Cellular Neurobiology / BIPN 140. FIRST MIDTERM EXAMINATION Fall, Please answer each question IN THE SPACE ALLOTTED (may be on next page).

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1 FIRST MIDTERM EXAMINATION Fall, 2016 GENERAL INSTRUCTIONS 1. Please write your name on ALL 6 pages. 2. Please answer each question IN THE SPACE ALLOTTED (may be on next page). 3. For full credit, state your assumptions and show your calculations. 1) _Antonia /10 pts 2) _Stephanie /20 pts 3) _Antonia /20 pts 4) _Milad /10 pts 5) _Susie /20 pts 6) _Peixi /20 pts TOTAL /100 pts 7)_Peixi /10 pts EXTRA CREDIT WAIVER: By signing this waiver I give permission for this exam to be left for me to pick up in the vestibule by the elevator on the 3rd floor of Pacific Hall. I understand that I may only pick up my own exam. I realize that the Division of Biological Sciences and its staff cannot take responsibility for exams, which may be stolen or lost, from this area. If I choose not to sign this waiver, I acknowledge that my exam will only be available for pickup 10:00 am-4:00 pm, Monday-Friday from the Exam Depot Window outside the north entrance to Pacific Hall. 1

2 1. You are studying a neuron with an arterial cuff around its axon, using your new microscope. However, you knocked over a bunch of chemicals onto the plate yesterday because of your coffee jitters (you ve been caffeinating all night to keep up with BIPN 140 readings). Anxiously, you look at the neuron again and see that a swelling has formed on one side of the cuff, as shown in the figure below: a) 2.5 pts Would you normally expect this swelling forming on only one side of the arterial cuff? Why? No, there should be swelling due to build-up forming on both sides of the cuff in a normal neuron. The formation of the build-up on both sides is the result of damming of orthograde and retrograde axoplasmic transport. b) 2.5 pts What type of axoplasmic transport can still be observed? Orthograde transport can still be observed, since there is build-up forming on the side of the cell body. Orthograde transport moves materials away from the cell body (towards the synapse). c) 2.5 pts At what rate can functioning axoplasmic transport propagate? Orthograde transport can propagate at either a fast or a slow rate. The slow rate moves materials at 1 mm/day. The fast rate moves materials at 400 mm/day. d) 2.5 pts Which motor protein has most likely been damaged by the chemicals? Dynein has most likely been damaged, since retrograde transport cannot be observed. There is no build-up of materials on the side of the synapse. 2. During a strange dive off the coast of La Jolla, you come across an underwater mermaid hippie commune. They are in the process of creating a power plant made completely of organic materials, using electric eels as batteries and GIANT squid giant axons as wires. Attached at one point through an electrode, the eel hyperpolarizes the membrane potentisl of the squid axon to 100 mv below its resting potential. a) 7 pts With a specific internal resistance of 50 Ω*cm and a specific membrane resistance of 100 Ω*cm 2, what is the length constant of this axon (in cm) if it has a 9 cm radius? λ=(r m /r i ) ^1/2, 2 pts and r m =Rm/(2*pi*a) 2 pts and r i =Ri/(pi*a^2). 2 pts So r m /r i = Rm*a/ (2*Ri). Plugging in the given values, λ=(100 Ω*cm 2 *9 cm/(2*50 Ω*cm))^1/2= 3 cm. 1 pt 2

3 b) 7 pts An energy efficient mermaid house requires at least a 5 mv difference from resting potential in order to operate. You decide to investigate the practicality of their power plant design. What is the maximum distance from an electric eel your mermaid friends could build their houses? Using the cable equation, Vx=Voe -x/ λ, 3 pts so -5 mv= -100 mv e -x/ 3cm, 3 pts solving for x. x= - ln(-5/-100)*3= 9 cm. 1 pt (This is not really a practical distance!) c) 6 pts There is considerable funding for any scientist able to increase the distance between house and eel while still providing enough voltage to power the house. You apply for funding to add myelin to the squid axon. Your grant states that myelin will increase the membrane and decrease the membrane of the axon. (Fill in the blanks). What combined effect do these properties have on voltage decay over length of axon travelled? Increase the membrane RESISTANCE 2 pts and decrease the membrane CAPACITANCE 2 pts. Combined, both of these properties slow voltage decay over length of axon traveled. 2 pts 3. You find a jar left in Geisel containing the most beautiful squid neuron that you have ever laid eyes on. Enchanted, you decide to keep it, and name it Spike There is a suspicious label on the jar that says that the neuron has to be stored at 20 o C,, otherwise half of its channels stop working. You carefully follow the instructions and decide to investigate some of the neuron s properties. First you measure the intracellular and extracellular concentrations of the following ions: Extracellular concentration (mm) Intracellular concentration (mm) Cl K Na Then you randomly voltage patch clamp a single ion channel and measure the currents at different potentials: 3

4 a) 3 pts What is the reversal potential of the unknown ion channel that you ve voltage clamped, and why? The reversal potential occurs at -70 mv, where the current flow is 0 pa. b) 4 pts Which ion channel are you most likely studying? How can you be sure? Show your work. Eion Reversal potential.!"!!"!" ECl- = 58log = 58 log =58log( )!"!!"#!"# = 58 x ( ) = mv ECl- is approximately -70 mv, which matches the reversal potential on the graph. Therefore, the channel is most likely a Cl- channel. c) 2 pts Is the ion flow for the unknown ion channel inward or outward at -30 mv? There is an inward ion flow. Negative ions (Cl-) flow into the cell, making the rest of the environment more positive. d) 3 pts What is the conductance (γ) for the unknown ion channel? i = γ(vm Eion) i γ = Vm Eion = 20 pa mv = 20 = 0.5 ns 40 e) 3 pts If the unknown ion channel is closed 400 times for 100 ms in 1 minute, what is the Popen? total time the channel is open Popen = total time =!"!(!""!.!) =!" =0.33!"!" f) 3 pts What is the number of conducting unknown ion channels at 36 o C, given that the total ion current (I) is 300 times greater than the single channel ion current (i)? I = Nfunctioning i Popen Nfunctioning = I i Popen = 300i i Popen = 300 Popen = g) 2 pts What is total number of the unknown ion channels? Ntotal = 2 Nfunctioning = = List the main classes of glial cells and their primary functions. 2 pts Astrocytes - blood-brain barrier formation; regulate ion flux 2 pts Oligodendrocytes - myelination in the CNS; one oligodendrocyte myelinates more than one axon 2 pts Schwann cells - myelination in the PNS; one Schwann cell forms one internode on a PNS axon 2 pts Ependymal cells - produce cerebrospinal fluid 4

5 2 pts Microglia - act like macrophages in CNS; scavenge and remove debris 5. Motivated by your neurobiology class, you hike down to Black s Beach and catch a fastmoving squid. You name it Squid Awesome and obtain the giant axon. a) You attach a battery and a switch to the axon. You close the switch, and an action potential is generated at the cathode. Why? 3 pts Action potentials are normally generated at the cathode. This is due to the negative charge at the cathode, depolarizing the membrane potential of the axon. b) You recall anode break excitation discussed in lecture, and would like to see an action potential generated at the anode. (i) Using this experimental setup, what would you do? (ii) Why would action potentials be generated at the anode? 3 pts (i) Close the switch for a sufficiently long time (for at least 0.1 s), and then open the switch. 4 pts (ii) At resting potential, ~10% of voltage-gated sodium channels are inactivated. When the switch is closed, positive charge is supplied at the anode, hyperpolarizing the axon. This removes the 10% inactivation of voltage-gated sodium channels. (2 pts) When the switch is opened and V m returns to the resting potential, sufficient G Na is now available to generate I Na that equals the I K through resting leak channels: the threshold has been lowered to the resting potential.. (2 pts) c) Using stimulating and recording electrodes, you discover a potassium current in a squid touch-sensitive neuron that has a conductance that increases as you hyperpolarize the membrane. (i) Is this current going into or out of the neuron? (ii) Which potassium current out of the four discussed in class would most likely be responsible for it (please write the complete name)? (iii) Draw an I-V plot for this current and label E K. 4 pts (i) Above EK this is an outward current (going out of the neuron) and below EK this is an inward current (going into the neuron). 3 pts (ii) It is most likely inward rectifier K+ current (I KIR ). 3 pts (iii) 6. a) After examining the Kyte-Doolittle plot, which has provided information regarding the structure of an unknown protein that you are investigating, you hypothesize that this protein may be a transmembrane channel protein. You would like to test your prediction, using the Xenopus oocyte as an expression system. With a cdna of this protein available, what experimental steps would you go through? 4 pts 1. Transcribe the cdna to obtain the corresponding mrna 5

6 2. Inject the mrna into the oocyte, which would express copies of the protein and insert them into the membrane b) You then proceed to record currents through the Xenopus oocyte at clamped voltages, and acquire the following results. What ions are likely to flow through these channels, and why? 4 pts At relatively depolarizing V m, the ion currents are flowing inward. Assuming standard extracellular and intracellular ion concentrations, it is likely that sodium ions flow through these channels, which are likely to be voltage-gated sodium channels. c) When you use site-directed mutagenesis to introduce mutations in the DNA sequence that replace arginine with alanine in the S4 segment of this channel, what changes in current will you record, and why? 4 pts The inward current will be smaller at each clamp potential. Since the charged arginine is replaced with the uncharged alanine, fewer charged amino acids in the S4 segment will mean that stronger depolarizations are needed induce the same conformational changes in these channels. d) Lastly, you would like to observe what happens if these channels don t undergo inactivation. What are two experimental modifications you could use to prevent channel inactivation and how would they achieve this result? 4 pts for each correct answer (2x4 pts) 1. Apply trypsin or papain intracellularly; these proteolytic enzymes will digest the intracellular loops of the channel protein. 2. Apply antibodies to the intracellular loops intracellularly, starting with antibodies to the III-IV domain intracellular loop. 3. Introduce a mutation in the IFM tripeptide in the III-IV domain intracellular loop. Extra Credit: 7. Recall the paper Protective Role of ATP-Sensitive Potassium Channels in Hypoxia-Induced Generalized Seizure by Yamada et al. a) 3 pts Which potassium channel subtype describes the K ATP channel? Inward rectifying potassium channel b) 4 pts Are these channels more likely to be open in the presence of high ATP or low ATP? How does this alter seizure activity during hypoxia? 6

7 They have a higher probability of opening in the presence of low ATP. Low ATP opens more K ATP channels which hyperpolarize the neuron, balancing other factors during hypoxia that promote depolarization and increased firing rates. In this way, the K ATP channel is protective against seizure activity. c) 3 pts Based upon data from K ATP channel knock-out mice, would you consider using an agonist or antagonist of the K ATP channel to restore normal firing rates in patients suffering from ATP insufficiency due to stroke? Briefly explain your answer. An agonist would be a better treatment. In mice lacking the K ATP channel, seizure activity is increased during ATP insufficiency as compared to wild-type mice. This is analogous to the use of an antagonist, and would exacerbate abnormal firing rates. On the other hand, an agonist could increase the activity of the K ATP channel, driving firing rates down towards normal levels. -The End- 7