Lecture 18: Metabotropic Receptors

Ionotropic and Metabotropic Receptors

ionotropic:

• rapid onset of effects

• rapid termination of effects

• 1:1 relationship between action and response

• effects limited by the type of ion channel that is part of the receptor protein

• often bind neurotransmitter in the uM range(low affinity)

• often located near site of NT release

metabotropic:

• slow onset of effects

• slow termination of effects

• >1:1 amplification of response by G-proteins and second messengers

• Diverse possible effects from a single NT due to a multitude of second messenger-mediated signaling pathways

• usually bind neurotransmitter in the nM range(high affinity so is activated by lower concentration)

  • far from AZ so you want them to be able to respond to small amounts of NT

• often located at some distance from site of NT release(perisynaptic)

Comparing the structure of ionotropic and metabotropic receptors

ionotropic:

• multiple subunits with transmembrane regions per subunit

  • ex. ACh: pentameric=5 subunits, 4 TM regions each

  • multiple subunits allows for variation

metabotropic:

• not multiple subunits; just one protein

• 7 TM segments with amino terminal extracellularly and carboxylic terminal intracellularly

• all have the same general structure

Metabotropic receptor organization

• intracellular: G-protein binding domain associated with segments 6 and 7

  • must have conservation here since they all bind to G-proteins

• extracellular: ligand binding sites

  • must have differences between receptors since different receptors bind different ligands

• NOT AN ION CHANNEL

Metabotropic receptors

• heterotrimeric G-proteins: 3 components(⍺, β, and ɣ subunits)

  • GTP binding proteins

• Some NT have ionotropic AND metabotropic receptors

  • ACh

• Some NT have ONLY metabotropic receptors

  • dopamine

• General actions:

  • activating enzymes(go on to turn on different intracellular messengers(protein kinases, lipids, etc.))

  • interact with ion channels when activated(change in membrane potential, change in probability of transmitter release if calcium channel is modulated)

Discovery of 2nd messengers

• second messengers were involved in the transduction of an extracellular signal with an intracellular signal

• activity of liver phosphorylase that changes with activity with various hormones and neurotransmitters → measure activity in a test tube

Experiment 1:

• liver homogenate(with pieces of cytoplasm, membrane, etc.) in a test tube → add epinephrine → increased liver phosphorylase activity

Experiment 2:

• spin homogenate test tube in centrifuge → can remove membrane from bottom → now only cytoplasm left components → add epinephrine → no change in liver phosphorylase activity without membrane present

• SHOWS: membrane is necessary for increased liver phosphorylase activity

• something must have happened on the membrane to cause the inc. change in Exp. 1

Experiment 3:

• isolate epinephrine treated cytoplasm from experiment 1 → take out that treated cytoplasm + add to test tube with cytoplasm with no membrane(experiment 2) → increased liver phosphorylase activity

• MEANS: activation of something on membrane that resulted in a cytoplasmic messenger(cAMP) that can change the function of liver phosphorylase

Epinephrine receptors

• metabotropic couples to heterotrimeric G-proteins

• when G-proteins are activated → activation of enzyme adenylyl cyclase which increases cAMP production -> turns on PKA which phosphorylates liver phosphorylase→ increased activity of LP

• EPI doesn’t enter the cell only binds to extracellular receptor site, no opening of ion channel but results of intracellular cytoplasmic signaling molecule that changes cellular function

G-proteins involved in activation of metabotropic receptors

• when receptor not bound to ligand(not activated), all subunits associated with each other

  • G⍺ associated with GDP

• At rest, G-protein has high affinity with GDP(not that much in the cell)

• Default state of receptors

Activation of G-proteins

• when the ligand binds → confirmational change in receptor protein → change affinity of G⍺ to have low affinity for GDP and high affinity for GTP

  • is an EXCHANGE of GDP for GTP not alteration

• now G-protein is activated and separates into 2 subunits

  • G⍺-GTP dimer: can be signaling molecule

  • Gβ-Gɣ dimer: can be signaling molecule

• G⍺ and Gɣ have lipid tails that stick into the membrane so they remain associated with the membrane

  • can move slightly but not fully free

Inactivation of G-proteins

• not just the opposite mechanism of activation, whole new mechanism

• G⍺ subunit has intrinsic GTPase activity: changes GTP to GDP

• Once it’s GDP, G⍺-GDP component has high affinity for Gβ and Gɣ so they all come together

  • after it’s reassembled, it reassociates with receptor protein → back to resting state

• Metabotropic effects last longer than ionotropic

Termination of metabotropic signaling

• GTPase activity is slow

• so it can be modulated by effector or RGS binding

  • speeds up GTPase activity and ends action of receptor a bit faster

Experiments that demonstrate role of GTP

• remove GTP from intracellular solution: no GTP in pipette solution in patch clamp

• use GTP-ɣ-S(nonhydrolysable form of GRP): activated forever

• Use GDP-β-S(high affinity for ⍺ subunit): affinity so high it won’t allow exchange

Antisense oligonucleotide

• antisense to mRNA(20-30 nucleotides)

• prevents translation from mRNA to protein

• Ex. effects of somatostatin and ACh to act on calcium channels and reduce calcium influc

• SOM uses alpha O1, beta 3, gamma 5

• ACh uses alpha O2, beta 1, gamma 4

• multiple types of subunits that combine and interact differently with different receptors

Macromolecular complexes can spatially segregate signaling cascades

• distance prevents crosstalk

Metabotropic receptors can cause changes in membrane potential

• neuron 1 releases ACh which binds to ionotropic receptor on cell B→EPSP

• Muscarinic and LHRH receptors are at perisynaptic and extrasynaptic sites=high affinity for their neurotransmitter

  • activation of either closes resting potassium channels

Metabotropic signaling sets background “tone” of cell and regulates effect of ionotropic signaling

A: stimulate 1 = Fast EPSP causing an AP

B: simtulate 2 = no response in B cell

C: stimulate 1 with 20 APs at 30 Hz = AP train followed by slow EPSP because with repeated stiulation of this presynaptic cell, you get enough ACh release it can diffuse out and activate these muscarinic ACh receptors and close K+ channels → slow EPSP

D: stimulate 2 with 50 APs at 30 Hz: release enough NT and dense core vesicles = enough NT to diffisue to B cell → activate LHRH = late slow EPSP because LHRH needs more diffusion and bc neuron 2 is stimulated which is further from B cell

E: stimulate 1 during late slow EPSP causes AP train because B cell is already depolarized from late slow EPSP