Lesson 8: Modulation of Synaptic Transmission-Second messengers

How do neurotransmitters act on ion channels?

Neurotransmitters can act directly or indirectly on ion channels, depending upon the way the receptor is coupled to the effector function.

What type of receptors typically mediate simple reflex behaviors and complex cognitive behaviors?

Ionotropic receptors.

What functions do metabotropic receptors modulate?

Metabotropic receptors modulate behaviors such as modification of reflex strength, helping to focus attention, setting emotional states, and contributing to long-lasting changes in neural circuits that underlie learning and memory.

What two main types of signaling do nerve cells use, and what additional role does chemical signaling play?

Nerve cells use both electrical and chemical signaling to receive and transmit information; chemical signaling also occurs within the neuron as part of its’ intracellular signaling cascade.

What fundamentally triggers signaling within neurons?

Extracellular chemical signals that bind to specific receptors on the cell membrane or within the cell.

Give examples of the extracellular chemical signals that initiate signaling within neurons.

Neurotransmitters, hormones, and trophic factors.

What is stimulated within the cell once an extracellular chemical signal binds to its receptor?

A cascade of events involving GTP-binding proteins, second messengers, protein kinases, ion channels, and other effector proteins.

How can intracellular signaling lead to longer-lasting changes in the cell?

By altering gene expression.

What is the ultimate purpose and nature of this intracellular signaling in the brain?

It is molecular signaling in nature and ultimately serves to regulate and modulate all brain functions, including circuits and systems, both temporally and spatially.

Where is chemical signaling used?

Throughout the body and inside cells, including neurons.

Aside from synaptic signaling, what other types of chemical signaling do cells use?

Cells also use paracrine and endocrine signaling, as well as autocrine signaling.

What is synaptic signaling?

Synaptic signaling is chemical transmission at the synapse, typically involving neurotransmitters.

How does paracrine signaling work?

it involves the secretion or release of chemicals onto a group of nearby target cells.

How is endocrine signaling characterized?

it is the secretion or release of hormones into the bloodstream, where they can act on targets throughout the body.

What is autocrine signaling?

it involves the secretion or release of chemicals onto the same cell that released the chemicals.

What three main components are required for chemical signaling?

A molecular signal, a receptor molecule, and a target molecule.

What is the role of the molecular signal in chemical signaling?

it transmits the information.

What is the role of the receptor molecule in chemical signaling?

it transduces the information.

What is the role of the target molecule in chemical signaling?

it mediates the cellular response.

In synaptic transmission, what serves as the signal, receptor, and target molecules?

Neurotransmitters serve as the signal (transmitting the information), neurotransmitter receptors act as receptor molecules (to transduce the information), and ion channels serve as the target molecules(mediating the postsynaptic response).

What are the typical steps involved in chemical signaling?

Recognition, transduction, transmission (of the second messenger signal to the effector), modulation of the effector (e.g., activation of protein kinases), a response, and termination of that response (usually by a feedback mechanism).

What are the three categories of signaling molecules based on their interaction with a cell?

Molecules secreted from other cells can be cell-impermeant or cell-permeant; others require direct cell-to-cell contact.

How do cell-impermeant molecules transmit signals?

They must bind to receptors located on the cell membrane.

Name some examples of cell-impermeant signaling molecules.

These include neurotransmitters, neurotrophic factors, peptide hormones, and some reproductive hormones.

How do cell-permeant molecules transmit signals?

Cell-permeant molecules can pass/go through the cell membrane and bind to receptors located inside the cell.

Name some examples of cell-permeant signaling molecules.

These include various steroids, thyroid hormone, and retinoids.

How do cell-associated signaling molecules transmit signals and where are they located?

They are located/positioned on the extracellular surface of a cell and must bind directly to like receptors on other cells via physical contact between the two cells.

• Binding of the signaling molecule causes a conformational change in the receptor protein, triggering a subsequent “signaling cascade” within the cell (whether the binding occurs at the cell membrane or inside the cell!)

• Channel-linked receptors (aka, ligand-gated ion channels) have the receptor and the transducing function as part of the same protein

• Enzyme-linked receptors (aka, catalytic receptors) are connected to an enzyme on the cytoplasmic side; there are typically protein kinases such as tyrosine kinases, that phosphorylate intracellular target proteins

• G-protein-coupled receptors regulate intracellular responses indirectly via an intermediate transducing molecule, in this case, GTP-binding proteins; there are hundreds of different G-protein-linked receptors

• Intracellular receptors (including nuclear receptors) are activated by signaling molecules that pass through the cell membrane; many of these receptors activate signaling cascades that produce new mRNA and proteins within the cell

What initial event occurs when a signaling molecule binds to a receptor protein?

Binding of the signaling molecule causes a conformational change in the receptor protein, triggering a subsequent signaling cascade within the cell. (whether the binding occurs at the cell membrane or inside the cell!)

What are channel-linked receptors?

(also known as ligand-gated ion channels) have the receptor and the transducing function as part of the same protein.

What are enzyme-linked receptors?

(also known as catalytic receptors) are connected to an enzyme on the cytoplasmic side; there are typically protein kinases, such as tyrosine kinases, that phosphorylate intracellular target proteins.

How do G-protein-coupled receptors regulate intracellular responses?

indirectly via an intermediate transducing molecule, in this case GTP-binding proteins; there are hundreds of different G-protein-linked receptors.

What are intracellular receptors?

They (including nuclear receptors) are activated by signaling molecules that pass through the cell membrane; many of these receptors activate signaling cascades that produce new mRNA and proteins within the cell.

What is the largest family of cell surface receptors?

G-proteins, with more than 1000 members.

Describe the general structure of most G-proteins.

Most have a similar structure despite their various ligands, composing a single polypeptide chain with 7 TM
\alpha -helical segments, an extracellular N-terminus, and an intracellular C-terminus.

What are heterotrimeric G-proteins composed of?

They are composed of three subunits: α, β, and γ with a variety of each subunit (e.g., there are at least 16 different α-subunits).

Which subunit of a heterotrimeric G-protein is responsible for binding and hydrolyzing guanine nucleotide GTP?

The \alpha subunit binds the guanine nucleotide GTP to hydrolyze it to GDP.

What are monomeric G-proteins also known as, and what was the first one discovered?

Monomeric G-proteins are also known as small G-proteins also convey signals from the cell surface to the cell interior.
\textit{ras} (rat sarcoma tumors) was the first monomeric G-protein discovered.

What are the common functions regulated by\textit{ras} proteins?

\textit{ras} proteins help to regulate cell differentiation and proliferation. (the virus form ras is defective, resulting in uncontrolled cell division). Other \textit{ras} proteins are involved with synaptic potentiation, vesicle trafficking, and protein and RNA trafficking out of the nucleus.

How is an inactive heterotrimeric G-protein characterized?

When GDP is bound, the \alpha subunit binds to both the \beta and \gamma subunits, forming an inactive trimer. (this is the inactive state)

What initial events lead to the activation of a G-protein upon ligand binding to a GPCR?

it causes the activated receptor to interact with the G-protein heterotrimer \alpha\beta\gamma to induce a conformational change that releases GDP to bind GTP. This allows the α subunit to dissociate from the βγ complex, activating the G-protein

Which components of an activated G-protein can bind to downstream effector molecules?

Both the \alpha subunit (after dissociating from the \beta\gamma
complex) and the \beta\gamma complex can now bind to downstream effector molecules.

How is G-protein signaling terminated?

Termination occurs when the \alpha subunit hydrolyzes GTP to GDP and organic phosphate.

What is the primary effect of neurotransmitter binding to a G-protein?

Binding of a neurotransmitter to a G-protein alters effector molecules, typically enzymes that produce second messengers.

Besides altering effector enzymes, what else can G-proteins activate?

ion channels.

How do G-proteins inhibit neuronal firing (action potentials), and which subunits are thought to mediate these inhibitory actions?

G-proteins can inhibit neuronal firing by activating ion channels (e.g., acetylcholine binding to mAChRs allows K^+ to cross the membrane). These inhibitory actions are thought to be mediated by the \beta\gamma subunits.

What effect do \alpha subunits of G-proteins have when binding to ion channels?

\alpha subunits can bind to ion channels, leading to the closing of voltage-gated Na^+ and Ca^{+2} channels, making it more difficult to generate an action potential.

Name some effector enzymes that G-proteins can alter.

Effector enzymes include adenylyl cyclase (AC), phospholipases such as phospholipase C (PLC), and phosphodiesterase.

What are the three different types of heterotrimeric G-proteins?

G_s (stimulatory), G_i (inhibitory), and G_q(activates phospholipase C).

What is the function of Protein Kinase A (PKA)?

Protein Kinase A (PKA) acts to transfer a phosphate to selected proteins, a process known as "phosphorylating"

What types of proteins are targeted by PKA phosphorylation?

ion channels, receptors, and signaling proteins.

How does phosphorylation by PKA affect targeted proteins?

it can affect either the localization of the protein or its substrate activity.

How are effector enzymes regulated in relation to G-proteins?

Effector enzymes can be stimulated or inhibited depending upon the G-protein involved.

Describe the process of phototransduction in relation to ligand-gated sodium channels and membrane potential.

In phototransduction, light activates PDE which breaks down cGMP to GMP, reducing the intracellular concentration of cGMP, causing the ligand-gated sodium channel to close and hyperpolarize the membrane

• Second messengers do not diffuse freely throughout the entire cytoplasm

• Second messengers are typically cytosolic messengers or membrane-associated messengers

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• Second messengers can be rapidly formed from precursors by enzymatic reactions

• Second messengers can be released quickly from intracellular stores (e.g., Ca⁺² ions from intracellular stores)

• Second messengers can be quickly inactivated or moved into a specific compartment (e.g., cAMP can be degraded to AMP, and Ca⁺² ions can be translocated into the ER)

• Second messengers can activate many different effector proteins (this leads to diversification and variability in signaling)

• Second messengers allow for signal amplification (e.g., a single molecule of norepinephrine (NE) can generate thousands of cAMP molecules)

What is a characteristic of second messenger diffusion within the cytoplasm?

Second messengers do not diffuse freely throughout the entire cytoplasm.

What are the typical locations or associations of second messengers?

They are typically cytosolic messengers or membrane-associated messengers.

How are second messengers formed rapidly?

From precursors by enzymatic reactions.

How are second messengers released from inside the cell?

Second messengers can be released quickly from intracellular stores (e.g., Ca^{+2} ions from intracellular stores).

How are second messenger signals terminated or regulated?

Second messengers can be quickly inactivated or moved into a specific compartment (e.g., cAMP can be degraded to AMP, and Ca^{+2} ions can be translocated into the ER).

What effect do second messengers have on effector proteins?

Second messengers can activate many different effector proteins, leading to diversification and variability in signaling.

What capability do second messengers provide for cellular signaling?

Second messengers allow for signal amplification (e.g., a single molecule of norepinephrine (NE) can generate thousands of cAMP molecules).

What is a typical example of a second-messenger pathway cascade?

The adenosine 3’, 5’-cyclic monophosphate (cAMP) pathway.

How is cAMP produced in the cAMP pathway, and what role does G_s play?

Once G_s is activated, it stimulates adenylyl cyclase (AC) to catalyze the reaction converting ATP to cAMP (when associated with the enzyme AC, G_s also acts as a GTPase, hydrolyzing GTP to GDP, which, in turn, inactivates the G-protein) (Such G-proteins typically remain active for only a few seconds)

What is the major target of cAMP?

The cAMP-dependent protein kinase A (PKA).

Describe the structure of PKA (protein kinase A) and how it binds cAMP.

PKA (protein kinase A) is composed of 2 regulatory subunits (R) and 2 catalytic subunits (C). It binds 4 cAMP molecules (2 cAMPs bind to each R subunit).

What happens to PKA when 4 cAMP molecules bind to it?

The binding of 4 cAMPs to PKA results in a conformational change in which the regulatory (R) and catalytic (C) subunits dissociate.

What is the function of the dissociated catalytic subunits (C) of PKA?

The now dissociated C subunits transfer a phosphate group from ATP to other proteins.

Name some types of proteins that PKA can phosphorylate.

PKA can phosphorylate proteins associated with voltage-gated ion channels, ligand-gated ion channels, synaptic vesicle proteins, enzymes involved with neurotransmitter synthesis, and proteins that regulate gene transcription.

How does a single neurotransmitter binding to its GPCR affect intracellular signaling?

It can lead to diverse amplification of that signal, influencing numerous other intracellular components and spreading throughout the cell.

When is signal amplification typical in the context of ligand and G-protein binding?

Such amplification is typical when the ligand binds and activates a G-protein where second-messenger molecules are involved.

How many G-proteins can a single activated GPCR influence?

A single activated GPCR can influence numerous G-proteins.

What is the main purpose of an ion channel in neurons?

to excite the neuron to fire an action potential or inhibit the neuron from firing an action potential (the ion channel acts as an on-off switch).

What is the effect of neurotransmitter binding to an ionotropic receptor?

Neurotransmitter binding to an ionotropic receptor always leads to the opening of the channels.

Where do the actions of ionotropic receptors typically occur?

These actions are mostly local, since such channels are typically confined to the postsynaptic region.

How do metabotropic receptors affect ion channels at a distance?

Because metabotropic receptors activate second messengers that are diffusible, they can act on ion channels at distances from the receptor.

What impact do the slow synaptic actions of metabotropic receptors have on neuronal firing and electrophysiological properties?

The slow synaptic actions of metabotropic receptors are normally not enough to cause a cell to fire an action potential. However, they can greatly influence the neurons’ electrophysiological properties!

How can Protein Kinase A (PKA) affect the NMDA receptor and what other proteins are involved in regulating this process?

PKA can phosphorylate the closed NMDA receptor, causing the channel to open (overcoming its inhibition). This mechanism typically requires other proteins including phosphatases (PP1 or PP2) which in turn turn off the “phosphorylation”.

Where can metabotropic receptors modulate neuronal activity?

Metabotropic receptors can modulate activity at the presynaptic membrane, the postsynaptic membrane, and on the cell body.

How do second messengers modulate activity in the presynaptic membrane?

In the presynaptic membrane, second messengers can modulate K^+ and Ca^{+2} channel activity as well as neurotransmitter release.

How do second messengers alter postsynaptic potential amplitudes in the postsynaptic membrane?

In the postsynaptic membrane, second messengers can alter postsynaptic potential amplitudes by modulating ionotropic receptors.

How can second messengers influence channels in the soma and dendrites?

In the soma (and dendrites), second messengers can influence resting and voltage-gated channels, altering the resting potential, threshold, and action potential duration.

What happens when glutamate binds to its' NMDA receptor?

It mediates the flow of Ca^{+2} into the cell, which leads to the activation of two kinases: CaMKII (in the cytoplasm) and CaMKIV (in the nucleus).

What is the function of CaMKIV in the nucleus?

CaMKIV phosphorylates (and activates) CREB (cyclic AMP response element binding protein), which then recruits various other proteins to activate RNA polymerase and initiate gene transcription.

How does dopamine signaling lead to CREB activation in the nucleus

Dopamine binds to a GPCR that activates adenylyl cyclase to generate cAMP, which, in turn, activates PKA; PKA can enter the nucleus and activate CREB

What is a common characteristic of second-messenger pathways activated by G-proteins, despite involving different G-proteins and effectors?

They share a common design or sequence.

How do the number of G-protein types compare to their effector targets?

The types of G-proteins are more numerous than the effector targets on which they act.

How do various effectors lead to changes in specific target proteins in G-protein signaling?

They do so either by generating a second-messenger that binds to a target protein or by activating a protein kinase that phosphorylates the protein.

How are some second messengers generated?

Some second messengers are generated by the hydrolysis of phospholipids in the inner plasma membrane.

What enzymes catalyze the hydrolysis of phospholipids to generate second messengers?

This hydrolysis can be catalyzed by phospholipase C (PLC), phospholipase D (PLD), and phospholipase A\text{2} (PLA\text{2}).

How are various lipases activated in the context of second messenger generation?

The various lipases can be activated by different G-proteins coupled to a variety of receptors.

Name one of the most common phospholipids hydrolyzed in second messenger pathways.

One of the most common phospholipids hydrolyzed is phosphatidylinositol 4,5-biphosphate (PIP\text{2}).

What products result from the hydrolysis of PIP\text{2} by PLC?

Hydrolysis of PIP\text{2} by PLC yields diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP\text{3}).

Which phospholipids are more abundant in the membrane?

Phosphatidylcholines are more abundant in the membrane than phosphatidylinositols.

What is the function of the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)?

The sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) serves as a storage compartment for intracellular Ca^{+2}; it pumps Ca^{+2} inside using ATP.

How is arachidonic acid (AA) generated in a cellular context?

PLA\text{2} can hydrolyze phospholipids to yield arachidonic acid (AA); arachidonic acid can be generated directly fromPIP₂ by PLA₂ or from DAG

What are the products of PIP\text{2} hydrolysis by PLC, and what role does Ca\text{+2} play?

Hydrolysis of PIP\text{2} by PLC yields diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP\text{3}), with Ca\text{+2} acting as a third second messenger.

What happens to DAG after PIP\text{2} hydrolysis, and what is its function?

DAG, being hydrophobic, remains in the inner membrane where it combines with protein kinase C (PKC) to phosphorylate both membrane-associated proteins and cytoplasmic proteins.

What is the role of IP\text{3} in calcium signaling?

IP₃ stimulates the release of Ca+2 from intracellular stores (e.g., the lumen of the smooth ER): the endoplasmic reticulum (ER) membrane expresses an IP₃ receptor that’s part of a Ca⁺² channel

How does a rise in intracellular calcium further amplify calcium release?

A rise in intracellular calcium can trigger the opening of calcium-gated channels in the plasma membrane and release additional Ca\text{+2} from internal stores by binding to the ryanodine receptor, which is located in the smooth ER and is gated by Ca\text{+2} (not IP\text{3}).