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57 Cards in this Set

  • Front
  • Back
Separate cell contents from the extracellular environment

Separate organnels from other cellular constituents
Membranes are biological barriers
Membranes provide ________ and _____ the flow of metabolites and other compounds in/out of the cell.
protection and control the flow of metabolites.
Amphiphatic lipids, such as phospholipids and sphingolipids

These lipids contain both polar and non-polar components.

When put in an aqueous medium, tend to self-associate into a spherical or a laminar structure.
Membranes are based on lipids
Has a hydrophobic core
Micelle
Lipid bilayer enclosing an aqueous core

Inject DNA into a cell
Liposomes
Membranes also contain proteins with a variety of functions

Carbohydrates can be bound to both the lipid and protein components

Different membranes have different compositions of protein, lipid and carbohydrate

Biological membranes are asymmetric
Membrane composition

Cannot flip from inner to outer membrane
Carbohydrates are mostly located on the ______ surface of a cell membrane
outer

Often serve as recognition sites for specific binding
Different compositions affect the membranes properties
Lipid/protein ratio matters
The inner and outer faces have different protein and lipid profiles.

Maintains entry into cells
The asymmetry of phosphatidylserine contributes to the membrane potential due to its negative charged.

Phosphatidylserine is a marker for apoptosis

An attempt to break voltage dependent linkages
Different membranes have different lipid and ______lipid compositions
phospholipid
Components can move laterally within a membrane face

Components move from one face to the other slowly, if at all.
Asymmetry impleis that both lipid and protein are 'fixed' on a given face
Fluidity is dependent upon ________ and _______
temperature and composition

Membranes are fluid structures
Membranes have a transition temperature at which they undergo a phase change
Fluidity increases with increasing temperature
- Increased saturation of fatty acid components decreases fluidity

- Increased cholesterol decreases fluidity

- Ca2+ binding to the ionic portion of lipid components can decrease fluidity. (provides chelator)
Fluidity is affected by composition, particularly the lipid composition

Fatty acids like to congregate tightly.

Unsaturated bonds form kinks and push them farther apart.
Polar head group associates with hydrophilic outer layer

Hydrophobic ring structure nestles into hydrophobic acyl chains.

Heaad group can hydrogen bond with phospholipid head groups in the hydrophilic region
Cholesterol

- Decreases fluidity caused by cis-double bonds

- Gets between saturated bonds and prevents them from congregating

Overall, fluidity decreases
- May contain hydrophobic transmembrane domains that traverse the membrane

- May be mostly on the inside or outside face of the membrane

- May be covalently bound to a lipid

- Removal requires drastic treatment (detergent, organic solvent), often with a loss of native structure
Integral proteins are bound within the membrane, including the lipid portion
- Mostly through polar interactions

- May be through interaction with an integral protein

- Easily released by ionic solvents
Peripheral proteins are bound to the surface of the membrane
- The glycosylphosphatidylinositol (GPI) anchor

- Attachment of myristic acid (C14:0) by N-terminus

- Attachment to FA (C14-18:0) by thioester bond with cysteine

- Attachment to prenyl groups through thioester bond with cysteine
Colvalent attachment of proteins to membrane lipids
Conjugation of a protein to an inositol.

Essentially integrated into membrane.
The glycosylphosphatidylinositol anchor (GPI anchor)
1. Specific enzyme catalysts
a. ETC, succinate dehydrogenase, carnithine acyltransferase

2. Transport of solutes across membranes
a. Transporters, translocases, translocators, permeases, pumps

3. Sites of recognition
a. Usually through bound carbohydrate
b. Surface immunoglobulins in cells of the immune system
c. Peptide hormone receptors

4. Structural component
a. Maintenance of cell shape or flexibility.
b. Cytoskeleton
c. Anchored to trans-membrane proteins
Functions of proteins in membrane
- Fluidity
- Flexibility
- Self-sealing ability
- Impermeability
- Sidedness
Characteristics of the Fluid Mosaic model membrane

Self-sealing:
Molecules are separated, then heal/repair itself
Integral and peripheral proteins and cholesterol

Carbohydrate binding
More properites of the FMM membrane

Implies sidedness.
Determined empiracally by measuring rates of lateral movement of specific lipids and other membrane components.

Observed their distributions
The Lipid Raft model

Counters the FMM of lateral diffusion
Membranes contain heterogeneous dirstributions of lipids and proteins.
Lipid Raft model

As opposed to homogenous distribution of the FMM
Domains with high concentrations of cholesterol, sphingolipids and localized concentrations of certain proteins.
These domains are called: Lipid Rafts

Rafts are small:
- Tens of microns in diameter
- Don't comprise the majority of the membrane
Certain _______ proteins (eg, receptors) have been shown to localize lipid rafts.
membrane proteins
This ________ fence model proposes that there are isolation of membrane subdomains encircled by proteins.
Picket fence

May involve the anchoring of these proteins to the cytoskeleton

May result inphase transitions between domains, due to differing lipid and cholesterol composition.
Raft organization appears to be dynaming and perhaps regulated.
Key point of Lipid Raft model
Testing has shown that certain molecules prefer to target these areas of a cell
Pockets of high cholesterol in a raft
1. Electrochemical transport
a. Chemical
i. Relative number of solutes on either side of a membrane
b. Electrical
ii. Inside of cell is negative, due to anions within the cell.

2. Passive transport

3. Active transport
Mechanisms of transport across membranes
Uncommon mechanism

Primarily for gases (CO2, O2)

Not for charged molecules
Simple diffusion (passive transport)
- Specific

- Saturable

- Inhibitable
Mediated transport

Much more like an enzyme catalyzed reaction
- Uniport

- Symport
Drags molecule against its concentration gradient, utilizes the high gradient of another molecule.
Ex: glucose transport into intestinal epithelial cells.

- Antiport
Na+/K+ ATPase
Types of mediated transport
Energetics of transport

dG = 2.3 RT log [S2]/[S1]
If dG is negative, transport is passive

If dG is positive, transport is active
Undergo a conformation change to move substrate across membrane

Functions like an enzyme
Transporters
Maintain an open cavity for passage of a substrate
Channels/pores

Channel:
Selective, usually gated.
Behave more like transporters

Pore:
Non-selective, larger opening
Commonly formed by amphipathic alpha helices

Allow passage by opening/clsoing ('gating') based on conformation changes

Gating may be dependent on:
a. Membrane potential (voltage-gated channels)
b. Binding of a specific agonist (ligand-gated channels)
c. Covalent modification (eg phosphorylation)
Ion channels

Clinical significance:
Cystic fibrosis is a deficiency in a chloride channel.
A member of the ATP binding cassette (ABC) family of proteins
CFTR found in epithelial cells of multiple organs
Channel is only active when phosphorylated.
a. Phosphorylation of the regulatory subunit (R) by PKA causes a confirmation change that allows ATP to bind to the adenine nucleotide binding domain (ABD) to initiate channel opening.
CFTR is a gated channel controlled by phosphorylation
Mutations that affect CFTR function result in the accumulation of thickened mucus in airways, leading to pathologies associated with cystic fibrosis

Cholera toxin activates CFTR phosphorylation, causing constituitve channel opening.
i. In the intestine, this causes dehydration.
Chloride efflux is important for the osmotic movement of water out of the cell to maintain mucous fluidity
CFTR required for osmotic balance in lung, liver, pancreas, GI tract, reproductive tract and skin.

CFTR defects can affect the function of any of these organ systems.

CFTR deficiencies have been attributed to >1400 mutations.

Most mutations result in unstable proteins that are degraded in the ER

The most common mutation (66% of cases) is a deletion of Phe 508, which causes improper folding of CFTR and its degradation by the cell.

Defects in CFTR cause varying degress of dysfunction of most epithelia.
CFTR affects epithelium in multiple tissues.

Phe 508
- Not a dysfunctional protein
- Protein isn't there
1. PKA phosphorylation of the RD renders NBDs available for ATP binding.
a. CFTR is unique among ABC transporters in containing an R-domain that controls ATP binding.

2. ATP binding causes NBD1/NB2 dimerization, and the conformational change that opens the channel.

3. Hydrolysis of ATP at site 2 releases of ADP, disrupting NBD dimerization and closing the channel.

4. The channel cycles approximately once per second.

5. Unclear how many Cl- ions pass through per cycle.
CFTR mechanism in action

Coordinated ATP binding of both cells

Hydrolysis of ATP and release of ADP:
Disrupts NBD dimerization and closes the channel
ATP-binding cassette transporters: A superfamily of transporters in all organisms that move diverse compounds across membranes.
a. Driven by a cycle of conformational changes mediated by ATP binding (opens) and hydrolysis (closes).

Most are active transporters, not gated channels like CFTR.

Originally described as ATP-dependent nutrient transporters in bacteria.
e.g. Maltose transporter

Also function in bacteria to pump endogenous virulence factors and antimicrobial agents out of the cytoplasm.
ATP-binding cassette transporters (ABC transporters)
- In humans, ABC family comprised of 49 transporters in 7 subfamilies (based on structural similarities, designated ABCA through ABCG) with diverse functions in various cell types and organelles.

a. Regulate local permeability at blood barrier in brain, spinal cord, and placenta.

b. Excrete toxins in the liver, GI and kidney.

c. Important for cellular lipid transport and metabolism.

d. Antigenic peptide processing/surface presentation in MHC.

e. Can modulate lipid composition of outer and inner layers of the plasma membrane.

f. Examples of ABC-transported molecules: cholesterol, lung surfactant, peptides,heme, bile salts, phospholipids, xenobiotics, toxins.
ABC transporters

Of note, Antigentic peptide processing/surface presentation in MHC
Broad substrate specificity of prokaryotic transporters contributes to the acquisition of multiple drug resistance (MDR) by pathogenic bacteria.

Serious problem with nosocomial infections (e.g. MRSA).
ABC transporters as drug pumps:
Multiple drug resistance (MDR) in bacteria & cancer cells
13 human ABC transporters have been associated with drug transport and resistance, primarily from the ABCC family.

Also designated as MRP (multidrug resistance protein) in this context.

Overexpression causes broad specificity resistance to drugs, due to their rapid efflux from the cell.

Can dramatically affect drug efficacy.

Particulary problematic for cancer treatment by chemotherapeutics.

Cancers can acquire this phenotype over type, due to the selection of drug resistant cells.

Each can generally transport multiple unrelated drugs.

Collectively shown to transport hundreds of different drugs compounds.
ABC transporters as drug pumps:
Multiple drug resistance (MDR) in bacteria & cancer cells
First/best characterized example is ABC-B1 (P-gp/MDR1), associated with the acquisition of Multiple Drug Resistance (MDR) in many cancers.

Overexpression confers high resistance to many classes of drugs Anthracyclines, vinca alkaloids, taxanes, etoposide, imatinib, colchicine, methotrexate, actinimycin D, et al.
ABC transporters as drug pumps:
Multiple drug resistance (MDR) in bacteria & cancer cells

Research is now directed toward inhibition of these pumps.
1) Substrate binding induces a conformational change that enhances ATP binding to NBD site 1.

2) Binding of ATP to site 1 stabilizes NBD interaction, facilitating ATP binding
to NBD site 2.

3) Binding of second ATP completes formation of the NBD dimer, and causes a conformational change in the MSDs that releases the substrate
on the opposite side.

4) The protein maintains outward facing orientation as site 2 ATP is hydrolyzed.

5) Release of ADP from site 2 resets the protein for inside face substrate binding.

Step 6? Not clear whether site 1 ATP hydrolysis and ADP release is also required to reset.
Current model of the ABC/ MRP transport cycle

Example: MRP1 (ABCC1) and leukotriene C4 (LTC4) transport
The Na+/K+-ATPase contributes to the electronegativity of the cytosol
(i.e. the membrane potential) due to the net efflux of positive charges.
Glucose transport through the cell
Glucose exits to plasma by passive mediated transport by this transporter
GLUT 2
- K+ is transported out of the cell to make room for Na+ influx

- Helps maintain electronegativity
Leakdown transporters (I.E. K+ transporters)
- Intracellular compartmentation is a defining characteristic of eukaryotic organisms.

- Compartmentation allows for the restriction of processes and reactions to physically separate environments within the cell.

a. Mediated by phospholipid membrane barriers between organelles and the cytoplasm.

b. Permits optimal conditions for subsets of processes without conflict.
i. Allows for disparate concentrations of molecules between compartments.

c. Provides an additional mechanism for regulation by controlling the interaction between compartments.
Compartmentation and shuttle properties
The inner mitochondrial membrane contains numerous specific transmembrane shuttles for the selective movement of small molecules between the mitochondrial matrix and the cytosol.
This is particularly important for intermediary metabolism with respect to the interaction between reactions that occur in the cytosol and those that occur in the motochondria.
Citrate Transporter

Citrate generated in mitochondria is exported to cytosol where it serves as a source of acetyl-CoA, in exchange for malate.
Metabolic pathways use multiple transporters
in shuttle systems
ADP/ATP and Phosphate Translocation
a. Adenine nucleotide transporter
b. Phosphate translocator
Metabolic pathways use multiple transporters
in shuttle systems
Glycerol Phosphate Shuttle & Malate/Aspartate Shuttle

Transport shuttles for reducing equivalents
Metabolic pathways use multiple transporters
in shuttle systems
Acylcarnitine transport (CPT I & CPT II)

A transport shuttle for acyl-CoA for oxidation in mitochondria
Metabolic pathways use multiple transporters
in shuttle systems