BL A512 -- Signal Transduction, Fall, 2002, Prof. Stark
Midterm exam -- Tuesday March 5, 2002, 100 points

1. In 1971, it was proposed that the separation of disks from the plasmalemma in the rod outer segment necessitated the intervention of an intracellular signal. At first, calcium ion was proposed, but later, another molecule was identified. What is this other molecule? Discuss the enzymes that make vs break down this mediator of visual transduction in the vertebrate rod. Also, discuss how these enzymes are regulated. (5 points)

cGMP, broken down by PDE when transducin alpha pulls two gammas off of alpha-beta, made by GC activated by GCAP (GC activating protein), and binding of Ca2+ inhibits this activation

2. Discuss the Shaker channel with respect to (a) how its speed and Hodgkin-Huxley parameters differ from those of the late K+ channel of the squid axon action potential, (b) how the molecular size and channel structure differ from the Na+ channel of the squid axon action potential, (c) where its activation is in the molecular structure, and (d) where its inactivation is located in the molecular structure. (4 points)

It is fast and it inactivates instead of being delayed with no inactivation. It is about 1/4 the size and is a tetramer instead of a pseudotetramer. Activation on S4 with a line of charged amino acids (every 3 or 4 amino acids) and this rotates and moveds. Inactivation is N-terminal stopper

3. (a) What does rhodopsin kinase do to (b) what residues at (c) what location in the rhodopsin molecule? That allows (d) what molecule to bind to rhodopsin to inactivate it? After all of these actions are reversed, there is still one thing that has to be done to regenerate the native rhodopsin so that it can be excited by light. (e) What is this? (5 points)

Phosphorylate, serines and threonines, near C-terminus, arrestin. Since rhodopsin eventually bleaches, 11-cis retinal must be reconnected (Schiff base to epsilon amine of lysine on 7th transmembrane span

4. Provide information centered around cyclooxygenase. (a) Give an example of an inhibitor. (b) What is cyclooxygenase's precursor? (c) What5 are the precursor's properties (d) What enzyme makes this precursor? (e) What is cyclooxygenase's product? (5 points)

The NSAIDs (non-steroidal anti-inflammatory drugs, aspirin, ibuprofen and now the prescription specific COX-2 inhibitors,viox and celebrex. arachidonic acie (20:4) (made by PLA-2) gets converted to prostaglandins

5. In addition to serving as black paint in the eyeball, what two supportive functions do retinal pigment epithelial cells provide to the rod outer segments? What happens to these cells after decades of providing this support? (3 points)

They phagocytose the tips of outer segments, They help in the vitamin A metabolism. They get filled with age pigment (lipofuscin) that fluoresces yellow to UV or blue excitation.

6. Adaptation is important in sensory systems. (a) Discuss the usefulness of fast adaptation in the phasic Pacinian corpuscle. (b) What ion regulates adaptation mechanisms in the vertebrate rod and (c) How does it get near the molecules it regulates (e.g. recoverin)? (d) What mediates adaptation in the bacterial aspartate receptor and (e) Why would this be important in this kind of chemotaxis? (5 points)

Phasic mechanoreceptors mediate a vibration sense that gives you a feel of texture when you run your fingertips over a surface. Ca2+ mediates adaptation. It comes in through the same nonspecific cation channels that the Na comes in through. Tar is methylated at 4 sites. They need to be comparing concentrations from one moment in time to the next to maintain a run before the next tumble

7. In 2000, Arvid Carlsson shared the Nobel Prize in Physiology and Medicine for his "discoveries concerning signal transduction in the nervous system." Specifically, he was involved in the discovery that dopamine was not just norepinephrine's precursor but also a transmitter. Tell the story of the famous disorder of dopamine deficiency. (a) The disease. (b) Where is dopamine in short supply? (c) What is given to help patients? (d) The precursor of what is given (which is also where the biosynthetic pathway begins) (e) A famous person who has the disease. (5 points)

Parkinson's disease is caused by low dopamine in the pathway from the substantia nigra to the striatum in the brain, an area that controls motor movements. You lave to give them the precursor (l-DOPA). Biosynthesis starts with the amino acid tyrosine. Mohammed Ali, Pope, Michael J. Fox

8. What is it that cAMP activates in its traditional signal transduction cascade? Describe the molecular arrangement for this activation. (3 points)

activates protein kinase A (A-kinase). Two cAMPs each bind to each of two regulatory (inhibitory) subunits releasing the two catalytic subunits from the tetramer

9. Describe the special situation thought ro apply to maintaining sufficient intracellular calcium ion concentrations if the small amount in the intracellular reticulum becomes depleted. (2 points)

For depletion in these stores, closely linked with a channel in the plasmalemma called trp (transient receptor potential), Ca2+ comes in through the trp channel

10. Discuss your heart beat from the standpoint of (a) the molecular geometry of the channel that carries the electrical signal from cell to cell, (b) the properties of the ventricular muscle action potential, (c) how that is seen in the electrocardiogram (EKG), and (d) what is at fault in well-known a genetic disorder where the repolarization of those cells is delayed. (4 points)

In the gap junction on the intercalated disk, 6 connexins on one cell are in register with the next cell's hexamer. There is a long action potential with depolarization at the QRS waves and repolarization at the T wave. Long QT syndrome is HERG (human ether-a-go-go related gene which is inwardly rectifying K+ channel regulating plateau of long action potential

11. Use what you know about intracellular vs extracellular ion concentrations and channel properties to explain why activating the GABA (gamma amino butyric acid) channel would lead to an IPSP (inhibitory postsynaptic potential). (2 points)

Cl- has gradient like Na+, but it is aa anion, not a cation, hence it would either hyperpolarize the cell or tend to hold it from depolarizing

12. Bacterial toxins have been useful in understanding. Tell signal transduction stories about toxins of (a) Vibrio cholerae (cholera toxin), (b) Clostridium botulinum (botulism), (c) Bordetella pertussis (whooping cough), and (d) Clostridium tetani (tetanus toxin). (4 points)

cholera ADP ribosylates G alpha stimulatory , won"t inactivate, cAMP goes up
pertussis affects G alpha inhibitory and t cannot be stimulated
botulism inhibits vesicle release, tetaus does the opposite

13. For the resting potential and the action potential, the membrane is sometimes represented as two batteries for the two most relevant univalent cations respectively. Discuss how the relative contributions of these two batteries is regulated by variable conductances to these two respective ions during the passage of the action potential. (4 points)

Na+ equilibrium potential approximates peak of action potential, where Na+ conductance becomes relatively high
At rest, the relatively high K+ conductance makes the membrane potential approximate the K+equilibrium potential

14. The striated neuromuscular junction and the neuroeffector junction of the sympathetic nervous system are models for the two respective mechanisms of neurotransmitter action. Describe the transmitter and its receptor in each of these examples. (4 points)

nmj - acetylcholine, nicotinic channel, 2 alpha subunits, one beta, one gamma, one delta
sympathetic, adrenaline, G-protein-coupled receptor

15. A paper is presented later in this course entitled "Mutation of the Stargardt's disease gene (ABCR) in age-related macular degeneration" where ABCR stands for "ATP-binding casette transporter-retina." Discuss how such a molecule might be related with a famous chloride channel you learned about. (2 points)

This is the CFTR = cystic fibrosis transmembrane (Cl-) conductance regulator

16. Experiments addressed toward identifying the way calcium ions regulated a potassium channel in Paramecium identified one of the most famous calcium binding proteins coded by a gene originally named after pantophobiac (pnt) mutants. Name and describe this calcium binding protein. (2 points)

calmodulin is about 140 amino acids and is z-shaped with two Ca2+ binding sites on each side of the molecule

17. What kind of a molecule is insulin and what happened to it after translation to make it take on that particular configuration? Like the receptor for EGF (epidermal growth factor), what type of enzymatic activity does insulin's receptor show? In 1987 Daniel Koshland likened this receptor to the aspartate receptor for bacterial chemotaxis. Why? (In what respect?) (4 points)

The proinsulin protein got chopped down to two peptide fragments linked to eachother by disulfice bridges
It is a receptor tyrosine kinase. It passes the membrane one or just 2 times

18. In addition to IP3 (inositol trisphosphate), PLC makes another "second messenger." What is this other signalling molecule and what does it activate? (2 points)

DAG stimulates PKC

19. Discuss the heterotrimeric G protein from the standpoint of (a) where it is in the cell, (b) what puts (and keeps) it there, (c) what its G protein-linked receptor interacts with, and (d) what function (also missing in small GTP binding proteins like ras) that receptor fulfills. (4 points)

on the inside of the membrane held there by fatty acids on the alpha and gamma subunits
receptor interacts with N and C terminals of alpha subunit
GEF = guanine nucleotide exchange

20. Carotenoids get chopped in half to make vitamin A which becomes the chromophore of the visual pigment rhodopsin. Without getting chopped in half, where do carotenoids (specifically lutein and zeaxanthin) reside in the eye? What useful function might they serve there leading drug companies to add lutein to multi-vitamin pills? (2 points)

they are the macular path in cells in the light path before light hits receptors
they block blue light, thought to be damaging, from the cones

21. OK, I'll start by reminding you of some alphabet soup: NSF = N-maleimide sensitive factor and SNAP = soluable NSF sensitive factor. There are two SNAP receptors (SNAREs). Where are they and what do they do? (3 points)

v-SNARE on vesicles and t-SNARE on the target membrane which happens to be the presynaptic plasmalemma

22. OK, I'll start with a similar offering in this question. CREB = cAMP response element binding protein. What is a response element? And what molecule is it on (i.e. where does CREB bind?) (2 points)

a response element is a short stretch of DNA upstream of the coding sequence

23. Discuss the functional geometry for manufacture of NO through excitation of an NMDA receptor. Specifically address (a) What is the natural ligand for the NMDA receptor? (b) What is the signal pathway from receptor to enzyme, (c) What keeps the necessary molecules near eachother? and (d) What molecule does NO regulate? (4 points)

the N-methyl d-aspartate channel uses glutamate, Ca2+ comes in, binds calmodulin, interacts with NOS. PDZ domain proteins hold things together, NO regulates GC

24. Why is coffee a mild stimulant? (2 points)

the action of adrenalin is potentiated since the cAMP phosphodiesterase is inhibited

26. Compare the calcium channels for (a) synaptic vesicle release and (b) the phosphoinositide signalling in terms of what activates the channel. Also, discuss the cellular localization and properties of two types of calcium channels involved in excitation-contraction coupling in striated muscle. (4 points)

for vesicle release, the channels are voltage gated while for the IP3 receptor has IP3 as a lighand
t-tubules Ca2+ channels get the action potential in to where it is needed, near the sarcoplasmic reticulum
in the sarcoplasmic reticulum there are ryanodyne receptors closely linked to the Ca2+ channels on t-tubules

27. What are the diverse functions and relative numbers of G protein-coupled receptors in the human body. Speculate on how this overall situation arose over an evolutionary time scale using, as an example, the evolution of color vision genes on the X chromosome in Old World Monkeys, great apes and humans. (4 points)

there are lots (1000) for hormones transmitters, vision, olfaction
genes get duplicated and diverge in function

28. In 1970, Sir Bernard Katz won the Nobel Prize in Physiology and Medicine for work in which he defined the quantum for synaptic transmission. What was this quantum of transmitter? How did lowering the concentration of extracellular calcium ions assist him in making this determination? (2 points)

a vesicle, and it was possible to see effects of individual or just a few quanta by decreasing Ca2+ since calcium is needed for transmitter release

29. What is the pheromone and the receptor for this pheromone for cell aggregation in the cellular slime mold Dictyostelium discoideum? (2 points)

cAMP stimulates a G protein-coupled receptor

30. When the glycoside ouabain effects a rapid block of the the Na+-K+ ATPase (sodium-potassium pump), the resting potential only changes a few mV (milli Volts). This is why the pump is called "electrogenic." Why is there a change of a few mV and how come the resting potential does not collapse completely and immediately? (2 points)

a few mV comes because there are 3Na+'s pumped per 2 K+'s.
The pump creates the ion gradients and these are robust,; voltages come from gradients ":seen" through channels

31. Structural work on the alpha subunit of the heterotrimeric G protein demonstrated switch I and switch II regions. Recently, it was shown that something made the positions of these switch regions to become more invariant. What was this something? (1 point)

GTP (when the gamma phosphare is chopped off, the switches are more variable)

32. What does the G-protein need to do to the phosphodiesterase in the rod to activate it? (In other words, how are the components of PDE altered?) (2 points)

pull off two inhibitory gamma subunits so the alpha beta enzyme is active

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