agonist results in desenstisation to variety of agonists
Receptor is uncoupled from cascade
Give some methods of desensitisation
Phosphorylation of receptor eg binding of adrenaliune to B adrenergic receptor activates G protein - while dissociated Gby subunit activates BARK a kinase which phosphorylates residues on carboxyl terminus of receptor
Receptor internalisation
Define pharmacokinetics
What the body does to the drug
What can changing receptor number do?
Changes agonist potency and affect maximal response
Why does binding to spare receptor have no effect one 100% effectiveness been reached ?
Lack of ability post receptor binding eg ions or cascade working at maximal capacity
What is orthosteric?
Binding act active site
Give three ways that a drug is administered? How are they altered in the body?
Oral - goes through first pass metabolism which alter the drug
Parenteral- intravenous and intramuscular which goes straight into extracellular fluid
Topical - cream into the site
Apart from administration what do drugs maker need to consider
Formulation - subset of oral administration
Compliance - liquid to children
What is theraputic ration
Lethal dose over effective dose for half the population
What does an incresed theraputic ratio lead to ?
Increased therpeutic window
What is bio-avaliablity?
Proportion of drug reaching systemic circulation unchanged - affected by first pass effect
Only applies to thing that are administered orally
Sometimes measured as area under curve of orally taken in drug divide the injected one
Describe first pass metabolism
May activate or not change drug but normally inactivated reducing the oral dose decreasing bioavaliability
How can first pass metabolism be avoided ?
Changing administration route - eg GTN given sublingually
Describe drug metabolism
Most by enzymes in the liver which are inducable and inhibitable by other enzymes
How are drugs excreted?
Only free drug filtered by glomerulus- may be activate lay secreted by tubule cells
Passive reabsorption only occurs to non ionised - depends on drug pka and urging pH- increases blood conc
Aspirin overdose make the urine alkali to increase removal
What happens to a drugs half life during renal failure?
Increased
How are drugs transported in the body? What effect does this have on amount of response ?
Bound to carrier proteins such as albumin
Only the free unbound drug can interact with the receptor
Why would you give larger concentrations of class two drugs? When is this used?
Given at dose greater than number of albumin binding sites - allow more of the class 1 drug to be free in the blood leading to a greater response at a lower dose When class 1 toxic at high doses due to side effects When class I has a high affinity to albumin
What type of drug is warfarin ?
Class 1 therefore must be careful with dose as narrow theraputic window
Describe first order drug elimination
Rate of elimination is proportional to drug conc
Half life is constant
Describe zero order drug elimination
Rate of elimination is contact because the enzymes are saturated -happens to all drugs at high doses
Must be very careful when administering 0 order drugs as conc will increase massively as no more can be removed
Describe the parasympathic nervous system
Rest and digest Craniosacral Use Ach at both ganglionic neurones Long myelinated pre ganglionic fibres short unmyelinated post ganglionic fibres Ganglia located in tissues
Describe the sympathetic nervous system
Fight or flight
Thoracolumbar
Preganglionic Ach and post ganglionic use NA
Short myelinated pre ganglionic fibres and long myelinated post ganglionic fibres
Ganglia in para vertebral chain
What are the exceptions in the sympathetic nervous system
sweat glands and hair follicles are cholinergic
NANC transmitter may be released - non adrenergic non cholinergic
Sympathetic neurones to chromaffin cells in adrenal medulla are only preganglionic
Describe nor adrenaline synthesis
Tyrosine , DOPA dopamine to adrenaline
Enzymes invoked tyrosine hydroxylase , DOPA decarboxylase and the Dopamine b hydroxylase
Describe signal termination of nor adrenaline
Uptake 1 - by Na dependent high affinity transporters
Uptake 2- 5% escapes
Within presynaptic repackaged or degradaded
What are NANC? Give some examples
Non adrenergic non cholinergic transmitters that may be co released with Ach or NA
Examples include- ATP, NO, 5 hydroxytryptamine and neuropeptides
Describe the function of chromaffin cells
Found in the adrenal medulla - sympathetic nervous innervation leads to release of nor adrenaline into the blood. They are considered only to have a pre ganglionic neurone therefore use Ach
What parasympathetic effects are there on the heart ? At what receptors?
Act at the atria on M2 receptors
Causing bradycardia and cardiac conduction velocity
What parasympathetic effects are there on the lungs ? At what receptors?
Bronchial/bronchiolar contraction - M3
What parasympathetic effects are there on smooth muscle ? At what receptors?
Increased intestinal mobility /secretion M3
bladder contraction and relaxation , Penile erection and ciliary muscle and iris sphincter contraction due to NO generation
What parasympathetic effects are there on glands ? At what receptors?
Increased sweat/ salivary/ lacrimal secretion at M1 and M3
What sympathetic effects are there on the heart ? At what receptors?
Atria and ventricles
Tachycardia and positive inotrophy
on B2 receptors
What parasympathetic effects are there on smooth muscle ? At what receptors?
Arteriolar contraction/ venous contraction A1 or B2
Bronchiolar, intestinal and urinal relaxation B2
Bladder sphincter contracition B3
Radial muscle contraction - A1`
What are the basic steps in neurotransmission?
1) uptake of precursors
2) synthesis of transmitter
3) vesicular storage of neurotransmitter
4) degradation of transmitter
5) depolarisation by propagated action potiental
6) Influx of ca in response to depolarisation
7) Exocytotic release of transmitter
8) diffusion to post synaptic membrane
9) interaction with post synaptic membrane
10) Inactivation of transmitter
11) reuptake of transmitter or degradation products
12) interaction with pre synaptic
Explain how Ach is synthesised?
Enzyme choline acetyltransferase
From choline ( essential in the diet) and acetyl CoA in the cytoplasm.
Producing Ach and coenzyme A
Explain how Ach is degraded ?
Enzyme - Acetyl cholinesterase
Produces- Acetate and choline
At what points could drugs be used to inhibit Ach action?
Anticholinesterases
Depolarising blocking agents
Non depolarising blocking agents
Presynaptic toxins
How do agents that interfere with cholinergic transmission usually act?
Interaction with cholinoreceptors or cholinesterase inhibitor to decrease rate of Ach degeneration
Describe the treatment of glaucoma?
Pilocarpine applied in the form of eye drops .
Pilocarpine is a muscarinic cholinoceptor agonist
Give some examples of some nicotinic cholinoceptor antagonists
Those that have preferential ganglion- trimethaphan
Neuromusclar blocking action - tubocurarine
Describe and name some muscarinic cholinoceptor antagonists
Hyosine - anaesthetic premedication- decreases brochial and salivary secretions, prevents reflex bronchioconstriction , reduces any bradycardia induced by anaesthetic and sedative effect
Local application of poorly absorbed muscarinc cholinoceptor antagonist- used to treat bronchoconstriction in asthmatics
Homatropine - causes pupillary dilation and paralysis of accomadation - facilatating opthalmoscopic examination
Give some function od cholinesterase inhibitors
Used to acutely reverse the effects of non depolarizing neuromusclar blocking agents used in anaesthesia, treatment of glaucoma and myasthenia gravis.
recently early treatment of alzheimers
What is the rate limiting step in the synthesis of NA?
Tyrosine hydroxylase
What allows the release of adrenaline into the blood?
Presence of phenylethanolamine N- methlytransferase in chromaffin cells
What enzyme allows conversion from dopamine to NA? Where is it found?
Dopamine B hydroxylase
Within synaptic vesicles
What do the vesicular transport systems recognise ? What does this allow for?
Dopamine and Nor adrenaline allowing reuptake and recycling.
What are the likely side effects of non selective muscarinic Ach receptor agonist?
Decrease heart rate and CO
Increased bronchoconstriction and GI tract perstalsis
Increased sweating and salvation
What is a variscosity? Where are they found?
Highly branching axonal network with numerous cell like bulges- varicosity- each of which is a specialised site for Ca dependent nor adrenaline release
Outline the stages following an Ca dependent exocytosis release of NA
NA diffuses across the synaptic cleft and interacts with adrenoceptors in the post synaptic membrane to intiate signalling in the effector tissue
NA interacts with pre synaptic adrenoceptors to regulate processes within the nerve terminal
Only a small window to influence adrenoceptors as rapidly removed by noradrenaline transporter proteins
What two enzymes are involved in metabolism of non taken up NA?
Monoamine oxidase
Catechol-O-methyltransferase
How can presynaptic G protein coupled receptors regulate neurotransmitter release?
Inhibiting Ca dependent exoytosis
G protein By subunit inhibits specific types of voltage gated Ca channels reducing ca influx and neurotransmitter release
What are the classes of drugs acting on adrenergic nerve terminals?
a methyl tyrosine A Methyl DOPA CarbiDOPA Adrenergic blocking agents Indirectly acting sympathomimetic agents Uptake 1 inhibitors
Describe the mechanism of action of a methyl tyrosine
Competatively inhibits tyrosine hydroxylase - blocks synthesis of NA.
Used to inhibit nor adrenaline synthesis in pheochromocytoma
Describe the mechanism of action of a methyl DOPA
taken up and converted into a methyl noradrenaline by DOPA carboxylase and dopamine B hydroxylase.
Accumulates in the synaptic vesicles - released by Ca mediated exocytosis but differs from NA as preferentially activates pre synaptic a2 adrenoceptors reducing transmitter release
Exploited in hypertension
Describe the mechanism of action of carbiDOPA
Inhibits DOPA carboxylase in periphery but not in CNS
Used as part of treatment for parkinsons
Describe the mechanism of action of adrenergic blocking drugs
Selectively concentrated in terminals by Uptake 1. They act via a variety of mechanisms, including a local anaesthetic action reducing impulse conduction and Ca2+ mediated exocytosis and repletion of NA from synaptic vesicles. Rarely used therapeutically because of severe side effects (postural hypotension).
Describe the mechanism of action of uptake 1 inhibitors
Comprise an important class of therapeutic agents, the tricyclic antidepressants. These agents exert their therapeutic actiosn centrally and their possible peripheral actions (e.g. tachycardia and cardiac dysrhythmias) are unwanted side effects.
What drugs act at cholinergic nerve terminals
- Nicotinic Cholinoceptor Antagonists
- Muscarinic Cholinoceptor Agonists
- Muscarinic Cholinoceptor Antagonist
- Cholinesterase Inhibitors
Describe the mechanism of indirectly acting symohomimetic agents
Structurally related to noradrenaline - weak agonists at adrenoceptors
IASA recongised and taken up into synaptic vesicles where they cause leak of NA- displacement can leak into synaptic cleft by unrelated mechanism.
Extent to leakage greatly enhanced by inhibition of noradrenaline degrading enzyme MAO.
Give some important uses of adrenoceptor agonists
selective b1 agonists - dobutamine- positive inotrophy and chronotrophic effect for use in circulatory shock (BUT prone to causing cardiac dysrythmias)
Selective B2 agonists - bronchoonstriction reversal in asthma
selective a1 agonist - nasal congestants - may be given in conjection with local anaesthetic injection to cause vasoconstriction and there slow spreading of anaesthetic
Selective A2 agonists - antihypertensive agents - through stimulation of inhibitory pre synaptic receptors which decrease NA release and centrally mediated action
Give some important uses of adrenoceptor antagonists
A adrenoceptor antagonist - cause peripheral vasodilation in treatment of peripheral vascular disease
Not hypertension as cause postural hypotension and reflex tachycardia
Selective a1 adrenoceptor antagonists used in treatment of hypertension
B adrenoceptor antagonists- hypertension, cardiac dysrhythmias, angina and MI- possible side effects bronchoconstriction, bradycardia , cold extremities, insomina and depression.
