BIOC 3560 final

What is a biological membrane?

A thin, flexible barrier made mainly of lipids and proteins that separates the cell or organelles from their surroundings.

Major functions:

Permeability barrier/compartmentalization

Communication

Energy conversion

Surface recognition

A phospholipid has a polar head and nonpolar fatty-acid tails.

In a membrane:

Water → HEAD — TAIL → ← TAIL — HEAD → Water

Important: The lipid tails interact with other lipid tails.

What is a phosphoglyceride/glycerophospholipid?

Definition:
A major class of membrane phospholipids.

Structure:

• Glycerol backbone

• 2 fatty acids

• Fatty acids attached by ester bonds

• Phosphate-containing head group derived from an alcohol

What is a sphingolipid?

Definition:
A membrane lipid built from a sphingosine backbone rather than glycerol.

Structure:

• Sphingosine

• One additional fatty acid

• Fatty acid attached by an amide linkage

What is a glycosphingolipid?

Definition:

A sphingolipid containing a carbohydrate head group.

Example:

The ABO blood groups depend on differences in glycosphingolipid carbohydrate head groups.

What is cholesterol?

Definition:
The major sterol found in animal cell membranes.

Structure:

• Four fused rings

• Alkyl side chain

• Small polar –OH group

What is a peripheral membrane protein?

Definition:
A protein associated with the membrane surface but not embedded deeply in the hydrophobic bilayer.

Interactions can include:

• Electrostatic interactions

• Hydrogen bonds

• Protein–protein interactions

What is an integral membrane protein?

Definition:
A protein strongly associated with the membrane.

Can be:

1. Lipid anchored

or

2. Membrane spanning

How do you distinguish them experimentally?

Detergent removes integral proteins.

Then:

Phospholipase removes it → lipid anchored

Phospholipase doesn't remove it → membrane spanning

Where is a myristoyl group attached?

Answer:
N-terminal glycine

Memory:

MYR → Gly at the beginning

where is palmitate attached?

Usually an internal cysteine residue.

Where are prenyl groups such as geranylgeranyl attached?

Answer:
A C-terminal cysteine, not an N-terminal cysteine.

Where are GPI-anchored proteins found?

Answer:
On the extracellular side of the plasma membrane.

Example:
GPI-anchored proteins are commonly associated with lipid rafts.

What amino acids are expected inside a transmembrane α-helix?

Mostly hydrophobic amino acids because they interact with hydrophobic lipid tails.

Examples:

• Leucine

• Isoleucine

• Valine

• Phenylalanine

What amino acids are common where the membrane head groups meet the fatty-acid tails?

Especially:

• Tyrosine

• Tryptophan

Charged residues such as Arg, Lys, Asp and Glu are mostly in the aqueous environment.

What is a β-barrel membrane protein?

Definition:
An integral membrane protein made from β-strands arranged into a barrel.

Found especially in:

• Bacterial outer membranes

• Mitochondrial outer membranes

Important properties:

• Backbone H-bonds occur between β-strands

• Side chains alternate hydrophobic/hydrophilic

• Hydrophobic residues face membrane lipids

• Hydrophilic residues face the aqueous pore

• Short β-strands do NOT show clearly on hydropathy plots

What amino acid receives N-linked carbohydrate?

Asparagine (Asn)

First sugar:

N-acetylglucosamine = GlcNAc

Memory:

N-linked → AsN

What amino acids receive O-linked carbohydrate?

• Serine

• Threonine

because they contain –OH groups.

First sugar:

N-acetylgalactosamine = GalNAc

Memory:

O-linked → OH → Ser/Thr

What Increases Membrane Fluidity?

Fluidity generally increases with:

• Shorter fatty-acid chains

• More cis-unsaturated fatty acids

Why?

Cis double bonds create kinks, preventing tight packing.

Saturated vs. Unsaturated

Saturated fatty acids

→ straight
→ pack tightly
→ favour ordered membrane

Unsaturated fatty acids

→ kinked
→ pack poorly
→ favour more disordered/fluid membrane

What does cholesterol do to membrane fluidity?

The effect depends on lipid composition/conditions.

The major concept is that cholesterol acts as a fluidity buffer.

It can prevent tightly packed saturated chains from interacting strongly, while in other membrane environments it can restrict lipid movement.

What is lateral diffusion?

Movement of a lipid sideways within the same leaflet.

Speed: Very fast.

Why is spontaneous flip-flop slow?

The polar/charged lipid head would have to pass through the hydrophobic interior of the bilayer.

Therefore spontaneous transbilayer movement is very slow.

Flippase

Outer leaflet → cytosolic leaflet

Moves especially:

• PE

• PS

Requires ATP.

Memory:

FLIP → IN

The lecture diagram identifies it as a P-type ATPase.

Floppase

Cytosolic leaflet → outer leaflet

Requires ATP.

It is shown as an ABC transporter.

Memory:

FLOP → OUT

Scramblase

Function:
Moves lipids in either direction between leaflets toward equilibrium.

So:

Inside ⇄ Outside

What is a lipid raft?

Definition:
A membrane microdomain with a different lipid/protein composition from the surrounding membrane.

Enriched in:

• Cholesterol

• Sphingolipids

Associated with:

• GPI-anchored proteins

• Other lipid-anchored proteins

• Caveolin

How can membrane movement be restricted?

Spectrin is part of the cytoskeleton and links to membrane proteins through ankyrin.

It acts like a fence/corral, restricting free lateral diffusion.

What are the four stages of intracellular membrane trafficking?

Memorize:

B → T → D → F

1. Budding/fission

2. Transport

3. Tethering/docking

4. Fusion

What do SNARE proteins do?

Mediate vesicle–target membrane fusion.

Important components:

• v-SNARE → vesicle

• t-SNARE → target

• SNAP25

Their helical domains interact to form a coiled-coil, bringing membranes together.

Examples of processes using SNAREs:

• Neurotransmitter release

• Insulin secretion

• GLUT transporter trafficking

• ER → Golgi transport

What Can Cross the Membrane?

Cross quickly:

• Hydrophobic molecules

• Steroid hormones

• O₂

• CO₂

• N₂

Cross slowly:

• Small uncharged molecules

• Glycerol

• Ethanol

Essentially cannot cross directly:

• Ions

• Sugars

• Amino acids

• Polar molecules

What is simple diffusion?

Movement of a substance:

High concentration → low concentration

No transporter and no ATP are required.

For an uncharged solute:

ΔG = RT ln(C₂/C₁)

If C₁ > C₂:

ΔG < 0 → spontaneous.

What is facilitated diffusion?

Movement through a membrane protein DOWN a concentration/electrochemical gradient.

No direct energy input is required.

Needed because many polar molecules cannot pass through the lipid bilayer directly.

Membrane Channel

Channel:

• Continuous pore

• Very rapid

• Not saturable in the same manner as transporters

• Highly selective

• Often gated

Examples:

• Na⁺ channel

• K⁺ channel

• Cl⁻ channel

• Aquaporin

Transporter

• Binds substrate

• Changes conformation

• Moves a set number of molecules

• Selective

• Saturable

Example:

GLUT1

GLUT1 Glucose Transport Steps

1. T1 conformation

↓

2. Glucose binds

↓

3. Conformational change

↓

4. T2 conformation

↓

5. Glucose released inside

↓

6. Another conformational change

↓

back to T1

What does Δψ mean?

Δψ describes the electrical potential difference across a membrane.

Typical plasma membrane:

Δψ ≈ −60 mV

Meaning:

inside is more negative than outside.

Free Energy of Ion Transport

For a charged solute:

ΔGₜ = RT ln(C₂/C₁) + zFΔψ

Two components:

Chemical gradient

RT ln(C₂/C₁)

Electrical gradient

zFΔψ

What is primary active transport?

Movement of a substance against its gradient using energy directly, usually ATP.

Na⁺/K⁺ ATPase

For every 1 ATP:

3 Na⁺ OUT

2 K⁺ IN

Both move against their concentration gradients.

Net effect:

one positive charge leaves the cell

→ contributes to negative interior.

Na⁺/K⁺ ATPase Transport Cycle

1. Pump binds 3 Na⁺ inside

↓

2. ATP phosphorylates pump

↓

3. Conformational change

↓

4. 3 Na⁺ released outside

↓

5. 2 K⁺ bind outside

↓

6. Pump is dephosphorylated

↓

7. Pump returns to original conformation

↓

8. 2 K⁺ released inside

What is secondary active transport?

Transport where movement of one substance down its gradient provides the energy to move another substance against its gradient.

It does NOT directly hydrolyze ATP at that transporter.

Example:

Na⁺–glucose symporter.

Intestinal Glucose Absorption Pathway

1. Na⁺/K⁺ ATPase

3 Na⁺ OUT / 2 K⁺ IN

↓

creates low intracellular Na⁺

↓

2. Na⁺ moves back into epithelial cell down its gradient

↓

through Na⁺–glucose symporter

↓

3. Glucose is carried into cell against its gradient

↓

4. Glucose exits basal side through GLUT2

↓

blood

K⁺ Channel Structure

Tetramer

Each subunit contains:

• 2 transmembrane helices

• Short helix

• Selectivity filter

It is about 10,000× more selective for K⁺ than Na⁺ according to your lecture.

How Does the K⁺ Channel Select K⁺?

Two important factors:

1. Size

The filter fits K⁺.

2. Carbonyl oxygens

Sequence:

Gly–Tyr–Gly–Val–Thr

Backbone carbonyl oxygens have partial negative charges that coordinate dehydrated K⁺.

The pore can collapse if the smaller Na⁺ enters.

What Does "Gated" Mean?

A gated channel:

1. Closed by default

↓

2. Opens in response to a stimulus

↓

3. Closes again after a short delay

Types include:

• Ligand-gated

• Voltage-gated

Voltage-Gated Na⁺ Channel Structure

Contains four homologous domains (I–IV) fused into one polypeptide.

Important helices:

Helix 4 = voltage sensor

Helix 6 = pore-forming helix

Voltage-Gated Na⁺ Channel Opening

At rest:

Inside negative

↓

positively charged helix 4 pulled inward

↓

channel closed

Then:

Depolarization

↓

Helix 4 moves outward

↓

movement transmitted to helix 6

↓

channel opens

↓

Na⁺ enters

↓

inactivation loop blocks channel

What is signal transduction?

Definition:
Conversion of information carried by a signal into a chemical change/cellular response.

Signal

→ receptor

→ intracellular process

→ response

Specificity

Definition:
A receptor selectively recognizes a particular signal molecule.

Example:
Epinephrine binds the β-adrenergic receptor.

Specificity can also occur because certain receptors are only present in particular cell types.

Amplification

Definition:
One activated signaling molecule activates many downstream molecules, making the signal much larger.

Example:

1 receptor

→ many G proteins

→ lots of cAMP

→ many activated enzymes

Signal amplification can occur by several orders of magnitude within milliseconds.

Modularity

Signaling pathways are constructed from reusable molecular components/modules.

This allows cells to combine signaling proteins in different ways.

Desensitization / Adaptation

Definition:
Reduced cellular response when a stimulus persists.

Example:
β-adrenergic receptor internalization.

When the stimulus falls sufficiently, the signaling system can become sensitive again.

Integration

A cell combines information from multiple signals to produce one appropriate response.

S A M D I

Specificity
Amplification
Modularity
Desensitization
Integration

What is a GPCR?

Definition:
A plasma membrane receptor containing 7 transmembrane helices that activates a heterotrimeric G protein.

The system contains:

1. GPCR

2. Heterotrimeric G protein

3. Intracellular effector enzyme producing a second messenger

Second messengers include:

• cAMP

• cGMP

• IP₃

Heterotrimeric G Protein

Contains:

Gα + Gβ + Gγ

Important:

Gα and Gγ are anchored to the membrane by lipid tails.

G-Protein Cycle

HIGH-YIELD PATHWAY

1. Resting state

Gα has GDP

→ inactive

↓

2. Ligand binds GPCR

↓

3. GDP leaves Gα

↓

4. GTP binds Gα

↓

5. Gα-GTP separates from βγ

↓

6. Gα-GTP activates effector

↓

7. Gα hydrolyzes GTP → GDP + Pi

↓

8. Gα-GDP reassociates with βγ

↓

Back to inactive state.

β-Adrenergic/Epinephrine Pathway

Memorize:

E → R → Gs → AC → cAMP → PKA → Response

Full pathway:

1. Epinephrine binds β-adrenergic receptor

↓

2. GPCR activates Gs

↓

3. Gα exchanges GDP → GTP

↓

4. Gα-GTP activates adenylyl cyclase

↓

5. Adenylyl cyclase converts ATP → cAMP

↓

6. cAMP activates PKA

↓

7. PKA phosphorylates cellular proteins

↓

CELLULAR RESPONSE

How Is the cAMP Signal Turned Off?

cAMP

↓

cyclic nucleotide phosphodiesterase

↓

5′-AMP

↓

PKA activation stops.

The epinephrine pathway diagram explicitly shows cAMP degradation reversing PKA activation.

β-Adrenergic Receptor Internalization PATHWAY

1. Epinephrine binds receptor

↓

2. Gβγ recruits βARK

↓

3. βARK phosphorylates receptor

↓

4. β-arrestin binds phosphorylated receptor

↓

5. Receptor–arrestin complex undergoes endocytosis

↓

6. Receptor is internalized

This is receptor desensitization.

Resting Neuron

High K⁺ inside

Low Na⁺ inside

Membrane potential:

≈ −60 mV

Action Potential Pathway

Start:

−60 mV

↓

Acetylcholine receptor opens

↓

Na⁺ enters

↓

Depolarization

↓

Voltage-gated Na⁺ channels open

↓

more Na⁺ enters

↓

≈ +30 mV

↓

Na⁺ channels inactivate

↓

Voltage-gated K⁺ channels open

↓

K⁺ leaves

↓

Repolarization

↓

≈ −75 mV

↓

K⁺ channels inactivate

↓

returns toward −60 mV

Nicotinic Ach Receptor Structure

Nicotinic AchR contains:

5 subunits

with

4 transmembrane helices per subunit

Composition shown in lecture:

2 α + β + γ + δ

The M2 helices line the channel

AchR Activation

Closed state:

Bulky hydrophobic Leu residues on M2 helices block pore.

↓

2 acetylcholine molecules bind

↓

M2 helices twist/rotate

↓

smaller polar residues line channel

↓

channel opens

↓

Na⁺ and Ca²⁺ enter

What is a receptor enzyme?

A plasma membrane receptor with:

Outside: ligand-binding domain

Inside: catalytic domain

Catalytic activity may include:

• Tyrosine kinase

• Guanylyl cyclase

Ligand binding activates the intracellular catalytic activity.

Insulin Receptor Structure

Insulin receptor is:

α₂β₂ tetramer

Insulin binds:

α subunits outside

β subunits contain:

intracellular tyrosine kinase activity

Insulin binding causes β-chain autophosphorylation and opens the active site.

Insulin Receptor Activation Pathway

1. Insulin binds α subunit

↓

2. Tyrosine kinase activated

↓

3. β chains autophosphorylate tyrosines

↓

4. Activation loop moves away

↓

5. Catalytic site becomes accessible

↓

6. Substrate proteins bind

↓

7. Substrate proteins are phosphorylated

What Is IRS-1?

IRS-1 = insulin receptor substrate 1

The activated insulin receptor phosphorylates IRS-1.

Phosphorylated IRS-1 becomes a docking platform for proteins containing SH2 domains.

Two important branches:

Branch 1

Grb2 → Ras → MAPK → gene expression

Branch 2

PI3K → PKB → GLUT4 + glycogen synthesis

MAPK Pathway

IRS → Grb2 → Sos → Ras → Raf → MEK → ERK → nucleus

Full pathway:

1. Insulin receptor phosphorylates IRS-1

↓

2. Grb2 SH2 domain binds phosphorylated IRS-1

↓

3. Grb2 SH3 domain binds Sos

↓

4. Sos activates Ras

GDP → GTP

↓

5. Ras-GTP activates Raf-1

↓

6. Raf phosphorylates MEK

↓

7. MEK phosphorylates ERK

↓

8. ERK enters nucleus

↓

9. ERK phosphorylates transcription factors

↓

gene expression changes

SH2 Domain

SH2 binds phosphorylated tyrosines.

Example:

Grb2's SH2 domain binds phosphorylated IRS-1.

PI3K's SH2 domain can also bind phosphorylated IRS-1.

SH3 Domain

SH3 domains bind proline-rich sequences.

Example:

Grb2 binds Sos through its SH3 domain.

PI3K PATHWAY

IRS → PI3K → PIP₃ → PKB → GSK3 / GLUT4

Full pathway:

1. Insulin receptor phosphorylates IRS-1

↓

2. PI3K binds phosphorylated IRS-1 using SH2 domain

↓

3. PI3K converts PIP₂ → PIP₃

↓

4. PKB binds PIP₃

↓

5. PKB becomes activated

↓

Two major effects:

Effect A — Glycogen

PKB phosphorylates GSK3

↓

GSK3 becomes inactive

↓

GSK3 cannot inhibit glycogen synthase

↓

glycogen synthase remains active

↓

↑ glycogen synthesis

Effect B — Glucose transport

PKB stimulates GLUT4-containing vesicles

↓

GLUT4 moves to plasma membrane

↓

↑ glucose uptake

What Does Insulin Do in Muscle?

1. ↑ GLUT4 at plasma membrane

→ ↑ glucose uptake

2. ↑ hexokinase synthesis

3. Activates glycogen synthase indirectly by phosphorylating/inactivating GSK3

Nuclear Hormone Receptor

A transcription factor directly activated by hormone binding.

Examples of steroid hormones:

• Estrogen

• Progesterone

• Cortisol

Steroid hormones have a four-linked-ring structure similar to cholesterol.

Thyroid hormones are instead built from iodinated tyrosine residues.

Example:

Thyroxine

Nuclear Hormone Signaling Steps

1. Hormone reaches target cell

↓

2. Hormone crosses plasma membrane

↓

3. Hormone binds intracellular/nuclear receptor

↓

4. Receptor changes conformation

↓

5. Hormone–receptor complex binds DNA

↓

at a Hormone Response Element (HRE)

↓

6. Transcription changes

↓

7. mRNA produced

↓

8. Translation

↓

9. New protein

↓

altered cellular function

What are zinc fingers?

Structural features in nuclear hormone receptors that allow them to bind specific DNA sequences.

Those DNA sequences are called:

Hormone Response Elements (HREs).