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Unformatted text preview: Homework 3 1. In which of the following ways do potassium channels in the squid giant axon differ from sodium channels? a. The potassium channels pass only a few ions per second. b. The potassium channels show little voltage dependence. c. The summing of the individual potassium channels does not reconstruct the macroscopic current. d. Once the potassium channels open in response to a voltage step command, they tend to remain open. e. All of the above 2. Which of the following is a major advantage to researchers of the Xenopus oocyte expression system? a. Xenopus is the only lower vertebrate whose genome has been sequenced. b. The unusually small size of the eggs makes patch-clamping relatively easy. c. The oocytes have many endogenous ion channels to which exogenous channels can be compared. d. The oocytes have quite thin membranes, which amplifies the ionic currents. e. It facilitates physiological characterization of modified ion channel genes. 3. Gap junctions may exhibit all of the following features except for the ability to a. pass small metabolites, including some second messengers. b. pass electrical current bidirectionally. c. pass electrical current unidirectionally. d. amplify small incoming electrical signals into large regenerative potentials. e. synchronize the activity of populations of nerve cells. 4. The capability of a nerve terminal to rapidly and dramatically produce very large changes in calcium levels is most dependent on the a. presence of calcium-selective ion channels. b. enormous gradient of calcium across the membrane. c. fact that calcium is a positively charged ion. d. fact that calcium is a divalent cation. e. All of the above are essential for producing large, rapid concentration changes. 5. Which of the following is an accepted criterion for defining a molecule to be a neurotransmitter? a. It must be present in the presynaptic terminal. b. It must be released in response to presynaptic electrical activity. c. It must exert an effect on the postsynaptic cell. d. All of the above e. None of the above 6. Miniature end-plate potentials, or MEPPs, are produced a. at miniature end-plates. b. by the smallest axons. c. in response to weak stimuli. d. by the smallest neurotransmitters. e. by spontaneous release of neurotransmitter. 7. Which of the following experiments would indicate a role for calcium in transmitter secretion? a. Observation of presynaptic depolarizing currents after blockade of sodium channels b. Voltage clamp experiments showing voltage-gated calcium channels in the presynaptic terminal c. Induction of transmitter release by injection of calcium into the presynaptic terminal d. Blockade of transmitter release by injection of calcium buffer into the presynaptic terminal e. All of the above 8. The two main families of neurotransmitter receptors are a. ligand-gated; ion-gated b. ionotropic; metabotropic c. voltage-gated; voltage-modulated d. cationic; anionic e. excitatory; inhibitory and 9. Which of the following statements about postsynaptic currents at the neuromuscular end plate is false? a. Depolarizing currents can be recorded from outside–out patches of postsynaptic membrane. b. Individual channels tend to stay open for no more than a few msec. c. Acetylcholine can induce openings of ligand-gated ion channels. d. The end plate potential is due to the opening of thousands or millions of channels. e. The end plate channels show a regenerative opening pattern that propagates an action potential along the length of the muscle fiber. 10. The most important factor determining whether a receptor-operated ion channel is inhibitory or excitatory is a. the ligand-binding properties of the receptor. b. whether the permeant ion is positively or negatively charged. c. whether the permeant ion’s reversal potential is positive or negative. d. whether the permeant ion’s reversal potential is positive or negative to threshold. e. None of the above . Short Answers 1. Describe briefly how each of the following can be used to learn about ion channels: X-­‐-­‐-­‐ray crystallography Provides the molecular structure and shows important features such as ligand binding sites, voltage sensors, pores, selectivity filters. Expression of mRNA in Xenopus oocyte This is will allow high levels of expression of the normal and/or mutated ion channel which can then be studied by other techniques such as patch clamping Patch clamping Able to detect the currents flowing through individual or many ion channels (depending on set-­‐up) while influencing the extracellular or intracellular environments and learn about the contribution of the ion channel to the maintenance or change in membrane potential. Mutagen esis Allows for the introduction of specific DNA changes in order to investigate important amino acids making up the ion channel. For example, mutations in ligand binding sites can show which amino acids are necessary for binding. Toxins Different toxins bind and affect ion channels in specific ways. These can be used to block or modulate channel opening, ligand binding, voltage sensing, etc. This gives important insight into structure and function of the ion channel. 11. What is patch clamping? Explain how it can be used to show that properties of voltage-­‐-­‐-­‐sensitive Na+ and K+ channels are responsible for the action potential. Patch clamping is a technique that uses a glass pipette with a small diameter that makes a tight seal with the membrane of a cell and measures currents across individual or many ion channels. As in voltage clamp, patch clamp can also control for membrane potential. Patch clamp can be used to show that voltage gated Na+ channels have quick inward currents when clamped at a depolarizing voltage step. These currents quickly go away suggesting inactivation of the channel even though the membrane potential is still depolarized. Voltage gated K+ channels show outward currents upon clamping at a depolarizing voltage step. The outward current persists until the voltage is returned to the resting membrane potential. The macroscopic currents (summed Na+ and K+ currents) appear similar to those obtained by voltage clamp experiments. 12. List the steps involved in chemical neurotransmission, from the synthesis of neurotransmitter through responses in the postsynaptic neuron. 1) 2) 3) 4) 5) 6) 7) 8) 9) Neurotransmitter is synthesized and packaged into vesicles An action potential invades the presynaptic terminal Depolarization causes opening of voltage gated Ca2+ channels There is an rapid influx of Ca2+ from outside to inside Ca2+ causes vesicles to fuse with membrane Neurotransmitter is released into cleft Transmitter binds to receptors on postsynaptic cell Post-­‐synaptic receptors cause channels to open or close Post-­‐synaptic current flowing inside post-­‐synaptic cell 10) Retrieval of membrane via endocytosis 13. You work in a human genomics biotech company whose goal is to identify and understand functions of proteins that when mutated cause human epilepsy. Your company has mapped an epilepsy gene to a putative ion channel (called channel x). Your boss gives you two genes to characterize. One is the wild type gene and one is the gene containing a mutation that leads to epilepsy in all patients that have this gene. She says that this gene is predicted to be an ion channels based on its protein sequence and predicted structures. Your job is to determine what kind of ion channel it is, and how the mutation affects channel function and hopefully to understand why patients who have this gene get epilepsy. 13a) Name two structural features common to all ion channels. 2 pts Multiple membrane spanning domains Center has a pore that allows ions through The first thing your boss tells you to do is to inject mRNA encoding channel X from normal individuals and the mRNA encoding mutated channel X into Xenopus oocytes and use voltage clamping in order to determine each proteins gating properties. ( Note: the ion concentrations across the Xenopus oocyte is similar to that of a squid giant axon.) 13b) Briefly explain what a Xenopus oocyte is and why they are used to study channels. Xenopus oocytes are large (1mm in diameter) cells that contain lots of protein synthesis machinery. One can inject RNA into them and they will express protein encoded by RNA. This system works great for ion channels, as one can voltage clamp and determine properties of a given channel. Below are conductance vs. time recording profiles at two different cell potentials from oocytes expressing either the wild type channel or the mutant version of channel X. Based on these graphs answer the following: 13c) Is this experiment using extracellular or intracellular recording? What is the approximate resting potential of the oocyte Intracellular – it’s voltage clamp, ~ -­‐50 to-­‐60mV 13d) Which of the following conclusions can be made from the voltage clamping experiment? Circle one answer. A) Only wild type channel X is voltage gated. B) Only the mutant channel X is voltage gated. C) Both the wild type gene and the mutant channel X are voltage gated. D) Neither the wild type nor the mutant channel X is voltage gated. E) None of the above is true. You next perform a patch clamp experiment to learn more about the properties of the ion channels and you get the recordings below. Note: for each channel there are 2 trials. The first pair are wild type channel X tracings and the second pair are mutant channel X tracings. Based on the above recordings answer the following. 13e) Why does each experiment give a slightly different microscopic current vs. time profile? 2 pts Channel opening is a probability. The probability of opening increases with depolarization. This also means that there is a chance (although small) of opening independent of the depolarization step or stimulation. This results in different microscopic currents. 13f) Are the microscopic currents inward or outward? 2 pt Inward. 13g) While patch clamping you find the channel X is sensitive to tetrodotoxin. conclude: Therefore you A) Calcium regulates the gating of channel X. B) Channel X is a sodium channel. C) Tetraethylamine (TEA) would also block the currents. D) Cs+ would also block the currents. E) None of the above conclusions could be made. 13h) Based on all of the above information, what kind of channel is channel X and how does the mutation affect channel X function? Where do you expect the mutation is located? Be as specific and concise as possible. Channel X is a voltage-­‐sensitive Na+ channel. The mutation affect channel X inactivation. The mutation is located in the inactivation particle or the part of protein that the inactivation particle binds to. 13i) How could having a mutation in channel X lead to epilepsy? Discuss with precision how the action potential profile might change in individuals harboring this mutation. Feel free to use diagrams to make your point. The mutant channel has poor inactivation. Na channels would stay open longer and would prolong the action potential. This would also result in a lack of undershoot phase leading to a smaller refractory period. This may cause action potentials to go backwards. In addition the cell potential will be closer to threshold resulting in what normally would be small changes in potential that shouldn’t trigger an action potential would now trigger them. This could cause inappropriate firing of action potentials and could lead to uncontrolled firing, spastic movements associated with epilepsy. ...
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