Lecture 14 Flashcards
passive transport
type of facilitated diffusion
transport of substrate along its concentration gradient
active transport
type of facilitated diffusion
move a substrate against its concentration gradient- requires an input of free energy
na- k pump
Na+ and K+ pump acts in all cells to maintain higher concentrations of K+ inside and Na+ outside
How to move Molecules using a Concentration Gradient Diffusion and Facilitated Diffusion
Ionophores, Ion Channels, Transporters
How to move material against a Concentration Gradient Active Transport – requires energy
Use Proton, Ion, or other concentration gradients
ATP-dependent transport
How to get really big stuff across a membrane
Clathrin-mediated Endocytosis – next quarter
How to Transfer Information Across a Membrane (2 Examples)
Insulin Receptor, G-Protein Coupled Receptors
hormones and signaling
act through second messengers- involve a 3- protien module- receptor, transducer (g-protein), and effector (adenylate cyclase or related enzyme)
To cross a membrane you need an energy source Energy is available from a concentration gradient
if ions are involved- there is also a membrane potential (delta V)
before Equil net flux
ΔG < 0
at equil no net flux
Negative ΔG -
Movement down concentration gradient
transport can be passive
Positive ΔG –
Movement against concentration gradient
requires energy (ATP or Concentration Gradients)
Transport must be an active process
To cross a membrane a molecule must be permeable to the lipid bilayer –
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
permeabilities
H2O Indole Glucose Na+ K+
water-5x10-3 indole- 2x10-4 glucose- 4x10-10 Na+ <1.6x10-13 K+ <9x10-1
Selective Permeability - Facilitated Diffusion
- Build a peptide cage to replace solvent shell & increase permeability
- Direction dependent on concentration difference
Valinomycin (Ionophore)
No energetic cost for binding K+
Increases Permeability of K+
30000x preference for K+ over Na+
Facilitated Diffusion: Ion Channels
The Potassium Ion Channel has a selectivity filter
K+ Channel is a tetramer
K+ Channel pore is lined with backbone carbonyls
Perfect diameter to
“solvate” K+
Facilitated Diffusion: Ion Channels
The Potassium Ion Channel has a selectivity filter
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
AquaPorins
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
Passive vs. Facilitated Diffusion
Facilitated Diffusion: • greatly increases Permeability • is highly Selective • depends on a limited # of proteins • rate of diffusion can be Saturated
permeability of water and glucose in synthetic and erythrocyte membrane
water- S- 5x10-3 E- 5x10-3
Glucose- S- 4x10-10 E- 2x10-5
Facilitated Diffusion
Family of Glucose Transporters
Glut1 permease
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
Facilitated Diffusion via Glucose Transporters
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
Glut1 –
erythrocytes
Glut2 –
Liver – generally transports glucose OUT for use by other tissue
Glut3 –
neurons – highest affinity for D-glucose
Glut4 –
adipose and muscle tissues
Stored in intracellular vesicles. Exposed on cell surface in response to
insulin
Primary Active Transport: Requires Energy
Maintain Concentration Gradients
P-type ATPase & ABC-transporters
ATP drives conformational changes
1) Open on cytoplasmic side
2) Open on extracellular side
ATP-dependent Transport - P-type ATPases
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
Ca+2 ATPase (SERCA)
pumps Ca+2 into sarcoplasmic reticulum
[Ca+2 cytoplasm] = 0.1mM [Ca+2 SR] = 1.5mM
Cytosol
[K+] = 140mM [Na+] = 12mM
Extracellular
[K+] = 4mM [Na+] = 145mM
Mechanism of P-type ATPases
2 main states:
changes in conformation driven by ATP
- 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.
ATP-dependent Transport – ABC Transporters
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).
Secondary Active Transport:
Intestinal Glucose/Na+ Symporter
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)
Symport
– move molecules in
the same direction
Terms apply to both active
and passive transporters
Antiport –
move molecules in
opposite directions
Terms apply to both active
and passive transporters
Moving Information across a membrane
Features of Signal Transducing Systems
• Modularity
• Adapt similar structures to respond to different signals
Moving Information across a membrane
Features of Signal Transducing Systems
• Specificity
• Specific receptor is responsive to a specific ligand
Moving Information across a membrane
Features of Signal Transducing Systems
• Amplification
• One ligand binding event outside a cell can activate 1000s of enzymes inside a cell – large, rapid response
Moving Information across a membrane
Features of Signal Transducing Systems
• Termination
• Mechanisms of turning off a signal
Receptor Tyrosine Kinases (RTKs) –
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
insulin
- Insulin is a peptide hormone – dispersed via circulatory system
- Binds to extracellular receptor domain
- Binding activates Tyrosine Kinase catalytic domain inside the cell
Receptor Tyrosine Kinases – Insulin Receptor
Ligand binding induces a conformational change
that brings kinase domains together
- Kinase self-activation (Auto-Phosphorylation)
- The start of a signaling cascade.
Receptor Tyrosine Kinases – Insulin Receptor
One branch of Insulin Signaling triggers glucose uptake in muscle cells
Phospholipid modifications recruit new kinases to the membrane
Transfer of Information linked to Cellular uptake of Glucose
(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
What is are the structural changes behind activation of the Insulin Receptor
Tyrosine Kinase?
Movement and phosphorylation of the Activation Loop open up the kinase active
site.
Information Transfer: G-Protein Coupled Receptors
Epinephrine binding outside stimulates cAMP production and Ca2+ influx
- 7-Trans-membrane receptor (blue)
- Ligand binding stimulates:
- Association of the G protein complex with the receptor
- 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)
Information Transfer: G-Protein Coupled Receptors
Signal Amplification - Generation of Second Messengers
- 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
Summary
Diffusion is Driven by Concentration Differences
• Understand the differences between Simple and Facilitated Diffusion
• Examples of facilitated Diffusion: Ionophores, Ion Channels, Glut1
Permease
Summary
Active Transport
- 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)
Summary
Movement of Information
• 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
