16. Intracellular Messengers - Part 1 "Diversity of Second Messengers"

What are first and second messengers in neurophysiology?

First Messenger:

• Ligand binds receptor on target cell (e.g., neurotransmitters, hormones)

• Initiates signal at cell surface

• Direct effect possible (ionotropic receptors open → no second messenger)

Second Messenger:

• Intracellular molecule that transduces the signal

• Often triggered by metabotropic receptors

• Activates signaling cascades → short- or long-term effects (e.g., metabolism, gene expression)

Flow:
Ligand (first messenger) → receptor → change in concentration of second messengers → signal cascade → cellular response

What’s the difference between direct and indirect neurotransmission?

• Direct transmission: NT binds ionotropic receptor → receptor itself is the channel → fast, direct gating.

• Indirect transmission: NT binds metabotropic receptor (e.g., G protein-coupled receptor, tyrosine kinase) → receptor activates a signaling cascade → channel affected indirectly → slower response.

What are the main types of second messengers and their role?

• Second messengers: Intracellular molecules that transduce signals from receptors to intracellular targets.

• Key examples:

  • cAMP, cGMP – cyclic nucleotides

  • IP3 – inosital trophosphate signaling

  • Calcium (Ca²⁺)

  • Gaseous molecules – nitric oxide (NO), carbon monoxide (CO) - NOT TESTABLE

• Function: Initiate signaling cascades in muscle and neurons; amplify or propagate extracellular signals.

What are the main types of cell surface receptors and key features of G protein-coupled receptors (GPCRs)?

• Channel-linked receptors: Ionotropic (e.g., nicotinic AChR) → direct gating, fast signaling.

• G protein-coupled receptors (GPCRs): Indirect, receptor separate from channel → triggers signaling cascade.

• Catalytic receptors: Activate enzymes → amplify signal through second messengers (involved in cell proliferation and differentiation).

• Intracellular/nuclear receptors: Alter gene expression (not a focus in this course).

What is the structure of the GPCR?

• 7 transmembrane helices (“serpentine”)

• Coupled to trimeric G protein (α, β, γ subunits)

• G protein binds GDP (inactive) or GTP (active)

• Can amplify signals via second messenger production.

How do G-proteins transmit signals from GPCRs to target proteins?

• Unbound state: GPCR not bound to agonist; G protein complex associated with GDP.

• Agonist/NT binding: NT binds → conformational change → GDP released, GTP binds α-subunit.

• Subunit activation: α-subunit (or βγ complex) separates to modulate effector proteins (e.g., channels).

• Signal duration: Active until α-subunit hydrolyzes GTP → GDP; subunits reassemble with GPCR.

• Key point: One agonist binding can activate the G protein complex for a duration determined by GTP hydrolysis.

What is the diversity and role of G protein subunits in GPCR signaling?

• Human genome ~23,000 genes → ~3.5% related to GPCR pathways.

• GPCR inactive when unbound or bound to antagonist.

• Agonist binding → activates G protein complex (GTP binds α-subunit).

• 27 Gα subunits, including:

  • Gs → stimulatory → activates adenylyl cyclase → ↑cAMP (second messenger)

  • Gi → inhibitory → inhibits adenylyl cyclase → ↓cAMP

  • Gq → involved in calcium signaling

  • G12/13 (not important for this course)

• Beta & gamma subunits: mostly regulatory; sometimes directly modulate channels.

• All G-protein can facilitate cellular signaling.

How does norepinephrine activate the beta-adrenergic receptor and downstream cAMP signaling?

• Norepinephrine binds beta-adrenergic receptor (a GPCR).

• GPCR-bound G protein sheds GDP → alpha subunit binds GTP.

• Beta & gamma subunits activate adenylyl cyclase.

• Adenylyl cyclase converts ATP → cAMP (second messenger).

• cAMP activates Protein Kinase A (PKA).

• PKA opens voltage-activated calcium channels.

• Overall effect: stimulatory, increasing cellular activity.

How does cAMP activate Protein Kinase A (PKA)?

• PKA = tetramer → 2 regulatory (R) subunits + 2 catalytic (C) subunits.

• At low cAMP: R subunits bind and inhibit C subunits.

• When cAMP increases: cAMP binds R subunits → causes conformational change.

• R subunits release the C subunits.

• Free C subunits = active PKA.

• Active PKA phosphorylates target proteins, including channels → promotes channel opening.

How do pacemaker cells in the heart generate an autonomous rhythmic heartbeat?

• Hyperpolarization opens HCN (“funny”) channels, causing slow Na⁺ influx → start of the pacemaker depolarization.

• Rising depolarization then opens T-type Ca²⁺ channels → pushes cell to threshold.

• L-type Ca²⁺ channels open at threshold → action potential upstroke.

• K⁺ efflux + Ca²⁺ channels closing → repolarizes the cell.

• Resulting hyperpolarization reopens HCN channels → cycle repeats → automatic rhythm.

How do the sympathetic and parasympathetic nervous systems regulate heart rate in pacemaker cells?

• Sympathetic (fight-or-flight): Norepinephrine ↑ funny current + Ca²⁺ currents → steeper pacemaker depolarization → faster heart rate (action potentials closer together).

• Parasympathetic (vagus nerve): Acetylcholine ↑ K⁺ efflux + ↓ funny current + Ca²⁺ currents → slower depolarization → slower heart rate.

• Heart rate is controlled by a balance of “gas” (sympathetic) and “brake” (parasympathetic), acting simultaneously on pacemaker cells.

How does sympathetic stimulation (NE) increase heart rate in pacemaker cells?

• NE binds β₁-adrenergic GPCR on pacemaker cells

• Gs protein activated → α-subunit stimulates adenylyl cyclase

  • Convert ATP → cAMP production

• cAMP + PKA both increase activity of L-type Ca²⁺ channels

  • Channels open more easily → more Ca²⁺ influx

  • Faster depolarization → higher action potential frequency

• Result: ↑ heart rate

How does parasympathetic stimulation (ACh) decrease heart rate in pacemaker cells?

• ACh released from vagus nerve → ACh binds muscarinic M₂ GPCR on pacemaker (SA node) cells

• GPCR activates G protein → βγ subunits dissociate

• βγ subunits open voltage-gated K⁺ channels → K⁺ efflux

• Hyperpolarizes membrane potential → slower depolarization toward threshold

• Slower pacemaker potential → decreased action potential frequency → lower heart rate

  • Gi subunit role: Inhibits adenylyl cyclase → reduces Ca²⁺ current

• Overall effect: Acts as a “brake” on sympathetic stimulation → balances heart rate regulation

**In lecture he kept saying vagal nerve.

How does the GPCR-mediated inositol phospholipid (IP3/DAG) signaling pathway work?

• GPCR activation: Ligand binds GPCR → α subunit of Gq protein separates from βγ subunits

• Gq protein: α subunit activates phospholipase C β (PLCβ)

• Substrate: PLCβ catalyzes PIP2 → DAG + IP3

• Second messengers:

  • IP3: Diffuses through cytoplasm → interacts with Ca²⁺ channels on ER/SR → Ca²⁺ release

  • DAG: Stays in inner membrane leaflet → activates protein kinase C (PKC) and D

• Outcome: Initiates intracellular signaling cascades → downstream effects (e.g., calcium signaling, protein phosphorylation)

• Notes:

  • PLCγ/tyrosine kinase pathways not tested in this course

  • Distinct from Gs/cAMP pathway; uses Gq/PLCβ → IP3/DAG instead of adenylyl cyclase

How does PIP₂ produce DAG and IP3 in Gq GPCR signaling, and what are their roles?

• PIP₂ (phosphatidylinositol 4,5-bisphosphate) is the substrate

• GPCR ligand binds → Gq α-subunit activates PLC-β

• PLC-β cleaves PIP₂ → DAG + IP3

  • DAG: lipid-based, stays in inner leaflet of membrane → activates PKC locally

  • IP3: soluble, diffuses through cytoplasm → binds ER/SR → releases Ca²⁺

• Outcome: DAG & IP3 act as second messengers for downstream signaling

• Note: PLC-γ (tyrosine kinase-associated) not relevant here

How does Gq GPCR signaling lead to second messenger responses?

• GPCR activated by ligand → Gq α-subunit exchanges GDP → GTP

• Gq α-subunit activates PLC-β

• PLC-β cleaves PIP₂ → DAG + IP3

  • DAG: stays in inner membrane → activates PKC

  • IP3: diffuses through cytoplasm → releases Ca²⁺ from ER/SR

• Outcome: DAG & IP3 serve as second messengers, initiating downstream cellular responses