Lecture 14

Question 1

passive transport

Answer 1

type of facilitated diffusion

transport of substrate along its concentration gradient

Question 2

active transport

Answer 2

type of facilitated diffusion

move a substrate against its concentration gradient- requires an input of free energy

Question 3

na- k pump

Answer 3

Na+ and K+ pump acts in all cells to maintain higher concentrations of K+ inside and Na+ outside

Question 4

How to move Molecules using a Concentration Gradient Diffusion and Facilitated Diffusion

Answer 4

Ionophores, Ion Channels, Transporters

Question 5

How to move material against a Concentration Gradient Active Transport – requires energy

Answer 5

Use Proton, Ion, or other concentration gradients

ATP-dependent transport

Question 6

How to get really big stuff across a membrane

Answer 6

Clathrin-mediated Endocytosis – next quarter

Question 7

How to Transfer Information Across a Membrane (2 Examples)

Answer 7

Insulin Receptor, G-Protein Coupled Receptors

Question 8

hormones and signaling

Answer 8

act through second messengers- involve a 3- protien module- receptor, transducer (g-protein), and effector (adenylate cyclase or related enzyme)

Question 9

To cross a membrane you need an energy source Energy is available from a concentration gradient

Answer 9

if ions are involved- there is also a membrane potential (delta V)

before Equil net flux
ΔG < 0

at equil no net flux

Question 10

Negative ΔG -

Answer 10

Movement down concentration gradient

transport can be passive

Question 11

Positive ΔG –

Answer 11

Movement against concentration gradient
requires energy (ATP or Concentration Gradients)
Transport must be an active process

Question 12

To cross a membrane a molecule must be permeable to the lipid bilayer –

Answer 12

Passive Diffusion driven by concentration differences

Diffusion across a membrane Correlates with size and water solubility

H20, CO2, & O2 readily cross the membrane

The concentration of water on both sides of the membrane is very high (55M).

Osmotic Pressure drives the movement of water – minimize the difference in solute concentration across the membrane

Question 13

permeabilities

H2O
Indole
Glucose
Na+
K+

Answer 13

water-5x10-3
indole- 2x10-4
glucose- 4x10-10
Na+ <1.6x10-13
K+ <9x10-1

Question 14

Selective Permeability - Facilitated Diffusion

Answer 14

1. Build a peptide cage to replace solvent shell & increase permeability
2. Direction dependent on concentration difference

Question 15

Valinomycin (Ionophore)

Answer 15

No energetic cost for binding K+
Increases Permeability of K+
30000x preference for K+ over Na+

Question 16

Facilitated Diffusion: Ion Channels

The Potassium Ion Channel has a selectivity filter

Answer 16

K+ Channel is a tetramer

K+ Channel pore is lined with backbone carbonyls

Perfect diameter to
“solvate” K+

Question 17

Facilitated Diffusion: Ion Channels

The Potassium Ion Channel has a selectivity filter

Answer 17

Flow of ions through a channel must be tightly controlled
• ion channels have open and closed conformations
• ligand-gated and voltage-gated ion channels

Exchange solvent shell with coordination by backbone carbonyl groups in the channel

Question 18

AquaPorins

Answer 18

Allows rapid movement of water across the cell membrane

Tetramer with four 2.8Å Pores
Engineered only for water
Excludes ions
Excludes H+ (H3O+)
Equalizes Osmotic Pressure without disrupting ion and H+ gradients

Question 19

Passive vs. Facilitated Diffusion

Answer 19

Facilitated Diffusion:
• greatly increases Permeability
• is highly Selective
• depends on a limited # of proteins
• rate of diffusion can be Saturated

Question 20

permeability of water and glucose in synthetic and erythrocyte membrane

Answer 20

water- S- 5x10-3 E- 5x10-3

Glucose- S- 4x10-10 E- 2x10-5

Question 21

Facilitated Diffusion
Family of Glucose Transporters
Glut1 permease

Answer 21

Glut1 permease: plasma membrane of erythrocytes
• 12 membrane-spanning helices
• Passive transport - driven by concentration gradient - reversible
• Specific for D-glucose
• Switches between 2 protein conformations (never an open pore)
• Allows for high rates of diffusion across the membrane
• Rate of diffusion shows saturation behavior

Question 22

Facilitated Diffusion via Glucose Transporters

Answer 22

Works well for red blood cells - require only small amounts of energy

Large family of transport proteins (14 members grouped into 4 classes)
Class I members: Glut1-4

Question 23

Glut1 –

Answer 23

erythrocytes

Question 24

Glut2 –

Answer 24

Liver – generally transports glucose OUT for use by other tissue

Question 25

Glut3 –

Answer 25

neurons – highest affinity for D-glucose

Question 26

Glut4 –

Answer 26

adipose and muscle tissues Stored in intracellular vesicles. Exposed on cell surface in response to insulin

Question 27

Primary Active Transport: Requires Energy Maintain Concentration Gradients P-type ATPase & ABC-transporters

Answer 27

ATP drives conformational changes 1) Open on cytoplasmic side 2) Open on extracellular side

Question 28

ATP-dependent Transport - P-type ATPases

Answer 28

P-type ATPases are primarily used to maintain ion gradients (also transport some lipids – eg. flippase) Phosphorylation & de-Phosphorylation of a critical Asp residue Phosphorylation drives protein conformational changes and ion transport

Question 29

Ca+2 ATPase (SERCA)

Answer 29

pumps Ca+2 into sarcoplasmic reticulum | [Ca+2 cytoplasm] = 0.1mM [Ca+2 SR] = 1.5mM

Question 30

Cytosol

Answer 30

[K+] = 140mM [Na+] = 12mM

Question 31

Extracellular

Answer 31

``` [K+] = 4mM [Na+] = 145mM ```

Question 32

Mechanism of P-type ATPases 2 main states: changes in conformation driven by ATP

Answer 32

* Digitalis and Ouabain bind to the E2 conformation of the Na+/K + pump * Proton Pump Inhibitors (like Nexium and Prilosec) are used to treat acid reflux by inhibiting the gastric proton pump.

Question 33

ATP-dependent Transport – ABC Transporters

Answer 33

Large Family of Transporters Transport amino acids, peptides, metal ions, lipids, bile salts, toxins and drugs against concentration gradients * Certain ABC Transporters are responsible for multi drug resistance * Defects in an ABC Cl- ion Transporter is associated with Cystic Fibrosis (build-up of mucus in the lungs).

Question 34

Secondary Active Transport: | Intestinal Glucose/Na+ Symporter

Answer 34

Secondary Active Transport One solute moving down its concentration gradient can transport another moving against a gradient (transporters in your intestine allows you to scavenge all the available glucose after a meal)

Question 35

Symport

Answer 35

– move molecules in the same direction Terms apply to both active and passive transporters

Question 36

Antiport –

Answer 36

move molecules in opposite directions Terms apply to both active and passive transporters

Question 37

Moving Information across a membrane Features of Signal Transducing Systems • Modularity

Answer 37

• Adapt similar structures to respond to different signals

Question 38

Moving Information across a membrane Features of Signal Transducing Systems • Specificity

Answer 38

• Specific receptor is responsive to a specific ligand

Question 39

Moving Information across a membrane Features of Signal Transducing Systems • Amplification

Answer 39

• One ligand binding event outside a cell can activate 1000s of enzymes inside a cell – large, rapid response

Question 40

Moving Information across a membrane Features of Signal Transducing Systems • Termination

Answer 40

• Mechanisms of turning off a signal

Question 41

Receptor Tyrosine Kinases (RTKs) –

Answer 41

Insulin Receptor Large family of plasma membrane receptors Extracellular ligand binding domain linked to an intracellular catalytic domain Insulin Receptor is a dimer. Other RTKs are monomers in the membrane. Ligand binding induces dimerization and activation

Question 42

insulin

Answer 42

* Insulin is a peptide hormone – dispersed via circulatory system * Binds to extracellular receptor domain * Binding activates Tyrosine Kinase catalytic domain inside the cell

Question 43

Receptor Tyrosine Kinases – Insulin Receptor

Answer 43

Ligand binding induces a conformational change that brings kinase domains together * Kinase self-activation (Auto-Phosphorylation) * The start of a signaling cascade.

Question 44

Receptor Tyrosine Kinases – Insulin Receptor

Answer 44

One branch of Insulin Signaling triggers glucose uptake in muscle cells Phospholipid modifications recruit new kinases to the membrane

Question 45

Transfer of Information linked to Cellular uptake of Glucose

Answer 45

(one of many cellular responses to insulin) - PDK1 (PIP3-dependent protein kinase) activation -> activation of additional kinases (signaling cascade) - promotes movement of Glut4 transporters, stored in the membranes of intracellular vesicles, to move to the plasma membrane - increased uptake and storage of Glucose

Question 46

What is are the structural changes behind activation of the Insulin Receptor Tyrosine Kinase?

Answer 46

Movement and phosphorylation of the Activation Loop open up the kinase active site.

Question 47

Information Transfer: G-Protein Coupled Receptors

Answer 47

Epinephrine binding outside stimulates cAMP production and Ca2+ influx * 7-Trans-membrane receptor (blue) * Ligand binding stimulates: 1. Association of the G protein complex with the receptor 2. Exchange of GTP for GDP on the α-subunit (orange) • GTP binding leads to dissociation of the complex and activation of other enzymes or channels (generate 2nd Messengers) • One ligand bound receptor can activate multiple Gα subunits • Slow hydrolysis of GTP inactivates α-subunit and causes re-assembly of heterotrimeric G-Protein complex (Built-in timing mechanism)

Question 48

Information Transfer: G-Protein Coupled Receptors | Signal Amplification - Generation of Second Messengers

Answer 48

* GCPRs are responsible for most of the cellular responses to: * Hormones, Neurotransmitters, Senses (Light, Olifaction, Taste) * Different Heterotrimeric G-Proteins in Different Tissues determine response * Types of second messengers * cAMP, Ca2+ * PIP2 converted by Phospholipase C into: * Diacylglycerol (DAG) and Inositol-1,4,5-trisphosphate (IP3) * 1/3 to 1/2 of all drugs on the market target GCPRs * Hypertension, cardiac arrhythmia, glaucoma, anxiety, migraine headaches

Question 49

Summary | Diffusion is Driven by Concentration Differences

Answer 49

• Understand the differences between Simple and Facilitated Diffusion • Examples of facilitated Diffusion: Ionophores, Ion Channels, Glut1 Permease

Question 50

Summary | Active Transport

Answer 50

* Transport against a concentration gradient requires energy * Understand the difference between Primary Active Transport (P-type ATPases) and Secondary Active Transport (Intestinal Glucose/Na+ Symporter)

Question 51

Summary | Movement of Information

Answer 51

• Receptor Tyrosine Kinases – Protein activation by Phosphorylation and phosporylation cascades * G-Protein-Coupled Receptors (GPCRs) * Generation of Second Messengers * Internal GTPase Clock to turn off signaling