7.06 Spring of PROBLEM SET #6

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1 7.6 Spring 23 1 of PROBLEM SET #6 1. You are studying a mouse model of hypercholesterolemia, a disease characterized by high levels of cholesterol in the blood. In normal cells, LDL particles in the blood are bound by the LDL receptor at the plasma membrane. The interaction of the receptor with the LDL particle is mediated by the Apo-B protein in the particles. The receptor-ldl particle complex is then endocytosed via clathrin-coated vesicle formation. LDL is transported to the lysosome, where it is broken down into useful products such as amino acids, fatty acids, and cholesterol. The LDL receptor is recycled to the plasma membrane. One of your mutant mice exhibits hypercholesterolemia symptoms, and you want to further characterize the molecular basis for the disease phenotype. Interestingly, you find that liver cells isolated from this mutant mouse have fewer LDL receptors at the plasma membrane. However, you do detect a lot of LDL receptor in the lysosomes of these cells. Intrigued, you decide to clone and sequence the LDL receptor from these mutant mice. You find that the receptor has no mutations. Now, you are really confused. But your baymate, Sumar T. Pants, comes up with a brilliant suggestion: he tells you to clone and sequence the Apo-B gene from these mutant mice. You re not quite sure where he s going with this, but he s a genius, so you do it. Sure enough, you find that the Apo-B protein has a single amino acid substitution. You check whether the formation of LDL particles is defective in these mice, and find that the particles appear to be present in the blood in above- normal numbers. a. Propose a hypothesis for the hypercholesterolemia observed in your mutant mouse taking into account all of the data you have thus far collected. (Assume that the Apo-B mutation is the only mutation in these mice). b. Provide a method to test your hypothesis. 2. Transport from ER to the Golgi apparatus involves the formation of COPIIcoated vesicles. The process is initiated by the recruitment of a small GTPbinding protein called Sar1 to the ER membrane. The vesicles are then transported to and fused with the cis-golgi membrane. This fusion process is regulated by another small GTP-binding protein called Rab1. You add the following drug to cells. What effects do you expect on COPII coated vesicle formation, trafficking, and fusion with target membrane? (NOTE: Please do not worry about the details about this question) a. A drug that inhibits the guanine exchange factor (GEF) required for the exchange of bound GDP with GTP on Sar1

2 7.6 Spring 23 2 of 6 b. A drug that inhibits the GEF required for the exchange of bound GDP with GTP on Rab1 c. A drug that greatly slows down the hydrolysis of GTP on Sar1 d. A drug that inhibits the GTPase accelerating protein (GAP) for Rab1 e. Nocodazole 3. You are studying the import of iron into cultured fibroblasts via apotransferrin (page 731 of MCB). One of your cell lines acquires a mutation that prevents efficient uptake of iron. You use immunofluorescence to stain the cells for the transferrin receptor, and see that the receptor is present both on the cell surface as well as in endocytosed vesicles. a. From this data, which two aspects of the transferrin cycle are NOT mutated in your mutant cells? b. You clone and sequence the transferrin receptor gene from your mutant cells. You find that the receptor is completely wild type. You also know that the transferrin molecules in your solution are normal. What else could account for the inability of the cells to absorb iron? 4. In 1939, Curtis and Cole showed that the action potential is caused by changes in membrane conductance and that its peak exceeds mv. This disproved an earlier hypothesis that, during the action potential, the membrane would become non-selectively permeant to all ions. a. Why does the result obtained by Curtis and Cole disproved the hypothesis that the membrane becomes non-selectively permeable to all ions during the action potential? A few years later, Hodgkin and Katz were studying the squid giant axon and noted that the intracellular Na + concentration was much lower than the external Na + concentration (sea water). They suggested that the positive membrane potential achieved during the action potential was due to a selective increase in Na + permeability and to test this hypothesis, they looked at the effect of decreasing the external [Na + ] on the action potential.

3 7.6 Spring 23 3 of 6 mv time 1% Sea water 5% Sea water 33% Sea water b. What is the effect of lowering the external Na + concentration on the action potential? Refer to amplitude and kinetics of the response. c. Using your knowledge of the Nernst Equation, estimate the maximum peak of the action potential if the experiment was done in Ringer s solution (14 mm Na + ; 3 mm K + ; 5 mm Cl - ). Assume the intracellular concentration of these ions are the following: 1 mm Na + ; 13 mm K + ; 125 mm Cl -. The Na + permeability explains the depolarization of the action potential but what accounts for the repolarization? For the membrane potential to return to its resting state, two things must happen: 1 open Na + channels must close, and 2 there must be a net outflow of positive charges to explain the repolarization. Hodgkin, Huxley and Katz proposed that K + efflux was responsible for the latter. To test the contribution of different conductances on the action potential, Hodkin, Huxley and Katz kept the voltage constant at different values and measured the currents generated. In this way, they showed that a voltage change of 65 mv to mv evoke an initial transient inward current followed by a delayed outward current. (By convention, in an inward current, net positive charges enter the cell, and in an outward current, net positive charges exit the cell.) (ma/cm 2 ) 1 mv Time (ms)

4 7.6 Spring 23 4 of 6 d. Indicate what ions are responsible for the initial inward current and the delayed outward current and design an experiment to demonstrate this. In the diagram above, plot your expected results. 5. The sea slug Aplysia punctata is a good model to study modulatory synapses. A facilitator neuron forms a synapse with the axon terminal of a sensory neuron that stimulates a motor neuron by releasing glutamate. Stimulation of the facilitator neuron leads to serotonin secretion which binds to serotonin receptors on the sensory neuron. This binding activates adenylate cyclase, triggering synthesis of camp in the sensory neuron. The subsequent activation of camp-dependent protein kinase leads to an inhibiting phosphorylation of the voltage-gated K + channel. This inhibition decreases the outward flow of K + ions that normally repolarizes the membrane of the sensory neuron after an action potential. See figure of MCB4ed (p.95). To demonstrate that serotonin leads to an inactivation of the voltage-gated K + channel, patch-clamp is performed on a sensory neuron of Aplysia in the presence and absence of serotonin. a. What would the readings look like if we measure single-channel currents by voltage-clamp in the absence and presence of serotonin. mv 1 pa 1 pa - serotonin + serotonin b. What would the readings look like if we measure whole-cell currents by voltage-clamp in the absence and presence of serotonin. (ma/cm 2 ) 1 - serotonin + serotonin mv Time (ms)

5 7.6 Spring 23 5 of 6 c. What would a sensory neuron action potential look like in the absence and presence of serotonin. mv time serotonin + serotonin The resulting prolonged membrane depolarization increases the Ca 2+ influx. This leads to a greater exocytosis of glutamate-containing synaptic vesicles in the sensory neuron, and hence greater activation of the motor neuron. d. How could you demonstrate that the serotonin addition to the sensory neuron ultimately leads to a greater activation of the motor neuron? Indicate the expected results of your experiment. 6. A Drosophila temperature-sensitive mutant, called paralytic (para), cause the fly to reversibly paralyze at temperatures > 33 C. Both paralysis and recovery of para mutant flies are extremely fast (in the order of seconds). At 37 C, no action potentials are elicited in the motor neurons innervating the thoracic flight muscles of para flies when stimulated with an electrode. You clone the gene encoded by paralytic and find it to be homologous to the a subunit of the voltage-gated Na + channel found in other species. This leads to you propose that at the restrictive temperature, the Na + channel is nonfunctional and that this is the cause for the absence of action potentials at 37 C. a. With this model in mind, what would the whole-cell currents look like in a voltage-clamp experiment at 2 C and 37 C in which you apply a depolarizing step of +65 mv?

6 7.6 Spring 23 6 of 6 (ma/cm 2 ) 1 2 C 37 C mv Time (ms) b. How can you prove that the Na + current is responsible for the differences in the currents above? Hint: can you use a specific inhibitor for the voltagegated Na + channel?