Receptors Flashcards
what are the three key sort of functions of ion channels?
- transport ions across the membrane
- allow Ca2+ influx for muscle contraction
- maintain membrane potentials
what are some common structural features of ion channels?
Alpha (⍺) helices - a right hand-helix conformation
Beta (β) sheets - strands connected laterally by at least two or three backbone hydrogen bonds, forming a sheet.
Subunits – single protein that forms with others to form protein complex
Transmembrane domain (TM) – protein that spans the width of the membrane from the extracellular to intracellular sides usually a helical shape
P-loop or pore – pocket where ion will bind
how are ion channels classified?
what is the earliest ion channel?
Classified by gating mechanism and ion selectivity (dependent on size and the Aa.s lining the pore)
All channels are evolved from pH-regulated K+ channel KcsA from bacterium streptomyces lividans
all ion channels have… (x2)
Transmembrane proteins typically with 2+ alpha helices
2-6 subunits (proteins combining to form the protein complex) surrounding the pore
explain the structure of a K+ channel (and the kind of ion channel it is)
its a simple ion channel -
2 Transmembrane domains, more tight on the cytosolic side of the bilayer (like a V) to form the ‘gate’ (but simple ion channels don’t have to be gated)
K+ must lose its water molecule to fit through (example of channel size contributing to selectivity)
Gate can be controlled by membrane potential, mechanical stress or ligands
what are the two main functions of voltage gated ion channels?
- Used to create action potentials via Na+ and K+
- Ca2+ influx for contraction
what structural features do voltage-gated ion channels have?
Additional S1 and S4 domains - detect voltage
Large polypeptides that extend into the cytoplasm - important for regulation of the channel
Plugging mechanism - literally a string of amino acid that blocks the pore when signalled to do so
what are transient receptor potential channels?
like voltage gated ion channels but they respond to chemicals and physical stimuli
for example TRPV responds to heat and capsaicin - spicy foods
give an example of an intracellular ligand-gated ion channel with detail on how it works
nucleotide gated channel -
C terminus has binding site for the cyclic nucleotide which opens gate (intracellular ligand)
N terminus (intracellular) has site for calmodulin to bind when there is enough Ca2+ in order to close gate
which ion channels have -
- p-loop
- the most a helices across the membrane
- S1 and S4
- plugging mechanism
- gated
- all, this is the pore
- voltage gated can have from 6-24 (simple channel has 2, TRP and ligand-gated have 6)
- voltage gated channel as these are the voltage sensing domains
- voltage-gated and TRP
- all (tho simple channels don’t have to be gated)
similarities and difference between the P2X family, glutamate family and nicotinic receptor family?
all Na+ and K+ selective (some can be Ca2+ selective)
each have different subunits within the families that can combine in many different ways
differences -
P2X = trimeric, ATP is the ligand
glutamate = tetrameric
nicotinic = pentameric (includes nAChRs, 5-HT, GABA)
disease caused by a mutation in nAChRs in the hippocampus?
autosomal dominant frontal lobe epilepsy
mutation causes slow unblocking of the channels which somehow causes an inc in NT release = seizures
what are some key features of nAChRs?
pentameric/cys-loop
4TM domains with intracellular and extracellular loop between M3 and M4
more ‘pliable’ - allow Na and K and Ca through
example of how subunits creating diversity in receptors is ideal for drug targets?
nAChRs are all over the brain and at NM junctions. treating for example tobacco dependency comes with the problem of not wanting to effect allllll the nAChRs and cause loads of side effects.
if the nAChRs involved in tobacco dependency have certain subunits this can hopefully be targeted
we know polymorphisms have been linked to tobacco dependency as well as resistance
glutamate receptors - how do these work?
main NT of the brain, has an inverted pore
ligand (glutamate) binds in clefts on the receptor that close as this happens like a clam, this closing is what pulls the pore open
glutamate receptors - how do they show diversity?
Three kinds, each with their own isoforms (AMPA with 4, NMDA and kainate each with 5 isoforms)
This diversity is generated by having more than one gene for glutamate receptors but also splicing/RNA editing
RNA splicing -
Each subunit for AMPA receptors has two isoforms, flip and flop. This is a result of alternate splicing of two exons
Flop = faster desensitisation rate and reduced current responses to glutamate than flip
how can RNA editing go wrong in glutamate receptors?
The M2 subunit - lines the pore so essential for selectivity - has an isoform called GluA2. This has a Q/R site - meaning a glutamine needs to be changed to an arginine. Without this editing pore is constantly open to Ca2+ = overexcited = seizures = early death in mice
what receptor is effected when a stroke occurs?
NMDA (involved in memory and neuronal plasticity) is overstimulated = too much glutamate = neuronal death
give key features of P2X receptors
3 subunits, 2 TM helices
large extracellular domain
3 ATP molecules need to bind to open channel
widely expressed
P2X 1 - 7 subtypes of subunits
GPCRs - what are some general aspects of their structure?
7 alpha helices span the membrane, linked by three intra and three extracellular loops
Protein binds at C terminal end
Movement of TM domains 5 and 6 allow room for G protein to bind
Differences in the extracellular domain = diversity/many possible ligands.
Around 25 families
what are the steps in the general mechanism of a GPCR?
Ligand binds and activates the receptor (exposing binding site for G protein)
G-protein binds to the receptor, and in doing so,
alpha subunit exchanges GDP for GTP
alpha subunit moves through membrane and binds to target ion-channel or enzyme
Elicits cellular response
explain how thrombin and protease activated receptors are an unusual kind of GPCR?
When platelets encounter tissue injury or damage, they release serine proteases such as thrombin. Thrombin cleaves the N-terminus of PAR-1 and PAR-4, exposing a new amino acid sequence that acts as a tethered ligand. This tethered ligand binds to and activates the receptor itself, leading to intracellular signalling events that ultimately result in platelet activation
what are G proteins?
Membrane anchored (they can move through the membrane tho), heterotrimeric - a, b and y subunits, has GTPase activity (to regulate itself)h
how do GPCRs create diversity?
Use different extracellular domains to respond to different ligands,
and different forms of g proteins attached (Gai, Gas etc…) to cause different downstream effects
how do GPCRs turn themselves off?
Ligand bound to receptor is released, so receptor turns off
Still need to deactivate the G protein -
Must hydrolyse the GTP back to GDP which takes around 15 seconds
It then returns to its inactive state
Often assisted by RGS proteins (regulators of G protein signalling proteins) in order to speed up or slow down this process depending on what is needed
long one - might want to write it down…
what are the 6 families of GPCRs and their effectors?
provide an example of the physiological responses each type elicits
Gi-a = inhibits adenylate cyclase (can result in closing of Ca2+ channels or opening of K+ channels)
negative feedback in neuronal synapses
Gs-a = stimulates adenylate cyclase to produce cAMP, which activates protein kinase A (in the case of adrenaline) can then phosphorylate loads of things.
B1 inc HR
B2 = smooth muscle relaxation
Gq-a = activates phospholipase Cb to turn PIP2 to DAG and IP3 (most common), causes calcium influx
smooth muscle contraction, vasoconstriction
Gt-a = activates cGMP phosphodiesterase to break down cGMP (its the G protein associated with rhodopsin)
G-olf-a = we know these, theyre the ones in the olfactory bulbs, sense of smell, they activate adenylate cyclase - cAMP - kinase of some kind - ion channels
G-13-a = thrombin/platelets one
what’s different when an ion channel is opened by a G protein cascade?
Ion channels opened as a result of a g protein are slower to open/close but the effect lasts longer. E.g. can be open for minutes rather than ms
scale of response caused by GPCR?
as it is a signalling cascade - one ligand activating one receptor can cause phosphorylation of millions of proteins
involvement of GPCRs in cholera?
Cholera - binds to a GPCR in the gut, Gs subunit - constantly open, so inc. in Cl and Na ion secretion = more fluid follows due to osmosis = diarrhoea and dehydration
involvement of GPCRs in whooping cough?
Whooping cough - inactivates GPCR with Gi, causing inc. cAMP and erosion of alveolar epithelium and lots of mucus causing coughing fits
mutations in GPCRs?
include an example
can be harmless, can be either loss or gain of function
uveal melanoma
Mutation in Gq subunit
Results in blocking of GTP hydrolysis so G protein is constantly activated, in growth pathways this leads to tumour formation
what are second messengers?
Small molecules inside cells that carry signals, activated by G protein
*Hydrophobic lipids confined to the membrane in which they are generated
* Small soluble molecules that diffuse through the cytoplasm (cAMP, cGMP)
* Calcium ions
adenylate cyclase structure?
10 isoforms, it has 12 transmembrane domains, 2x 6 domains (like a duplication)
Activated by Gs and inhibited by Gi
Each of the two sets of 6 transmembrane domains has a catalytic domain, when the two combine the molecule is activated
signalling cascade caused by adrenaline (Gs-a)?
Ligand binds, a subunit swaps GDP for GTP
Moves through membrane and activates adenylate cyclase
Adenylate cyclase turns ATP into cAMP
cAMP activates PKA which can activate many proteins dependent on the cell
In the case of adrenaline:
PKA phosphorylates phosphorylase kinase (PK) using ATP
PK activates glycogen phosphorylase, which leads to breakdown of glycogen to be used by the cell for fight or flight response
how is a signal turned off for Gs?
Agonist dissociates
Gas has GTPase activity and hydrolyses it back to GDP
cAMP will be broken down by phosphodiesterase
Enzymes involved will get dephosphorylated
cGMP mechanism?
litch same as cAMP but activated by guanylate cyclase
cascade for Gq?
Generates two kinds of second messengers
Phospholipase C targets the phospholipid PIP2, breaking it down into IP3 which can then move through the cytoplasm, while DAG, the other part, is hydrophobic and remains in the membrane
DAG binds to and activates protein kinase C, PKC
Lipid kinases can add phosphate groups back to the lipids (to DAG for example to make PIP2 again)
IP3 is a ligand for IP3 receptors, which open calcium channels on ER
how are there different kinds of PLC?
different isoforms of PLC, allowing for variety
They all have an x and y catalytic subunit, but have different regulatory domains.
Different regulatory domains = binds to different phospholipids in the membrane = different cascade
how does DAG activate PKC?
When DAG binds to PKC, a ‘pseudosubstrate’ dissociates from PKC, creating a space to allow other proteins to bind and become activated
Gq cascade regulation?
Phosphorylation of PLC can provide negative feedback for the GPCR signalling
why and how is intracellular calcium kept low?
kept at about 100nM by ATP driven pumps so that the cells have capacity to respond to stimuli using calcium influx
calcium pumps on ER membrane help maintain low cytosolic Ca2+
how is a cell’s calcium store of the ER replenished?
STIM is located in the ER membrane and serves as a calcium sensor. When ER calcium levels decrease, STIM undergoes a conformational change and translocates to ER-plasma membrane junctions.
At the plasma membrane, STIM interacts with ORAI proteins, leading to the opening of the ORAI calcium channels. This allows extracellular calcium to enter the cytoplasm and be taken up by the ER, thus replenishing the depleted calcium stores
how does desensitisation of GPCRs occur?
otherwise known as tachyphylaxis,
Receptor kinases - GRK - binds to the binding pocket of where the G-protein usually would, so that receptor can no longer respond
B-arrestin can also phosphorylate the catalytic domain of the receptor and also stop the G-protein from binding
Can then internalise and degrade the receptor
e.g. can occur if salbutamol is taken too much
