Test question: polar head group with 2 acyl chains —for lipid membrane
Fall 2013 Study Guide for Plasma Membrane I and II (Dr. Smas) (One page):
1. What type of molecule underlies the basic architecture of the plasma membrane? Why are they called amphipathic? What type of molecular interactions underlie assembly and maintenance of the lipid bilayer? Why is the plasma membrane termed "fluid mosaic"? In what ways can integral or peripheral proteins associate with or embed in the plasma membrane? What about in the specific case of "glycophorin A"?
Basic Architecture of Plasma Membrane: Phospholipid —Glycerol with 2 Acyl Group, and a phospate-oh polar group
Amphipathic: Polar/hydrophilic heads and hydrophobic tails
Assembly and Maintenance —formed and maintained by multiple noncovalent interactions (esp. hydrophobic association of tails) o Membrane formation due to amphipathic nature of membrane lipids o Hydrophobic interactions by exclusion of water drives formation of E stable structures o Van der Waals attractive forces b/w hydrocarbon tails favor close packing o Electrostatic and H-Bond attractions b/w polar head groups and H20 molecules
Fluid Mosaic: Fluid due to hydrophobic acyl groups in the Hydrophobic core that allow for lateral movement of proteins. Mosaic because it consists of many different macromolecules: glycoproteins, phospholipids, cholesterol, peripheral and integral proteins, etc.

Fluid=unsaturated FA with Kinks (disrupt stack)  Excessive fluidity=membrane destruction

Viscous=Saturated FA (can stack better)
Fx of Fluidity: influence arrangement of PRO and lipids, promote assembly/disassembly of PRO subunits and signaling complexes in membrane, change membrane permeability
Integral and Peripheral Proteins:
Integral Proteins —enters/spans hydrophobic core via single or multiple pass ( 
-Helix)
Peripheral Proteins —more loosely associated w/ membrane, do not enter hydrophob. Core
Glycophorin A: utilizes

-Helix to span the membrane
Fx: RBC integral membrane glycoprotein —produces MN blood group
Mech:

-Helix contains polar peptide bonds and charged AAs however it is oriented so that it hydrogen bonds with itself, instead of the hydrophobic core and the exterior side chains spanning the membrane are noncharge/hydrophobic. A sequence of 20-25 AA, Rich in hydrophobic/nonpolar
AA with and

-helical strucutre often acts as transmembrane segment.
2. Why is the hydrophobic
 � -helix a highly suitable structure that allows transmembrane proteins to span the plasma membrane? What types of "R" groups/amino acids might you predict to be present or absent in this type of
 � -helix?

-Helix contains polar peptide bonds and charged AAs however it is oriented so that it hydrogen bonds with itself, instead of the hydrophobic core and the exterior side chains spanning the membrane are noncharge/hydrophobic. A sequence of 20-25 AA, Rich in hydrophobic/nonpolar
AA with and

-helical structure often serve as transmembrane segment.
Likely AAs: Alanine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tyrosine, Tryptophan
Unlikely AAs: Proline (Breaks helix), Histidine, Arginine, Glutamine, Aspartate (POLAR AAs)
3. Cholesterol in the membrane leads to a diminution/abolishment of the phase transition. Think of it as having a “buffering” effect on membrane fluidity. What are the effects of increasing cholesterol content at: Above the Tm? Below the Tm? of an artificial simple membrane. How would membrane fluidity/Tm be affected by alteration of the degree of bond saturation or the length of the fatty acyl chains?
Fx: disrupts regular interactions of fatty acyl side chains, makes membranes less likely to undergo phase transition, buffer against fluidity change

Add Cholesterol Below Tm:

Below Tm the membrane is gel-like /more solid

The lipid side chains are tightly packed and orderly

Indrocing kinked cholesterol disrupts this= increased fluidity
Add Cholesterol Above Tm :

Above Tm, membrane is more fluid-like

Lipid side chains are disorganized and moving

Cholesterol acts to limit/restrict the overall free moevement of the lipid side chains due to its planar shape (steroid nucles) = decrease fluidity
Increasing Tm/Right Shift:
 longer Acyl Chain

More saturated
Decreasing Tm/Left Shift:

Shorter Acyl Chain

Desaturated
Membrane Fluidity

Adding saturated FA=decrease fluidity

Adding unsaturated FA=increased fluidity (kinks decrease stacking)
4. If you wanted to "extract/remove" an integral membrane protein (like "glycophorin A") away from the lipid bilayer, what types of experimental conditions/treatments might you employ?
You would need to remove it with a detergent that could disrupt the lipid bilayer since it is embedded or anchored within the phosholipid bilayer. However, a peripheral protein can be
removed with salt/pH change.
5. Give one example of how changes in the lipid composition of the extracellular and intracellular faces of the plasma membrane might signal cells for destruction, specifically the role of phosphatidylserine.
In RBC phosphatidylserine normally exists on the inner/cytoplasmic leaflet, however when the RBC ages, the phosphatidyl serine moves to the outer/exoplasmic leaflet.

Serves as an alternate pathway for recognition and destruction by macrophages in
the Spleen.
Cells undergoing apoptosis expose phosphatidyl serine on the exoplasmic leaflet, this triggers macrophage recogntion and destruction of apoptotic cell
—general example of how PS function as signal for destruction
6. Using "glycophorin A" as an example, at what stage and where in the cell during protein synthesis are the sugars attached (glycosylation). Where on the protein do they occur (in general terms, which
3 types of amino acids)?
Glycophorin A confers blood type of RBC. It becomes glyocsylated on its exoplasmic portion while it is processed in the Lumen of the ER or Golgi. In general, the glycosylation can be N-Linked or Olinked to the protein.

N-linked —Asparagine

O-Linked —Serine or Threonine

7. What types of substances are least likely to pass through the plasma membrane without a transporter protein? Give an example of a ligand-gated and a voltage-gated ion channel (as presented in class). What are their key structure/function features?
Gases (CO2, N2, O2), small uncharged polar molecules (Ethanol), water via osmotic pressure, and urea* Depending on cellular/organ setting
Note: All cells must maintain EC gradient to fx, gradients can serve as form of E to drive cell processes. While ion channel opening responds to spec. signals, ion channel flow rate and direction rely on EC gradient present across plasma membrane. Selective permeability creates the concentration gradient. Sum of Free Energy (Delta G) determines direction & rate of ion flow.
Facilitated Diffusion:
1) Voltage-Gated Ion Channel: Na+ and K+ Channel

Na+ ECF=140mM ICF=10mM

K+ ECF=4mM ICF=140mM a. Structure: facilitates rapid passage (10^7-10^8 ions/s), transient alteration of ion membrane permeability can rapidly and precisely control signailing b. K+ Channel has positively charged lysines-- S4 Segment = voltage sensing c. Function: Propagation of Nerve Impulses —action potential d. Selectivity Filter —provides specificity for channel based on thermodynamic favorability of solvation vs. desolvation of a particular ion e. Electrostatic Repulsion rapidly pushes ions through the transporter f. Ball & Chain Model for INACTIVATION
2) Ligand-Gated Ion Channel: Nictotinic Acetylcholine (Ach) Receptor (allow Na+ & K+ Passage)
a. Location: Neuromuscular Junction
b. Function: Propagation of nerve impulse at synaptic cleft
c. Structure: 5 subunits, each with bulky hydrophobic Leucine side chain, and small
polar AA side chains
d. Mechanism:
i. Closed State —all Leucines face center of channel—no ions can pass
ii. Open Sate —Binding of Acetylcholine  Conformational Change: Helical
subunits slide and rotate (E Favored) exposing their polar AAs to the center
iii. Large flux of Na+ into cell, and K+ too
iv. Transient depolarization of membrane/action potential  Propagation  muscle
contraction
8. In the example of a "voltage gated K+" channel, the first 20 or so amino acids form a globular region referred to as "ball". How is this "ball" involved in function of this ion channel?
The ball allows for rapid inactivation of the K+ Ion channel, allowing quick control of ion entry.
Initially, the channel is closed and the ball is bound to the inactivation domain.
Signal via membrane depolarization (S4 segment sense change in voltage) causes it to open
Next, The ball swings up and creates a physical block in the channel (at inactivation domain), preventing ion entry.
Finally, as charges dissipate, the chain relaxes, the ball is removed and the channel resets.
Q: If ball was incorrectly formed? –Channel would always be open
Q: If Inactivation domain had AA mutation? –It wouldn’t experience conformational change, nonresponsive
9. What is the function of the "Na+/K+ ATPase", at which point in the function do " cardiotonic steroid " drugs act, and with what result?
The Na+/K+ ATPase is a P-Class Primary Active Transporter (ATP-Powered pump) Rate: 1x10^3 /s

Structure: Two subunits per transporter--

and

.

Does actual pumping,

probably locates pump in membrane?
Generates Ion Concentration Gradient necessary for:

Controlling cell volume

Driving transport of sugars and AA

Establishing and maintaining EC gradient in all cells

This EC gradient and K+ Leak channels maintain the membrane charge differential-Membrane
Potential (at rest = -60mV)
Important source of potential energy
Hydrolyzes 25-70% of cytosolic ATP in cells
Mechanism of pump:
1) Cytoplasmic Na+ binds to the protein  stimulates phosphorylation of protein by ATP (covalent bond)
2) Phosphorylation causes the protein to change conformation
3) Conformational change expels Na+ outside of cell and promotes binding of extracellular K+ to protein
4) K+ triggers release of phosphate group
5) Loss of phosphate restores original conformation
6) K+ is released into cell and Na+ sites are receptive again
Cariotonic Steroid Drugs: Digitalis and Ouabain
Tx: of Congestive heart failure
Fx: Increases strength of heart contraction w/o increasing heart rate (inc. stress)
Mech Background: Phosphorylation and dephosphorylation cycle of the

subunit is required for conformational changes necessary for proper pump function.
Mechanism of drug:
1) Ouabain inhibits dephosphorylation of E-2-P form (step 3  4), pump locked-non fx. w/ K+ still bound
2) [Na+] increases inside cardiac muscle cells
3) The increase in Na+ leads to an increase in [Ca2+] in cell, b/c Transporter for Ca2+ is dependent on Na+
4) calcium mediated signals act to increase the contraction and strength of heart muscle contraction
10. What is the underlying defect in "Myasthenia Gravis "? What model is proposed for the conformational change in this receptor that allows for controlled ion passage? What is the role of auto-antibodies in this disease?
Channelopathy:
Defect: Acetylcholine Receptor —at NMJ (junction b/w nerve endings and muscle fibers)
Sx: Skeletal Muscle weakness (receptor normally fx in muscle contraction)
Mech: Can involve impaired binding of Ach or destruction/blocking of the Ach receptors via
Autoantibodies —autoimmune disease
Conformational Change: sliding and rotating of helical subunits exposes small polar AA to core and allows passage of ions for AP propagation
Tx: 1) Acetylcholinesterase inhibitors  decrease breakdown of Ach, have more receptors to receive ligand 2) Immunosuppressives —to decrease effect of autoimmune disease/antibodies blocking Ach receptors
11. In insulin-sensitive cells, such as a fat cell, what is the role and location of insulin, glucose, insulin receptor, and "GLUT4" in regard to the uptake of glucose into the fat cell? What type of transport mechanism is this (i.e. For insulin-stimulated glucose uptake)? What is meant by translocation of GLUTt4 to the plasma membrane? Where does the insulin come from? Where is the insulin receptor located? As occurs during insulin stimulated glucose uptake, does either insulin or the insulin receptor actually enter the fat cell or does it function via “signal transduction” at the extracellular face of the plasma membrane? (HINT: THE ANSWER
IS NO, Glucose enters through GLUT4, insulin stays outside and interacts with the extracellular region of the insulin receptor, in a form of “signal transduction”)
Facilitated Diffusion —Carrier Mediated

Insulin comes from pancreatic

-cells and binds to insulin receptors on the extracellular surface of the plasma membrane in Insulin sensitive cells (muscle and adipose with Glut 4). This results in signal transduction  Glut 4 protein is stored in preformed intracellular vessicles, the vessicles move to the membrane, their lipid bilayer fuses with the lipid bilayer of the plasma membrane and Glut 4 protein is now on the cell surface. Glut 4 then facilitates the uptake of extracellular glucose.
**INSULIN NEVER ENTERS CELL, INSULIN RECEPTOR IS NOT INTERNALIZED
**The Glut 4/Insulin activity was designed to expediate the response of cells to increase blood glucose, if this relied on transcriptional regulation/gene regulation it would take so long that the glucose would already have been excreted.
VOCABULARY
Facilitated Diffusion-3 Main Features in comparison w/ simple diffusion:
1) Significantly Increased rate
2) Transport is specific
3) Transport is limited by the number of available carrier PROs  this results in them behaving similar to enzymes with regards to Vmax (rate of glucose uptake, [glucose] and # transporters
Signal Transduction —only the biomolecular signal, not the molecule itself transduces the membrane
12. Of the examples presented in detail in lecture, which types/classes of transporters are directly involved in binding and hydrolyzing ATP? Give two examples. How is one of these involved in drug resistance in cancer chemotherapy? What does the ABC stand for with this type of transporter? How is another (CFTR) involved in cystic fibrosis? What ion is transported by CFTR? What is the key distinction between passive and active transport mechanisms?
I. Primary Active Transport—Transporter itself hydrolyzes ATP
A) P-Class or P-Type: ATP Powered Pumps
Ex) Ca2+ ATPase—muscle SR, H+/K+ ATPase--stomach, Na+/K+ ATPase—ALL CELLS
Drugs—cardiotonic steroid drugs (digitalis and ouabain for Congestive Heart Failure)—See Q #9
B) ABC-Type: ATP Binding Cassette
Fx: Transport small molecules, ATP hydrolysis coupled with solute movement
Ex) MDR1 and CFTR
Mechanism: Cell naturally fluctuates b/w open and closed, ligand binds from cell interior  conformational change increases affinity for ATP  2 ATP bind to interior portion of protein  release of substrate to exterior
MDR1 and Cancer
 Drugs used to tx cancer and malaria consist of small planar drugs that can diffuse into cells, esp. cancer
 MDR1 gene is often amplified in tumor cells and this causes increased activity of pump
 MDR1 pump uses ATP E to export the drug from the cells cytosol  Drug FAILS to exert benefits
 Cells become chemo resistant  uncontrollable tumor growth/spreak  poor prognosis
NOTE: these cells naturally exist in the liver for pumping out toxins
CFTR and Cystic Fibrosis – channelopathy
“Cystic Fibrosis Transmembrane Regulator”
Cystic Fibrosis— chronic and progressive autosomal recessive genetic disease of bodies mucosal glands
Mutation: deletion in CFTR Gene, most common—F508 loss due to deletion of 3 bp, deltaF508 CFTR exists
 Normally, CFTR, located on the Apical surface of epithelial cell plasma membrane, fx to pump Cl- out of cell
 Mutant CFTR (deltaF508) can not reach cell surface  build up of Cl- in cell  Na+ and H20 enter cells from extracellular space
 Loss of H20 from extracellular space = thick, dehydrated mucus  respiratory tract cilia function is now defective and patient is more prone to infections

II. Secondary Active Transport—Unfavorable/uphill flow of one molecule coupled w/ downhill flow of another
REQUIRE PRIMARY TRANSPORTER FOR ATP HYDROLYSIS AND FAVORABLE GRADIENT
Ex) Sodium/glucose symporter  sodium gradient produced by Na+/K+ ATPase, Na+ flows in down its gradient, allowing glucose to enter against its gradient
13. What types of interactions occur at an ion channel "selectivity filter"? In simple terms, for a K+ channel, why is passage of K+ energetically favored while passage of Na+ is not? What is the role of the hydration shell?
Facilitated Diffusion —Ion-Channel Mediated: Voltage-Gated Channels (K+, Na+, Ca2+ etc)
Selectivity Filter—location in channel where the channel pore size becomes too small for the hydrated (shell) form of the ion to pass through. At this point shedding of the shell must be thermodynamically favored in order for it to occur. This favoring occurs when the resolvation within the channel (via polar side chains) provides a greater decrease in energy than does the desolvation, or removal of the water shell.
K+ Channel: (K+) resolvation > desolvation =Favorable, for (Na+) desolvation E>resolvation =Unfavorable
Na+ channel is single polypeptide chain, while K+ Channel consists of 4 sep. polypep chains (both w/  helices)
Process of Transport through K+ Ion channel:
Restrictive site/selectivity filter has four binding sites in a row. In the cell interior when K+ ions are high, they propagate around these sites and eventually one enters. After this binding of the second ion creates electrostatic repulsion, that pushes the first ion out towards the cell exterior. This process then repeats.
14. How would an RBC with an intact cytoskeleton and plasma membrane respond to isotonic, hypertonic, or hypotonic saline (as covered in class)? What about a RBC from a patient with "hereditary spherocytosis "?
Where is "glycophorin A" and "spectrins" in relation to the RBC plasma membrane
A RBC that is placed into an isotonic solution= NO change, Hypertonic solution = shrinkage (water would move out to attempt to mediate the increased extracellular ion concentration), Hyoptonic solution = swelling/lysis (water would move in to decrease higher molarity inside the cell)
Hereditary Spherocytosis
Mutation: genes for spectrin, ankyrin
Fx: Weakened interaction of peripheral and integral memberane PROs, change cytoskeletal
Architecture
Detection: Osmotic Fragility Test —In Hypotonic solutions normal RBC will stay intact, however if Pt has hereditary spherocytosis you will see increased fragility of RBC and they are more likely to lyse/rupture in Hypotonic solution










