Lecture 18 Metabotropic Receptors

Comparing the structure of ionotropic and metabotropic receptors

Ionotropic:

Ach specifically- has 5 different subunits with 4 diffferent domains- all come together to make recpetor

metabotropic receptor: no subunits, one protein

• 7 transmembrane segments- all of them

• amino terminus extracellular, carbon - inner membrane

Info about metabotropic receptors - what segments are best conserved and why

intracellular part has g-protein binding domain

• 6-7th transmembrane segment

This means that this part of the channel has a good amount of conservation because all g proteins bind here in diff metabotropic receptors

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RECEPTOR IS NOT AN ION CHANNEL!

does not pass current and no pore for things to go through

it transduces extracellular signal to intracellular

Structure of metabotropic channel- g proteins - what can it do to ionotropic channels

g-proteins are heterotrimeric- has alpha, beta, gamma components

GTP binding proteins- bind to GTP and

LIGANDS: many neurotransmitters for ionotropic also bind to metabotropic

activation of metabotropic receptor can modulate the activity of ion channels:

• changes in membrane potential

• change amount of transmitter release

• change shape of AP

Discovery of second messengers experiment pt 1,2,3

transduce extracellular signal and connect it to intracellular response

Experiment:

• observed activity of liver phosphorylase (enzyme)

• increase and decrease activity based on hormones and neurotransmitters

• measure activity in test tube- convenient

1. Make liver homogenate ( mixed up tissue)— no in tact cells there anymore

2. add epinephrine

3. increases liver phosphorylase activity

Experiment 2

1. spin in centrifuge

2. memrbane pieces are removed from solution bc they are heavy

3. have all components of cytoplasm

4. add epinephrine

5. no change in liver phosphorylase activity

Experiment 3:

6. take some epinephrine-treated cytoplasm

7. increase in liver phosphorylase activity

MEANS: epinephrine did something to the membrane that resulted in a cytoplasmic messenger that can modulate LP function

What is actually happening in liver cells when epinephrine is added

1. G- proteins are activated

2. activate adenylyl cyclase

3. cAMP

4. PKA- protein kinase A

5. liver phosphorylase

*epinephrine does not enter cell

not opening an ion channel

results in activation of intracellular cytoplasmic signalling molecule

G-proteins involved in activation of metabotropic receptors

When receptor not bound by ligand

• alpha, beta, gamma subunits are all bound together

• G alpha associated with GDP

before GDP turns into GTP, present in low concentrations in the cell (at rest)

• g protein high affinity for GDP (alpha subunit)

Activation of g-proteins step by step

1. ligand binds, causes conformational change in receptor protein

2. changes g-alpha affinity for GDP- now high affinity for GTP

• EXHCHANGE: as soon as affinity for GDP swtiches, g-alpha binds to GTP

3. now g-protein is activated— can go do its individual stuff- can separate into its individual components

4. G-alpha GTP can be signalling molecule

5. G-beta gamma dimer can also be signalling molecule (do not come apart)

   • one or all of them can be doing things- gives variety to signalling pathways

Which g-proteins remain associated with the membrane-what is the significance of this

look at slide

after activation as well

• they are NOT fixed, they can move within the membrane

• stay associated with membrane in general region by receptor

• non-soluble cytoplasmic proteins

• There are so many

Sig:

• so many signalling pathways in the cell

• each activated by g-proteins

• if you apply NE to cell, and activates NE receptors, g protein should NOT turn on all the pathways

• that is why it is important that it has a general vicinity

Inactivation of G-protein and time vs ionotropic receptor

** Not the opposite of activation

1. G-alpha subunit has INTRINSIC gtp-ase activity

   • means GTP-ase hydrolyzing GTP

2. hydrolysis changes GTP to GDP

3. G-alpha has high affinity for GDP

4. reassociates with membrane receptor

5. back to resting state

metabotropic effects last longer and takes longer

• secondary reaction to the activation of g proteins lasts longer than the receptor inactivated

• for iontropic receptors, after channel done with conducting ions, the effect is not seen

• seconds vs a few miliseconds to open channels

Modulation of metabotropic termination

GTP to GDP hydrolysis is normally slow

1. sped up sometimes through g-alpha molecule binding to effector

2. Regulation of G-protein signalling (RGS)- they are not effectors, they regulate GTPase activity of galpha

   • they know how long g-protein signalling lasts

Experiments to demonstrate role of GTPS

WHOLE CELL EXPERIMENT

• cell that you are studying will be replaced by contents of the patch electrode

• do not put GTP in patch clamp to eliminate it

voltage clamp experiment - depolarize and turn on ca2+ current

apply neurotransmitter

ca2+ current decreases in amplitude

is it GTP mediated?

methods:

• remove GTP from intracellular solution

• use GTP-gamma-S (non-hydrolyasable form of GTP)

• USe GDP-beta S (high affinity for alpha subunit)

Without GTP, g-proteins cannot do anything

• if you no longer get signal without GTP, g proteins are required in cell

With GTP-gamma S

• once GTP is activated, it is on forever

• cannot turn the signal off

GDP beta S

• will replace endogenous GDP for binding to Ga

• will not exchange for GTP

• oppsite affect of GTP gamma S

what is crosstalk

crosstalk- one NT activating everything

what is the source of variability for G-proteins and how to find which different g-proteins are coupled to which different receptors

there are multiple types of alpha, beta and gamma subunits in g proteins

• like a handful

one type of receptor coupled to a specific type of alpha, beta, gamma

HOW TO IDENTIFY DIFF GPROTEINS:

• insert antisense strand (opposite of mRNA into the cell)

• antisense to mRNA for protein

• around 30 pairs long

• binds to mRNA so it is no longer single-stranded

• double stranded mRNA cannot be translated

• cell does not make protein anymore

• wait a day or two to have previously made protein expire/degrade

• activate g-protein coupled receptor- see if still works

IF Gene you chose to do mRNA from was caused activation, signalling pathway wont work now**

Spatial segregation of G-protein subunits

macromolecular complex- all within same vicinity

because the g-proteins cannot move large distances from where they are intially hinged, they cannot activate any other complexes they are not directly coupled with

can avoid cross talk within cell

Metabotropic receptors can cause changes in membrane potential

picture- autonomic sympathetic synapse

synapse between preganglionic and post

synapses also from spinal cord that synpases on diff type of cell (C cell)

From sympathetic chain ganglia:

1. synapse releases Ach

   • effect: bind to nicotinic ach recpetors (ionotropic)

   • results in EPSP

B cell:

metabotropic receptors located at perisynaptic and extrasynaptic sites

high affinity for neurotransmitters- do not need to be in the cleft

both have negative effect on potassium channels- depolarizing the cell

• Muscarinic ach receptors

• LHRH receptor

• K+ channels- CLOSE

How do you activate the metabotropic receptors on the b b cell and what does the EPSP look like

look at chart to see what happens with stimulation to other cells

when stimulate B cell with ionotropic signalling at HGH FREQUENCY get enough acetylcholine release that it can diffuse out of the synpase and activate metabotropic receptors

close k+ channel, slow EPSP

shows that ionotropic fast and other is low

causes slow ionotropic action potentials as well

Stimulate 2:

At even higher stimulation: release NT and dense core neurotransmitters

• late, slow EPSP- more action potentials and more diffusion for LHRH to reach its receptors

What is value of late, slow EPSP

stimulate 1 during late and slow EPSP- you will get a train of action potentials instead of just getting one

it increases the strength of synapse

there are many situations where you need significant activation of fight or flight