Membranes Part 1 & 2

Module 2 Week 5

In the amphipathic design of membranes what are the outer and inner leaflets enriched with respectively?

Outer: Phosphotidyl-choline, sphingomyelin, and glycolipds

Inner: Phsphatidyl-serine (negative), phosphatidyl-ethanolamine

What is Cholesterol needed for in membrane?

Strengthen/ Stiffen bilayer while allowing mid bilayer to remain fluid

How do phospholipids move in bilayer?

Lateral: polar heads move w/n SAME leaflet very fast

Rotation/flexion: Hydrophobic tails bending or rotating within SAME leaflet to adjust packing

Flip flop: Very long process, rare without enzymes moving polar head through hydrophobic core to other side

4 Function Classes of membrane associated proteins & their functions:

Transporters: move solutes across bilayer

Receptors: Sense extracellular signals and initiate intracellular cascades

Anchors: Link cytoskeleton to ECM

Enzymes: Catalyze reactions at the membrane surface

Explain Sodium Potassium Pump

Extracellular contains high Na and low K

Intracellular contain low Na and high K

Moving Na out and K in against gradient requires ATP for energy (active transport)

ATP phosphorylated into ADP + P, pyruvate binds to receptor/pump creating conformational change that lets 3 Na out and 2 K in pyruvate removed from receptor/pump creates conformational change allowing 2 K in

Explain Epinephrine pathway

7-pass receptor, Adrenal medulla, Triggers: stress, fear, exercise

1. Epinephrine binds to G protein-coupled receptor (beta-adrenergic)

2. Alpha G subunit separates from the receptor and replaces GDP with GTP, activating it

3. Alpha subunit activates adenyl cyclase, which develops an affinity for ATP, converting it to cAMP

4. cAMP activates Protein Kinase A

5. PKA phosphorylates phosphorylase, activating it to break down glycogen into glucose to be released into bloodstream

Explain Glucagon pathway

7 pass receptor, alpha cells in pancreas, low glucose levels trigger

Glucagon binds to glucagon receptor on liver cells, G protein activated, alpha subunit activates adenyl cyclase, adenyl cyclase makes cAMP, binds to PKA which activates multiple enzymes to glycogen phosphorylase (turn glycogen into glucose), glycogen synthase (stop glycogen production), stimulate gluconeogenesis

Increasing glucose in blood

Explain PDGF receptor & 4 functions

PDGF released in response to injury or cell growth demand, binds to PDGF receptor (tyrosine kinase), receptor dimerizes (attaches to another PDGF receptor), they psophorylate each other, attracts other signalling proteins for proliferation (MAPK), survival (AKT), and migration & growth (PKC/Ca)

Explain Adenylate Cyclase

Second Messenger enzyme activated by alpha subunit of G protein to turn ATP into cAMP

Explain cyclo-oxygenase-1 & 4 functions

Archidonic acid released activates COX-1 receptor, converts prostaglandin into PGH2 which is the precursor to other products responsible for clotting, protecting stomach lining and regulating blood flow and kidney function

Define Integral Proteins & Removal Method

Transmembrane Proteins

Embed 1 or more hydrophobic alpha helices and beta barrels across bilayer requiring detergent for removal

Define Peripheral Proteins & Removal Method

Non-covalently bounded to lipid head groups or with other proteins and can be removed with pH change or salt

Covalent Lipid Anchor Types:

Leaves polypeptide either on cytosolic or exoplasmic side

Fatty Acid: Myristate (14:0) or palmitate (16:0) attached to N-terminal Gly

Prenyl: Farnesyl (C15) or Geranyl (C20) chains added to C-terminal Cys

GPI: complex glycolipid added to C-terminus to display the protein on extracellular surface

Explain translocation of PKC

signaling pathway makes diacylglycerol, PKC binds to DAG non covalently and becomes anchored to membrane (peripheral protein), activating it

Explain Phospholipase A2 translocation

Stimulus (injury) —> Intracellular Ca rises —> Ca binds to Phospholipase A2 —> cPLA2 changes shape moving to membrane —> PLA2 cleaves phospholipids releasing arachidonic acid—> arachidonic acid converted to prostaglandins

Example of restricting movement of integral proteins by anchoring cytoplasmic portion to intracellular cytoskeleton

Spectrin-actin networks form scaffold under membrane maintaining shape

Ankyrin binds to Band 3 restricting Band 3s lateral movement

Glycophorin & protein 4.1 anchor cytoskeleton to membrane stabilizing vertical interactions

Example of restricting movement of integral proteins by tight junctions keeping them in specific domains

Epithelial cells are held together by tight junctions so Protein A able to move within surface but not into portion of membrane abuts adjacent cells

How to see lateral diffusion of protein FRAP & Heterokyrons?

FRAP- dye and bleach are see if fluorecent returns yes = mobile no = anchored

Heterokyrons: 2 different cells fuse together, see how integral proteins redistribute move freely = evenly distribute across membrane or stay separated = anchored or. restricted movement

Glycocalyx, Epithelial Cells, & 5 Functions

Outer leaflet of membrane rich in carbohydrates, some covalently bonded to glycolipids and glycoproteins, other peripheral glycoproteins

Epithelial cells have to glycocalyx parts with different components one of which is facing basil lamina the other the free epithelial surface

Contributes to protection, hydration barrier, cell-cell recognition, filtration, immune interactions

ECM & 5 Main functions

Bigger/ more complex than glycocalyx, main components are integrin, fibronectin, collagen, proteoglycan, influence development, migration, proliferation, shape and function of cells that contact it

Intracellular (Cytosol) Composition (4)

A variety of negatively charged groups from carboxyl and phosphates on:

• Bicarbonate, phosphate, proteins, and nucleic acids

Composition of ions in Intracellular (Cytosol) vs Extracellular (outside cell)

• Potassium

• Hydrogen

• Sodium

• Free Magnesium

• Free Calcium

• Chloride

Intracellular:

• High K+ & H+ (pH 7.2)

• Low Na+, Mg2+, Ca2+, Cl-

Extracellular:

• High Na+, Mg2+, Ca2+, Cl- (major anion)

• Low: K+ & H+ (pH 7.4)

Membrane Permeability:

Free to pass small nonpolar molecules:

• O2 and CO2

• Paartially Permeable to small, uncharged polar molecules:

  • Water & Ethanol

  • Glycerol diffuses less rapidly

IMPERMEABLE to ions of ALL sizes and large polar molecules

• Glucose

Transporter Proteins move water soluble molecules across bilayer:

• Substrates into the plasma membrane

  • Nucleotides

  • Sugars (glucose)

  • Amino Acids

  • Ions Na+ out and K+ in

• ADP into mitochondria; ATP out of mitochondria

• H+ or H3O into Lysosomes to maintain acidic pH

Channel Proteins

• Passive Diffusion

• Form pores for ION flow

• Regulated by ligand gated channel or voltage gated channels

  • ie) Acetylcholine Receptor = Ligand-Gated Channel

Acetylcholine Receptor

• Ligand-Gated Receptor/neurotransmitter

  1. Neurons bind to receptors on muscle or nerve cells

  2. Quaternary Structure changed to move hydrophobic side chains outwards

  3. Allow passage of Na+ in and K+ out depolarizing membrane

• Opposite of Na+/K+ pump & passive diffusion

Carrier Proteins or Transporters

• Transport small molecules/ solutes across membrane

• 2 Confirmations: opening outside or opening inside

• Binds 1 type of molecule (ie. Glucose) or several related molecules (ie. aromatic amino acids)

• Passive Transport

Symporters vs Antiporters

Carrier Proteins:

Symporters —> 2 different molecules SAME direction

Antiporters —> 2 different molecules DIFFERENT direction

Cotransport of Glucose and Na+

• Symporter

• When Na+ flows down a concentration gradient, it yields energy

• Transporter uses that energy to move glucose uphill (active transport), simultaneously

Cotransport Na+/K+ ATPase

• Antiporter (3 Na+ out & 2 K+ in)

• Uses energy from the hydrolysis of ATP to ADP + P

• Used in conjunction with:

  • Glucose/Na+ —> Pump returns Na+ out into extracellular space

  • Na+/Ca+: 3 Na+ in and 1 Ca2+ out (when Calcium builds up in cell)

  • Acetylcholine —> Restore electrochemical gradient by pumping Na+ out and bringing back lost K+ ions

Isotonic vs Hypertonic vs Hypotonic

• Isotonic: Total molecules in = Total Molecules out

• Hypertonic: Total Molecules In < Total Molecules out = Shrink

• Hypotonic: Total Molecules In > Total molecules out = Swell/Bursts

How does the cell maintain isotonic environment?

What does medication like Ouabain do?

• Higher concentration of organic molecules inside which creates an osmotic gradient only balanced by:

  • Opposing gradient of high Na+ & Cl- out of cell

  • Na+ pumped out by Na+/K+ pump —> net effect to pump out cations

  • Cl- stays in extracellular because of negative membrane potential

• Inhibit Na+/K+ pump, which creates a hypotonic environment, causing cells to burst

ABC Transporters:

Use energy from ATP to move substances out of cell

• Specific molecule binds to transporter from inside causing dimerization of the ATP domains

• 2 ATP molecules bind to domains providing energy and opening the channel

• Substrate released against concentration gradient (Active Transport)

• 2 ATP hydrolyzed and resets transporter

ABC Transporter Examples

• Multi-Drug Resistant Pump: Pumps hydrophobic drugs out of cell (Overexpressed in cancer cells making them resistant to cytotoxic drugs)

• CTFR: Regulates Cl- movement in epithelial cells

• Another moves cholesterol out of cell to high density lipo proteins

Receptor Mediated Endocytosis & LDL

• Transports large molecules into cell (ie. cholesterol and iron)

• LDL & Cholesterol Example

  • LDL transport particle with esterified cholesterol packed in coat of protein, phospholipid, and free cholesterol

  • Protein Apo-B100 binds to receptor on cell surface

  • LDL complex moved into cell by clatharin-coated vesicles and fuse with lysosome

  • LDL complex hydrolyzed into amino acids, cholesterol and fatty acids

  • Receptor and associated lipid bilayer recycled

Gap Junctions Structure

• 6 molecules of connexin embedded in each of the membranes, forming 2 connecting hemichannels

• Larger than membrane channels

• Stay open longer than ion channels

• Non-Specific

Gap Junctions Function & Examples

• Move polar (ions, sugars, amino acids, metabolites and nucleotides with molecular weight < 1000

• cell communication during embryo/fetal development and trophoblast proliferation

• nourishment of cells distant from blood vessels

• heart muscle where connections between cells ensure rapid/ synchronous responses to stimuli

Caveolae & Rafts

• Lipid Rafts rich in cholesterol and sphingolipids that help organize and group important proteins for signaling or transport

  • Caveolae: flask looking and facilitates signaling, endocytosis, protection from stress

  • Rafts: floating patches that facilitate sorting and signaling