Module 3: Electrical & Synaptic Signaling

Neurons

• send and receive electrical signals (nerve impulses)

• have specialized extensions like axons and dendrites

Nervous System

• responsible for sending and receiving signals (nerve impulses) throughout the body

• these impulses travel along nerve cells (neurons) using their specialized plasma membrane

What are the 2 nervous systems that vertebrates have?

1. Central Nervous System

   • consists of the brain and spinal cord

   • its the control center for processing information

2. Peripheral Nervous System

   • includes sensory and motor neurons outside the CNS

   • connects CNS to the rest of the body

2 types of cells in the nervous system

1. Neurons

2. Glial Cells

Glial Cells

• support and protect neurons

• different types have specific functions

3 types of neurons

1. Sensory Neurons

   • detect and respond to stimuli

   • carry information from sensory receptors to the CNS

2. Interneurons

   • act as messengers and processors

   • connect neurons within the CNS and interpret incoming information

3. Motor Neurons

   • transmit signals from the CNS to muscles and glands

   • cause muscle contraction or glandular secretion

5 types of glial cells

1. Microglia

   • fight infections and remove debris

2. Oligodendrocytes

   • found in CNS

   • forms the myelin sheath that wraps around axons, increasing signal speed

3. Schwann cells

   • found in PNS

   • forms the myelin sheath that wraps around peripheral nerves

4. Astrocytes

   • regulate the blood-brain barrier: controls what substances from blood can enter brain tissue

5. Ependymal cells

   • ciliated, epithelial cells that are filled with cerebrospinal fluid

4 parts of the neuron

1. Cell Body

   • contains the nucleus and organelles

2. Dendrites

   • receive electrical signals

3. Axons

   • conduct electrical signals

4. Synapse

   • functional part of how neurons communicate

   • connection point between a neuron’s axon terminal and another cell

4 features of the axon

1. Axoplasm

   • the cytoplasm inside an axon

2. Myelin Sheath

   • where vertebrates are wrapped around

   • discontinuous - wraps segments of the axon, leaving gaps

3. Nodes of Ranvier

   • gaps between myelinated segments

   • electrical signals jump from node to node for faster transmission

4. Nerves

   • bundle of axons

   • not a single neuron

Membrane Potential

• the difference in electrical charge across a cell’s plasma membrane

• all cells have it, but in neurons, it is especially important for signalling

Resting Membrane Potential

• when a neuron is not sending a signal, it said to be “at rest”

cells at rest:

• have excess negative charge on the inside of the cell

• have excess positive charge on the outside of the cell

this charge difference creates the resting membrane potential

Why is there a membrane potential?

1. Uneven Ion Distribution (ion gradients)

   • cells maintain different concentrations of ions inside vs outside

   • inside the cell (cytosol)

     • high potassium

     • low sodium and chlorine

     • large trapped anions (proteins, DNA, RNA)

   • outside the cell

     • high sodium and chlorine

     • low potassium

   • this difference creates a potassium ion concentration gradient

     • potassium wants to move out of the cell

2. Selective Membrane Permeability

   • the membrane is more permeable to potassium than sodium due to leak channels

   • so potassium tends to diffuse out of the cell, leaving behind negatively charged anions that cannot leave

   • this causes the inside to become more negative

3. Na+/K+ pump (active transport)

   • pumps 3 sodum out and 2 potassium in

   • this maintains the ion gradient and contributes to the negative charge inside

4. Electroneutrality & Counter-ions

   • in solutions, ions are balanced - positive and negative charges pair up

   • inside: potassium balances trapped anions

   • outside: sodium is balanced by chlorine

   • this results in resting membrane potential

     • inside becomes negatively charged relative to the outside

Uneven Ion Distribution (ion gradients)

• cells maintain different concentrations of ions inside vs outside

• inside the cell (cytosol)

  • high potassium

  • low sodium and chlorine

  • large trapped anions (proteins, DNA, RNA)

• outside the cell

  • high sodium and chlorine

  • low potassium

• this difference creates a potassium ion concentration gradient

  • potassium wants to move out of the cell

Electroneutrality

• in any solution, there is overall electrical balance

• for every positive ion, theres a matching negative ion nearby

Counter-ions

• oppositely charged ions that pair with given ion to maintain neutrality

Inside the Cell

• there are trapped anions (negatively charged proteins, DNA, RNA)

• potassium acts as their counter-ion to balance the charge

Outside the Cell

• sodium is the main positive ion (cation)

• chlorine acts as its counter-ion

even though the overall charge in each fluid compartment stays neutral, the selective movement of ions across the membrane creates the charge difference → membrane potential

2 factors that resting membrane potential depends on

1. Ion Concentrations

   • the cytosol (inside) and extracellular fluid (outside) have different ion compositions

     • inside: high potassium, low sodium, and trapped anions (proteins, DNA, RNA)

     • outside: high sodium, low potassium

2. Selective Membrane Permeability

   • the membrane is more permeable to potassium than to sodium

   • this selective movement of ions creates a charge difference across the membrane

2 main ion channels that maintain membrane potential

1. Leak Channels

   • always open ion channels in the membrane (not gated)

   • allows ions to move passively along their concentration gradients

   • they require no energy

   • more potassium leak channels than sodium

   • potassium diffuses out → leaves behind negative anions → results in negative resting membrane potential

2. Na+/K+ Pump

   • maintains ion gradient via active transport

   • pumps 3 sodiums ions out, and 2 potassium ions in

   • requires ATP

   • compensates for the small leak of sodium into the cell

   • helps restore and maintain resting potential

What is the effect of ions on membrane potential?

Potassium (K+)

• diffuses out

• makes the inside more negative (hyper-polarization)

Sodium (Na+)

• diffuses in

• makes the inside more positive (de-polarization)

Chlorine (Cl-)

• diffuses in, but is repelled by negative membrane potential

• only enters with positive ions

• helps balance charge

Electrical Excitability

• certain cells (like neurons and muscle cells) can respond to a stimulus by generating an action potential

Action Potential

• rapid change in the membrane potential of an excitable cell

• changes from negative → positive → negative in a very short time

3 membrane transport proteins that facilitate action potentials

1. leak channels

2. Na+/K+ pump

3. voltage-gated channels

   • voltage-gated potassium channels

     • multi-meric proteins (4 subunits)

   • voltage-gated sodium channels

     • large monomeric protein (4 separate domains)

Voltage-Gated Ion Channels

• essential for generating action potentials in excitable cells

1. Ion Specificity

   • each channel is selective for a particular ion

   specificity depends on:

   • size of the central pore

   • how the pore chemically interacts with the ion

2. Channel-Gating

   • channels open rapidly in response to a stimulus

   • after opening, they close again

   • this open-close process is called channel-gating

3. All or None Behaviour

   • channels are either fully open or fully closed

   • there is no partial opening

4. Channel Inactivation

   • after opening, many voltage-gated channels enter a second closed state → called channel inactivation

   • an inactivating particle physically blocks the channel

   • this ensures that the channel cannot reopen immediately

How is an action potential generated?

• depolarization brings the membrane to a threshold potential

  • this threshold is typically reached when a stimulus causes enough sodium channels to open

• once threshold is reached, an action potential is initiated

• action potentials generate electrical signals across an axon

  • rapid electrical depolarization and repolarization occurs

  • caused by sodium flowing in, and potassium diffusing out

Depolarization Phase

• triggered when the membrane potential reaches threshold

Positive Feedback (Hodgkin cycle):

• voltage-gated sodium channels open

• sodium rushes in

• membrane becomes more positive

• this continues rapidly until the peak of the action potential is reached

Repolarization Phase

• after the peak of the action potential, the membrane must return to its resting negative state → repolarization

• sodium channels become inactivated (temporarily blocked)

  • shuts down sodium influx

• voltage-gated potassium channels open

  • open more slowly than sodium channels

• potassium ions exit the cell

• restores the negative charge inside

• the delay between sodium channel inactivation and potassium channel opening creates the falling phase of the action potential

  • this timing determines shape and speed of the action potential

What is the role of voltage-gated channels?

• the movement of sodium and potassium ions during the action potential is controlled by:

  • voltage-gated sodium channels: open first → sodium enters the cell → causes depolarization

  • voltage-gated potassium channels: opens second → potassium exits the cell → causes repolarization

• once triggered, the action potential spreads along the axon

  • the depolarized region causes nearby sections of the membrane to also depolarize

  • this wave of activity travels away from the origin → propagation

  • the entire process happens in just a few milliseconds

6 stages of action potential

1. Resting state

2. Sub-threshold depolarization

3. Depolarization phase

4. Repolarization phase

5. Hyperpolarization phase

6. Refractory period

   • absolute refractory period

   • relative refractory period

Resting State

• at rest, voltage-gated sodium and potassium channels are closed

• the neuron is not actively firing an action potential

• there are more potassium leak channels compared to sodium ones

  • as a result, the membrane is about 100x more permeable to potassium than to sodium

Sub-Threshold Depolarization

• occurs when the depolarization is too small to trigger an action potential

• the membrane becomes slightly less negative, but not enough to open voltage-gated sodium channels

Hyper-Polarization Phase (undershoot)

• occurs right after repolarization, before the membrane returns fully to its resting potential

• voltage-gated potassium channels stay open a bit too long

  • this causes extra potassium to leave, making the inside more negative

• increased potassium permeability as the channels are still open

• once voltage-gated potassium channels close → returns to resting potential

Refractory Period

• brief time after an action potential during which a neuron cannot or is less likely to fire another action potential

2 types of refractory periods

1. Absolute Refractory Period

   • sodium channels are inactivated

   • cannot trigger another action potential

   • the membrane must repolarize (return to resting potential) to remove inactivation

2. Relative Refractory Period

   • sodium channels are capable of opening again

     • but difficult to trigger an action potential

   • membrane is in hyperpolarization (more negative)

     • far away from threshold value

Passive Spread of Depolarization

• when one point of the membrane depolarizes, that positive charge can spread to adjacent regions

• as depolarization spreads away from origin

  • the signal weakens with distance and cannot travel far

• to transmit signals over long distances, the action potential must be actively regenerated along the membrane

Why don’t action potentials weaken along the axon?

• when one part of the axon depolarizes, the local positive charges spread to nearby regions

  • this passive spread decreases in strength with distance

  • it cannot carry the signal very far

• to send signals long distances, the neuron regenerates the action potential at each point along the axon

  • this is called active propagation

How is a signal transmitted in a neuron?

• incoming signals are transmitted to a neuron at the synapse

  • the synapse is the point of contact

  • the signals depolarize the dendrites

  • this depolarization spreads passively over the membrane to the axon hillock (base of the axon)

    • this is where action potentials are most easily generated

Action Potential in Non-Myelinated Nerve Cells

• in non-myelinated axons, the action potential moves continuously along the membrane

  1. stimulation causes sodium channels to open → sodium rushes in → depolarization

  2. the membrane polarity reverses locally (inside becomes positive)

  3. this local depolarization spreads to nearby regions

  4. if nearby depolarization is above threshold, inward movement of sodium occurs

  5. the original region opens potassium channels → potassium diffuses out → repolarization

  6. process repeats along the axon → propagated action potential

Action Potential in Myelinated Nerve Cells

• action potential jumps from one node to the next

  • this is called saltatory propagation

  1. Nodes of Ranvier

     • gaps between myelinated segments

     • contain high concentrations of voltage-gated sodium channels

     • action potentials are regenerated here

  2. Myelin Sheath

     • act as insulators, prevents ion leakage

     • increases speed of signal transmission

     • made by Oligodendrocytes in CNS, Schwann cells in PNS

  3. Paranodal Regions

     • adjacent nodes

     • has adhesive proteins that attach glial and axonal membranes

  4. Juxtaparanodal Regions

     • next to paranodes

     • contains potassium channels (where repolarization occurs)

Saltatory Propagation

• when action potential jumps from one node to the next

  • occurs in myelinated nerve cells

Myelin Sheath

• found in myelinated nerve cells of action potentials

• formed by oligodendrocytes in CNS & Schwann cells in PNS

• not continuous, has regular gaps (nodes of Ranvier)

  • made of multiple layers of membrane that wraps around the axon

• acts as an insulator, prevents ion leakage

• decreases

• increases conduction speed

  • electrical signals travel farther and faster

• enables saltatory propagation

  • action potentials jump from one node of Ranvier to the next

Nodes of Ranvier

• gaps between myelinated segments

  • only found in myelinated nerve cells

• contain high concentrations of voltage-gated sodium channels

• where action potentials are regenerated

Paranodal Regions

• adjacent nodes

• has adhesive proteins that attach glial and axonal membranes

• found in myelinated nerve cells

Juxtaparanodal Regions

• next to paranodes

• contains potassium channels (where repolarization occurs)

• found in myelinated nerve cells

What happens when the signal reaches the end of the neuron?

• when the signal reaches the end of the neuron, it triggers synaptic transmission

  • this is how neurons communicate with the next cell

• this transmission happens in two ways:

  • Electrical synapses

  • Chemical synapses

2 types of synaptic transmissions

• nerve cells communicate with each other and other cell types at synapses (specialized contact points)

  1. Electrical Synapses

  2. Chemical Synapses

Electrical Synapses

• direct connection via gap junctions

• ions (sodium, potassium, calcium) flow directly from the pre-synaptic cell to the post-synaptic cell

  • these ions move through the junctions between the cells

• no delay in signal transmission, occurs very fast

Chemical Synapses

• the pre-synaptic neuron and post-synaptic neuron are not connected via gap junctions

  • they are separated by a small gap, called synaptic cleft

  • pre-synaptic neuron terminal sends signal chemically to the post-synaptic neuron

Neurotransmitter

chemical messengers that:

• are stored in synaptic boutons in the pre-synaptic neuron

• are released when an action potential reaches the axon terminal

• diffuse across the synaptic cleft

• bind to receptors on the post-synaptic cell

• convert chemical signals into electrical responses for the next cell

  • this stimulates an action potential

3 criteria for neurotransmitters

1. induce the appropriate response when introduced to the synaptic cleft

2. occur naturally in the presynaptic neuron

3. be released at the right time when the pre-synaptic neuron is stimulated

2 types of neurotransmitter receptors

1. Excitatory Receptors

   • causes depolarization of the post-synaptic neuron

2. Inhibitory Receptors

   • causes hyperpolarization of post-synaptic cell

5 examples of neurotransmitters

1. Acetylcholine

   • most common neurotransmitter in vertebrates

   • excitatory neurotransmitter in cholinergic synpases

2. Catecholamines

   • used in adrenergic synapses

   • ex: dopamine, norepinephrine and epinephrine

3. Amino acids & their derivatives

   • ex: histamine, serotonin, GABA, glycine and glutamine

4. Neuropeptides

   • short amino acid chains formed by proteolysis of precursor proteins

   • ex: enkephalins - inhibit neuron activity in brains, involved in pain perception

5. Endocannabinoids

   • lipid derivatives that inhibit pre-synaptic neuron activity

   • ex: THC from cannabis

How are neurotransmitters transmitted?

• elevated calcium levels stimulate the secretion of neurotransmitters from pre-synaptic neurons

1. Action Potential Arrives

   • electrical signals reach the axon terminal (synaptic bouton)

2. Calcium Channels Open

   • depolarization causes voltage-gated calcium channels to open

   • increased calcium ions flow into the synaptic bouton from outside the cell

   • neurotransmitters are stored in bouton of neurosecretory vesicles

3. Calcium Triggers Vesicle Fusion

   • calcium binds to protein called synaptotagmin

   • this activates SNARE proteins

     • v-SNARE on the vesicle

     • t-SNARE on the plasma membrane

   • these proteins help the vesicle dock and fuse with the presynaptic membrane

   • docking occurs at the active zone

     • where synaptic vesicles and calcium channels are closely positioned

4. Neurotransmitter Release

   • neurotransmitters are released by exocytosis

     • neurosecretory vesicle fuses with the plasma membrane

5. Alternative: Kiss-and-Run Exocytosis

   • in rapid signalling, neurons may use a faster method

     • vesicles briefly touches the membrane

     • releases some neurotransmitters

     • then pulls back and reseals

     • this conserves vesicles and allows rapid reuse

6. Membrane Recycling

   • to maintain membrane size, the neuron performs compensatory endocytosis

   • vesicle membrane is retrieved and recycled

How are neurotransmitters detected?

• neurotransmitters are detected by receptors on the post-synaptic cell membrane

• these receptors convert chemical signals into electrical responses

2 types of neurotransmitter receptors

1. Ligand-Gated Ion Channels

   • also called ionotropic receptors

   • neurotransmitter binds → channels open → ions flow → fast response

2. G-Protein Coupled Receptors

   • also called metabotropic receptors

   • neurotransmitters binding triggers indirect signalling via messengers → slower, but long-lasting response

3 examples of neurotransmitter receptors

1. Nicotinic Acetylcholine Receptor (nAchR)

   • ligand-gated sodium channels

   • acetylcholine binds → sodium rushes in → depolarization

2. GABA Receptor

   • ligand-gated chlorine channels

   • GABA binds → chlorine enters → hyperpolarization → inhibits action potential

   • decreased chances of an action potential being generated

3. NMDA Receptor

   • ionotropic glutamate receptor

   • permeable to sodium and calcium after glutamate binding

Agonists

• substance that binds to the receptor and activates them

• they trigger depolarization

• cannot be rapidly inactivated → longer-lasting effects

Antagonists

• substance that binds to neurotransmitter receptors

• blocks their activation

• compete with normal neurotransmitter, preventing depolarization

Examples:

• Acetylcholine antagonists

  • snake venom neurotoxins

• NMDA antagonists

  • used as anesthetics

Why must neurotransmitters be inactivated quickly?

• after a neurotransmitter is released into the synaptic cleft, it must be removed quickly

• if not, the post-synaptic cell would be over-stimulated or over-inhibited

3 inactivation mechanisms

1. Re-uptake

   • neurotransmitters are pumped back into:

     • the pre-synaptic neuron

     • or nearby support cells

2. Degradation - Acetylcholinesterase

   • acetylcholinesterase breaks down acetylcholine into:

     • acetic acid

     • choline

3. Diffusion

   • neurotransmitters diffuse out of the synaptic cleft

Post-Synaptic Potentials (PSPs)

• when neurotransmitters bind to receptors, they cause small voltage changes in the post-synaptic potential of the membrane

Summation

• most single EPSPs are too small to trigger an action potential on their own, so they integrate signals from multiple neurons

2 types of post-synaptic potentials (PSPs)

• EPSP

  • excitatory: depolarization

• IPSP

  • inhibitory: hyperpolarization

2 types of summation

1. Temporal Summation

   • one neuron fires multiple action potentials rapidly over time

   • EPSPs add up → can reach threshold and cause an action potential

2. Spatial Summation

   • multiple neurons fire at the same time

   • more likely that an action potential is induced

4 types of chemical signals

1. Endocrine Signalling

   • long-distance

   • hormones are released into the bloodstream

   • they travel to target tissues far away

2. Paracrine Signalling

   • short-distance

   • the signal diffuses through the local extracellular fluid

   • affects nearby cells only

3. Juxtacrine Signalling

   • requires physical contact between sending and receiving cells

4. Autocrine Signalling

   • self-targeted

   • the cell produces and responds to its own signal

How are signals perceived or detected by cells?

• cells detect and respond to signals through receptor-ligand interactions, which trigger signal transduction pathways

Ligand

• signalling molecules that binds to specific receptors on or inside the target cell

• examples: hormones and neurotransmitters

• the receptor binds to the ligand through its binding site (or binding pocket)

• co-receptors help stabilize ligand-receptor binding on the cell surface

Signal Transduction

• the ability of a cell to respond to ligand-receptor binding through altering its behaviour or gene expression

• ligand binding is just the first step

  • produces additional molecules within the cell → second messengers

  • triggers a cascade of intracellular events

Receptor-Ligand Binding Dynamics

• similar to how enzymes bind to substrates

• when a ligand binds, the receptor is said to be occupied

• as ligand concentration increases, the saturation of occupied receptors is reached

Receptor Affinity

• the strength of ligand-receptor interaction

  • this involves the ligand concentration in solution and the number of occupied receptors

2 types of signal termination

Cells can shut down signals in 2 ways:

1. Reduce free ligand levels

2. Reduce sensitivity or amount of receptor

this ensures that cells respond to changes, not constant signals

Signal Amplification

• cells can amplify a small signal into a large response

• one ligand-receptor interaction can stimulate the production of many molecules needed for the next step

  • this multiplication of the signal effect → signal amplification

• example: one epinephrine molecule → releases millions of glucose molecules from glycogen in liver cells

4 types of signalling pathways

1. Ligand-gated ion channel

   • hydrophilic ligand

   • ligand binds → ion channel opens → allows ions to flow in/out → changes membrane potential

2. G protein-coupled receptor (GPCR)

   • hydrophilic ligand

   • ligand binds → activates G protein inside the cell → G protein triggers second messengers

3. Receptor kinase

   • hydrophilic ligand

   • ligand binds → receptors dimerize or auto-phosphorylate → activates intracellular signalling proteins

4. Nuclear receptor

   • hydrophobic ligand (can cross the membrane)

   • ligand enters the cell → binds to receptor → receptor-ligand complex binds to DNA → changes gene expression

G Protein-Coupled Receptors (GPCRs)

• cell surface receptors that respond to external signals (ligands)

• ligand binding changes the shape of the receptor

• this activates a G protein, which then triggers a cellular response

• example: opioid receptors - narcotic drug

Structure of G protein-coupled receptors (GPCRs)

• made of 7 transmembrane alpha-helices

• connected by alternating extracellular loops (binds ligand) and intracellular loops (interact with G proteins)

• the ligand-binding site is specific to each receptor

G Proteins

• guanine nucleotide-binding proteins

• act like molecular switches (ON or OFF)

  • the state depends on whether they bind to GTP or GDP

2 types of G proteins

1. Small monomeric G proteins

2. Heterotrimeric G proteins

   • made of 3 subunits: Gα (largest subunit), Gβ, and Gγ

   • Gα binds GDP or GTP (this controls activity)

   • G_s are stimulators of signal transduction

   • Gⁱ are inhibitors of signal transduction

G Protein Activation

• Ligand (messenger) binds the GPCR → receptor changes shape

• GPCR binds to G protein → Gα releases GDP and binds to GTP

• Gα detaches from G_βγ subunit, which are permanently bound together

• Gα or Gβγ (depending on the protein) activates target enzymes

• G proteins remain active if Gα subunit is bound to GTP and separated from G_βγ subunit

G Protein Inactivation

• Once Gα subunit is hydrolyzed from GTP to GDP

  • Gα becomes inactive

  • Gα rebinds to Gβγ

  • the signal stops

Regulation of G Proteins

• some Gα subunits are slow at GTP hydrolysis

• RGS proteins (regulators of G protein signalling) help by:

  • acting as GTPase-activating proteins (GAPs)

  • speeding up inactivation of Gα

Roles of α and βγ subunits in signalling

• both subunits can lead to the release or formation of second messengers

Gα (activated G protein)

• can activate enzymes like adenylyl cyclase and phospholipase C

Gβγ (dissociated G protein)

• can activate G protein-receptor kinases (GRKs)

Secondary Messengers

• small molecules inside the cell that help pass on the signal from the receptor

• they are made or released in response to an external signal (neurotransmitter or hormone)

• they amplify the signal molecule into a large response

cAMP (cyclic AMP)

• second messenger made from cytosolic ATP by adenylyl cyclase

• triggered by Gs protein

• inactive until binded to Gsα protein

• helps control many processes inside the cell

• main function: activate Protein Kinase A (PKA) enzyme

  • separates regulatory and catalytic subunits

• PKA can now phosphorylate other proteins

  • specifically on serine or threonine amino acids

  • uses ATP as phosphate source

cAMP Activation & Inactivation

Activation:

• a ligand molecule (signal) binds to GPCR on cell membrane

• this activates Gs protein

• the Gsα subunit switches GDP for GTP, becomes active

• Gsα detaches from the Gβγ subunit

• active Gsα-GTP binds to and activates adenylyl cyclase

• adenylyl cyclase turns ATP into cAMP

• because G proteins remain active for a short time, they can respond quickly to changing conditions

Inactivation:

• Gsα hydrolyzes GTP to GDP → becomes inactive

• adenylyl cyclase (enzyme) stops making cAMP

• any remaining cAMP is broken down by phosphodiesterase (PDE)

Grab A Cold Pepsi (cAMP signalling pathway)

• G → G protein

  • activated by ligand-bound GPCR

• A → adenylyl cyclase

  • enzyme activated by G protein

• C → cAMP

  • secondary messenger made from ATP

• P → Protein Kinase A

  • activated by cAMP

IP₃ & DAG Pathway (aka Phospholipase C Pathway)

1. Ligand binds to GPCR

   • this activates a specific G protein (usually Gq)

2. Gq activates phospholipase C (PLC)

   • PLC cuts a membrane lipid called PIP₂ into:

     • IP₃ → water soluble

     • DAG → stays in the membrane

3. IP₃ moves into the cytosol

   • binds to IP₃ receptors (ligand-gated calcium channels)

     • calcium is released into the cytosol

4. DAG + calcium activates Protein Kinase C

   • PKC phosphorylates proteins on serine/threonine residues

     • this triggers cellular response

2 ways that cells control calcium

• calcium ions help regulate cellular functions

• calcium ATPases maintains low calcium concentration by:

  1. pumping calcium out of the cell

  2. storing calcium inside the ER lumen

How does calcium work in cell signalling?

• Calcium enters the cytosol in 2 ways:

  1. From outside the cell → through calcium channels → in the plasma membrane

  2. From inside the cell (ER storage) → through IP₃ receptor channels → in the ER membrane

• Signals bind to GPCRs or other receptors

  • these activate phospholipase C (PLC)

  • PLC produces IP₃, which opens calcium channels in the ER

• Calcium ions rush into the cytosol, triggering intracellular response

  • calcium can bind directly to effector proteins, changing their activity

  • example: calmodulin mediates calcium-activated processes in the cell

Calmodulin

• effector protein that mediates calcium-activated processes in the cell

• structure: like a rope with a hand at each end

  • each end binds two calcium ions

  Function:

  • calcium enters the cell (from outside or from ER storage)

  • calcium binds to calmodulin → this causes a shape change

  • the new shape is the active calcium-calmodulin complex

  • calmodulin wraps around target proteins w/ calmodulin binding sites

  • this activates or modifies the target protein’s activity

Protein Kinase-Associated Receptors

• receptors that have dual function:

  • able to detect a signal (ligand)

  • act as enzymes called kinases

• ligand binding stimulates their kinase activities

• signals are transmitted via phosphorylation cascade

  • a chain reaction of protein activations

2 types of kinases

1. tyrosine kinases

2. serine / threonine kinases

Growth Factors

• messengers in the blood serum that simulate growth

• example: PDGF (platelet-derived growth factor)

  • found in blood serum after clotting

  • binds to its receptor, which is receptor tyrosine kinase

Receptor Tyrosine Kinases

• receptors on the cell surface that trigger growth or division inside the cell

• contains one long protein chain w/ one transmembrane segment

  • extracellular part (outside): ligand-binding domain

  • cytosolic part (inside): tyrosine kinase domain

• sometimes, the receptor and tyrosine kinase are two separate proteins

2 activation mechanisms of receptor tyrosine kinases (RTKs)

• ligand binding triggers RTK activation (like growth factors)

  • this starts the signal transduction process

  1. Dimerization

  • two RTKs pair up (dimerize) after ligand binding, and they phosphorylate each other

  • example: fibroblast growth factor (FGF)

  2. Clustering

  • ligand binding causes multiple receptors to cluster together

  • example: epidermal growth factor (EGF)

Autophosphorylation

• when receptor tyrosine kinases (RTKs) are of the same type, they phosphorylate themselves

How do receptor tyrosine kinase (RTKs) start a signal cascade?

1. ligand binds with receptor tyrosine kinase → RTKs dimerize and auto-phosphorylate

2. these phosphorylated tyrosines recruit cytosolic proteins

3. the cytosolic proteins recognize and attach to these phosphotyrosines

   • they use amino acid domains

   • example: SH2 domain → Src Homology

Ras

• small G protein that regulates reproduction of cells

• central target of the receptor tyrosine kinase signalling (RTK)

• can bind to GDP or GTP

  • but only active when bound to GTP

• helper protein called SOs (GEF protein) helps Ras acquire a GTP molecule

How does Ras initiate signalling?

• Ras initiates signalling through the RTK-Ras-MAPK pathway

  1. RTK Activation

     • a growth factor binds to RTK

     • RTK dimerize and autophosphorylates

  2. Adaptor Recruitment

     • for Sos to become active, it must bind to GRB2 protein

       • which has an SH2 domain

  3. Ras Activation

     • Sos activates Ras by exchanging GTP → GDP

     • this activated Ras triggers a series of phosphorylations

  4. Raf Phosphorylation

     • activated Ras phosphorylates Raf, a protein kinase

  5. MEK Phosphorylation

     • Raf phosphorylates MEK, serine and threonine residues in protein kinase

  6. MAPKs Phosphorylation

     • MEK phosphorylates MAPks (mitogen-activated protein kinase)

  7. MAPK Enters the Nucleus

     • MAPK phosphorylates transcription factors

     • these factors enter the nucleus to alter gene expression

     • for example: factors Jun, Ets → regulates growth and division

Great Sushi Rolls Require Meticulous Munching

GRB2 → Sos → Ras → Raf → MEK → MAPK

Inactivation of Ras

• once Ras is in the active state, it must be inactivated to prevent constant cell growth signalling

• GAPs (GTPas activating protein) stimulate Ras to hydrolyze GTP → GDP

• GDP-bound Ras is now inactive

Activation of Phospholipase Cγ (PLCy)

• alternative RTK signalling pathway

• some RTKs activate Phospholipase Cγ instead of Ras

• PLCy binds to the phosphorylated RTK via SH2 domain

• this leads to production of IP₃ and DAG

How did scientists explain RTK signalling?

• scientists used mutations to uncover the steps in RTK signalling

• mutations can be induced in the receptors themselves or in “downstream” signalling components