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6 Cards in this Set
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The voltage clamp method was considered a breakthrough in science when Kenneth
Cole invented it in the 1940s. This method helped Hodgkin and Huxley in their experiments trying to understand the basis of changes in conductance during an action potential. Explain how the voltage clamp method works and what it measures. |
The voltage clamp is a method by which the membrane voltage can be imposed at a specific voltage (the command voltage). A feedback amplifier compares the membrane voltage to the command voltage and injects current to make the membrane voltage equal to the command voltage. The injected current is
measured and is equal in magnitude but opposite in sign to the current produced by the ion channels. |
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One of the challenges in Hodgkin and Huxley 's experiments was to separate INa and IK. from the total INa+ IK. What is choline and what role did it play in their original experiments? How did they
measure INa and IK? |
Choline is a water-soluble B complex vitamin that has many physiological purposes in the body such as structural and signaling roles in the cell membrane as well as being a precursor to acetylcholine that is a neurotransmitter. The
reason H-H used choline in this experiment was to replace the extracellular Na+ since they both have a charge of positive 1 and are water soluble. This allowed them to first record INa in normal saline, and then introduce a saline in which enough Na+ was replaced by choline to set ENa=Vc so that the driving force for 2Na+=0 and they could record IK in isolation. They then subtracted IK from Itotal to find INa. Itot-IK=INa. |
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Hodgkin’s and Huxley’s results have been confirmed by pharmacological studies with
drugs that specifically affect INa and IK. What drugs were used in these experiments and how did they measure the two currents? |
They used drugs to selectively block either the Na+ channels or the K+ channels. Tetrodotoxin (TTX) blocks Na+ current and allows IK to be isolated. Tetraethylammonium ions (TEA) block K+ current and allow INa to be isolated.
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Four classes of ion channels were discussed in lecture. Name these 4 classes and
give a short description of their roles in the nervous system. |
A. Leak Channels K+ selective which make the resting potential negative. Na+ selective which make the resting potential positive.
These channels are not affected by voltage, ligands, or stimulation. B. Voltage-dependent channels Important for action potentials C. Ligand-dependent channels Important for synaptic transmission where ligands (neurotransmitters) bind to postsynaptic neurons to allow ions to flow across the plasma membrane. D. Sensory stimulus -dependent channels. Such as light and sound. |
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What are the 2 ways that Dr. Spitzer described in lecture to determine the ion selectivity of the channel? If you apply TEA and you find that the channel activity is
unaffected, what can you eliminate? If you apply TTX and you find that the channel activity is eliminated at all voltages, what do you conclude? |
Find the reversal potential/Eion of the channel in single channel recording by applying various voltage commands and measuring the current – when the current changes direction (sign), then you have found the reversal potential. This is the most unambiguous if the channel is only permeable to one type of ion. (To test the possibility of a channel being permeable to more than one ion, you would
replace the ions in the solution with an impermeable charged molecule, and then see how the reversal potential is affected.) If you apply TEA to the channel preparation and there is no change, then either it isn’t a voltage gated K+ channel OR it’s a potassium channel that’s not TEA sensitive. If TTX eliminates the channel activity, then that (corroborating reversal potential of about +55mV) would support the conclusion that the channel is a voltage-gated Na+ channel. |
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You are performing a voltage clamp experiment with a squid giant axon. You are interested in finding the amplitude of the current produced by potassium ions. What can you do to find this value? What are some advantages and disadvantages (if any) your
approach presents? |
Use tetrodotoxin (TTX) in order to block INa and to isolate IK. This approach is very easy and convenient. However, it is not 100% reliable. Some INa are not blocked by TTX.
OR: Replace Na+ ions with an impermeant cation (choline) to set ENa to VC, causing the driving force on sodium to be 0. Then clamp to VC and record IK in isolation. This approach is more difficult to perform, but is 100% reliable. |
