Lecture 14 + 15

Question 1

Inotropic Receptors

Answer 1

Used in rapid chemical transmission

Post-synaptic ligand-gated ion channels

Question 2

Metabotropic Receptors

Answer 2

Used in slow chemical transmission

Activation of membrane-expressed receptors
• Receptors couple with intracellular signaling proteins
• Often directly or indirectly modulate the activity of ion channels

Question 3

The GABAa receptor is __________

Answer 3

The GABAA receptor is ionotropic

Question 4

The GABAb receptors is __________

Answer 4

The GABAB receptors is metabotropic

• Attenuates Cav channel activity, thus reducing pre-synaptic Ca2+ influx and vesicle
exocytosis

Question 5

Metabotropic synaptic processes tend to be ______ but _______ lasting than ionotropic ones

Answer 5

Metabotropic synaptic processes tend to be slower but longer lasting than ionotropic ones

Question 6

G protein- coupled receptors (GPCRs)

Answer 6

Account for most of the SLOW synaptic processes in the nervous system

Question 7

Structure of GPCRs

Answer 7

• Ligand-activated transmembrane proteins with 7 trans-membrane helices
• Associate with cytoplasmic G-proteins via cytoplasmic regions
• III-IVandV-VIloops
• C-terminus

Question 8

Function of GPCRs

Answer 8

G proteins mediate intracellular signals

Question 9

Humans have ~______ GPCRs

Answer 9

Humans have ~800 GPCRs

Question 10

What Ligands activate GPCRs?

Answer 10

Amine NTs such as dopamine, serotonin, histamine,
norepinephrine, and nucleotides such as adenosine
• Bind in pockets between transmembrane helices

Neuropeptides
• Bind in outer regions of transmembrane helices

NTs such as ACh, GABA & L-glutamate
• Bind specialized external ligand-binding domains in GPCR N-termini

Other ligands include chemical odorants, light- activatable ligands (i.e. rhodopsin GPCRs bind retinal), cell-cell contact via extracellular proteins, and external proteolysis

Question 11

GPCRs and pharmacology

Answer 11

GPCRs are a major pharmacological target for human disease
• Both in the nervous system and elsewhere in the body

Approx. 60% of currently prescribed drugs target GPCRs

Thus, GPCRs are important ALL OVER the body

Question 12

Ligand activation of GPCRs causes…

Answer 12

G protiens to activate

Question 13

G Protien Activation

Answer 13

Binding causes exchange of GDP for GTP on the α subunit

α-GTP acts as an intracellular second messenger
• Slow and long lasting

Gβγ complex moves short distances along the membrane, and directly interacts with membrane proteins to modulate their function (e.g. ion channels)
• Less slow and less long lasting

Question 14

G Protien Structure

Answer 14

α,β & γ subunits

Question 15

Promiscuous Coupling

Answer 15

The coupling between specific GPCRs and G proteins is somewhat variable

Question 16

In the CNS, GPCR-G protein coupling tends to be _______

Answer 16

In the CNS, GPCR-G protein coupling tends to be more specific

Question 17

Types of G Proteins:

Gs

Answer 17

↑Adenylyl cyclase –> ↑ cyclic AMP –> ↑ protein kinase A

Question 18

Types of G Protiens:

Gq

Answer 18

↑ Phospholipase C –> inositol triphosphase –> Ca Diacylgylcerol –> protein kinase C

Question 19

Types of G Proteins:

Gi

Answer 19

↓ Adenylyl cyclase –> ↓ cyclic AMP –> ↑ K channels opened –> inhibition

Question 20

Types of G Proteins:

Go

Answer 20

↓ Ca channels shut –> ↓ transmitter release

Question 21

Sympathetic Nervous System

Answer 21

“Fight or Flight”

Question 22

Parasympathetic Nervous System

Answer 22

“Rest or Digest”

Question 23

Parasympathetic Nervous System:

The Vagus Nerve - Slows Heartbeat

Answer 23

• Cholinergic neurons in the vagus nerve innervate pacemaker cells in the heart sinoatrial (SA) node
• ACh release at vagus-SA node cell synapses leads to a slowing down of rhythmic AP firing
• Thus cardiomyocyte APs and contraction also slow (i.e. decrease heart rate)

Question 24

Mechanism for reduced AP frequency in SA node cells

Answer 24

involves activation of a transient K+ current

Ionophoresis of ACh, mimicking synaptic release at vagus nerve endings, creates a hyperpolarization that stops SA cell APs
• At lower physiological concentrations, ACh serves to slow the heart beat

V-clamp recording → ACh activates a K+ channel that lasts for > 1 second
• Considerably slower than ionotropic synaptic responses which lasts milliseconds

Question 25

Activation of the K+ current occurs via ACh activation of____________________.

How does this happen?

Answer 25

Activation of the K+ current occurs via ACh activation of the M2 muscarinic GPCR

Activated Gi protein βγ subunit travels a short distance along the membrane and directly interacts with a G-protein activated inward rectifying K+ (GIRK) channel

Question 26

David Clapham - Provided proof that the Gβγ subunit modulates the GIRK channel

Answer 26

Used Inside-Out Recording

1. ACh in the pipette led to increased single channel activity
   • Thus the membrane contains the necessary mechanisms for K+ channel activation (you don’t need stuff that’s in the cytoplasm)
2. Application of soluble in vitro expressed Gβγ to the bath mimicked the effect of “extracellular” ACh application (i.e. inside the pipette)

Question 27

Soejima and Noma - showed that the GPCRs and their G proteins must be quite close to the GIRK channels

Answer 27

Used Cell-Attached patch method

ACh has to be in the pipette to increase the activity of recorded channels
• ACh in the bath had no effect on the channel in the patch

Indicates very close association between the GPCR, the G proteins and the GIRK channel

Question 28

GIRK Channels

Answer 28

Also active at neuron-neuron synapses

Activated by NTs that target GPCRs coupled to Gi/Go proteins
• e.g.ACh,GABA,glutamate,dopamine,norepinephrine

Question 29

GABAb receptors

Answer 29

GPCRs that couple with Go G proteins

Question 30

Gβγ complex

Answer 30

The activated Gβγ proteins are in close proximity to the Cav2 channels
• i.e. membrane-delimited signalling

The Gβγ complex slows down Cav2 channel activation

The Gβγ complex is STUCK in the membrane

Question 31

Gα proteins

Answer 31

activated Gα proteins are liberated into the cytoplasm

Question 32

α-GTP

Answer 32

Acts as an intracellular second messenger
• Slow and long lasting,
• Amplified due to cascade activation of downstream signaling components

Question 33

In cardiomyocytes, activation of β- adrenergic GPCRs…

Answer 33

• Causes a widening of the cardiac action potentials due to ↑Cav1 channel activity
• Increased Ca2+ influx leads to stronger contraction
• Voltage lamp recording of Cav1 current reveals stronger current