At one concentration of k of 5 mm you find that the

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Unformatted text preview: is dependent on the concentration of extracellular K+ used in the experiment. At one concentration of K+ of 5 mM, you find that the membrane potential is -80.5 mV. From this information, calculate the intracellular K+ concentration. E = - 0.0805 V, E = 0.059 log (Kout/Kin) Kout/Kin = 10(E/0.059) = 0.043 Kin = Kout / 0.043 = 115.7 mM 5) You are doing a summer internship in a lab that studies a recently discovered receptor called RKR. It is a G-protein coupled receptor that is expressed in kidney cells and found to be mutated in patients with a kidney disease. You sequence the receptor in 100 patients and find that the mutations fall into two classes, A and B. Unlike wildtype RKR, both mutants fail to increase cellular levels of cAMP. (i)The ligand for the RKR receptor is called MC. How could you find out if the mutant receptors are expressed on the surface of kidney cells? First you would transfect cDNAs for wt and mutant RKRs into cultured human kidney cells. These receptors would have been tagged e.g. using GFP, and then you would incubate the ligand with the cells and then look under the microscope to see whether the receptor is localized at the membrane and you would examine the relative levels of expression between mutant and wild type. (ii)You find that mutant A is localized to the membrane at approximately the same levels as wildtype but mutant B has very low levels of RKR receptor on the surface. Mutation A lies in the conserved C3 loop, which is known to be important for binding and activation of the Ga subunit. How could you determine whether Ga becomes activated in cells expressing mutant A? One way to do this would be to use the FRET system mentioned in class....
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