Midterm Essay Answers

Midterm Essay Answers - Q1 (Sheet attached with diagrams of...

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Q1 (Sheet attached with diagrams of the experiment) A) Experimental techniques and pharmacological agents used a. Pharmacologic agents i. Pipette filled with: 1. CsCl, EGTA, MgCl2, Tris-creatine-PO4, MgATP, Tris-GTP ii. Recording solution: 1. TEA-Cl, NaCl, BaCl2, CdCl2, HEPES b. Experimental techniques i. Voltage-clamp at -90 mV ii. Reference step to 0 mV iii. Fixed time delay at -90 mV to produce full recovery iv. Conditioning step to +30 mV v. Variable recovery interval at -60 mV, t vi. Test step to 0 mV B) How the experiment works a. Percent availability of channels will be calculated as the peak current brought forth by the test step, normalized by the peak current brought forth by the reference step, plotted as a function of the recovery interval, t. i. Possible ambiguity- Available channels will be defined as those which never inactivated, those still open, and those that have recovered from inactivation.
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step to 0 mV (arbitrarily determined, above threshold stimulus) is given causing an action potential to fire. Measuring the peak current evoked by the first test step, I 1, and plotting the peak amplitude of each n th test step, I n , as a function of time, t, will bring us to a point at which I n = I 1, MAX . At this point it is evident that all of the Na channels have fully deinactivated, and are functional in producing an action potential equal in current magnitude to that of the first test pulse, I 1 . The patch-clamp/voltage-clamp technique brings the membrane to a hyperpolarized state of -60 mV immediately following the reference step, in order to allow for recovery. Percent availability of channels will be calculated as the peak current brought forth by the test step, normalized by the peak current brought forth by the reference step, plotted as a function of the recovery interval, t. Once I n = I 1, MAX , all of the Na channels have become fully deinactivated. The channel gate opens with depolarization and closes with repolarization. The difference between the times it took to reach these conformational states will tell us the time of recovery from inactivation for Na channels. To further show that the responsible currents are passing through Na channels, we
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Midterm Essay Answers - Q1 (Sheet attached with diagrams of...

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