The procedure uses frog cytes as an in vivo expression system. To quantify the transport activity of potassium counter transporting P-type A, TP, ais, or other trace element membrane transporters first express the transporter protein in zap lavis. Cytes then induce cation uptake by incubating the cells in flux solution containing a certain concentration of the transported cation for a fixed time and temperature.
Alternatively, perform the uptake Experiment under membrane potential control using the two electrode voltage clamp technique. After washing the cells homogenize, individual oocytes in one milliliter of millipore water. Next, measure the samples on an atomic absorption spectrophotometer equipped with a transversely heated graphite atomize furnace.
Ultimately tracer flux analysis by atomic absorption. Spectometry can be used to determine transport or inhibitor kinetics, the voltage dependence of transport and transport, stoic geometry, and may eventually be used for pharmacological screening. The main advantage of this a a s technique is that it's an accurate, cheap, and safe alternative to conventional tracer flux experiments with radioisotopes.
It also offers precise measurement of the influence of membrane potential on transporter function. Though this method can provide insight into structure functional relationships of P-type A TPAs, it can also be applied to other membrane transporter proteins specific for copper sink or other trace elements. Prepare the opus cytes and perform CRNA injection as described by Richards and Demsky to elevate the intracellular sodium concentration for functional measurements of sodium potassium.
At TPAs. First, transfer the cytes into sodium loading buffer and incubate on ice for 45 minutes. Then place the cytes to recover in post loading buffer for at least 30 minutes on ice.
Now to block the endogenous sodium potassium at TPAs, incubate the cytes in post loading buffer containing 100 micromolar lobbing for 15 minutes. At room temperature, prepare micro electrodes by pulling Bos silicic capillaries using a two step micro pipette puller. Fill the glass electrodes from the back with three molar potassium chloride and mount it over the silver, silver chloride wire of the micro electrode holders of the two electrode voltage clamp set up.
Next, insert the glass micro electrodes and two sodium chloride agar bridges into the recording chamber pre-filled with measuring buffer. Switch the voltage clamp amplifier into the off mode. Check the resistance of each glass electrode separately.
Also, check offset potentials of voltage and current electrode on the appropriate TEVC amplifier display. In case of discrepancy from zero, adjust the offset with the corresponding offset regulators of the amplifier. Now place an oocyte in the middle of the chamber and penetrate the membrane gently with the current and the voltage electrode.
Check the potential reading of the amplifier for the resting potential of the cell. Switch the amplifier to VC mode using the P clamp software. Clamp the cell to a predefined membrane potential.
Monitor the amplitude and stability of the leak current perfuse the oocyte with rubidium containing solution for a defined time. And record the pump current with p clamp software. Make sure that rubidium uptake is completely stopped afterwards by perfusing the cell with rubidium free solution for 30 seconds.
Now stop the two electrode voltage clamp experiment. Remove the oocyte from the recording chamber and proceed to the washing. Step 4.4 to analyze rubidium uptake with atomic absorption.
Spectrometry with the data. Perform an offset current subtraction and evaluate the time integral of the pump current using the clamp fit program of the P clamp package. To prevent oocyte damage, cut and melt a 200 microliter pipette tip to about two millimeters in diameter.
Then for each oocyte to be measured, prepare a 1.5 milliliter einor tube filled with one milliliter of millipore water. Arrange three Petri dishes with rubidium free washing buffer and one dish with millipore water. Now transfer five pre incubated cytes simultaneously into a 3.5 centimeter Petri dish filled with rubidium flux buffer incubate under temperature control.
Next, rinse the cytes simultaneously in the first dish with rubidium free wash buffer. Proceed to the second and third washes and then rinse gently in millipore water. Now transfer each oocyte individually into the prepared eph tubes filled with one milliliter of millipore water Homogenize each oocyte using a 200 microliter pipette tip until the solution is homogenously turbid.
Turn on the valve Argonne gas supply. Insert an appropriate hollow cathode lamp in the arbitrary socket position and equilibrate the instrument for 15 minutes. Then start the wind lab 32 control software and allow 15 minutes for equilibration of the lamps.
Verify that the transversely heated graphite atomizer tube is in good order. Then select the THGA furnace technique in wind lab 32. Prepare calibration samples with at least five rubidium lithium concentrations between 10 and 50 micrograms per liter, dissolved in millipore water and one blank probe of millipore water only.
Now choose the appropriate initialization file termed method file. Enter the positions and concentrations of the calibration samples and the blank probe. Select the appropriate autos sampler.
Measure the calibration probes. Now transfer each oocyte homogenate into individual 1.2 milliliter polypropylene sample cups, and place these test tubes into the autos sampler tray. Create a sample information file to identify the probes in the autos sampler positions.
Then adjust the sample volume to 20 microliters and start the instrument after completion of the run. Check for probes in which the measured amount of rubidium exceeds the maximum of the calibration curve. Dilute these samples appropriately and measure them again for the electrogenic sodium potassium.
A TPAs rubidium fluxes can be determined in two electrode voltage clap experiments aimed at the correlation between net charge transport and rubidium transport. Here, integration of the current signal yielded 7, 840 nku of total transported charge. The ratio between total charge and rubidium flux was 0.47 a value consistent with the three sodium to two potassium transport stoichiometry of the sodium potassium APAs.
The reproducibility of this A a s technique on single cell experiments is shown by results of repeated experiments producing a linear correlation between total charge and rubidium transport with a slope factor of 0.49 for the electro neutrally operating proton potassium. A TPAs the concentration dependence of rubidium uptake and its sensitivity towards the specific inhibitor S CH 2 8 0 8 0 can be determined. These measurements in different batches of oocytes show a background uptake into unin injected oocytes of less than 10%of the maximally detected rubidium uptake, and this is insensitive to the addition of SCH 2 8 0 8 0.
By contrast, the fluxes in proton potassium atpa expressing cytes are reduced to background level. Upon addition of 10 micromolar s CH 2 8 0 8 0 plotting the specific uptake values allows calculation of the apparent affinities of proton potassium ATP's turnover transport for rubidium extracellular sodium results in a decrease in the apparent affinity for rubidium consistent with competition for the extracellular facing cation binding pocket during transport. The combination of rubidium uptake experiments under membrane potential control by two electrode voltage clamp and a a s determination can also be applied to determine the voltage dependence of rubidium transport by proton potassium APAs.
Under different pH conditions, rubidium flux experiments can be used to investigate functional effects of proton potassium APAs mutations. For instance, analyses of the rubidium transport activity of proton potassium APAs mutants with different C terminal deletions reveal that rubidium uptake activity gradually decreases with the number of deleted amino acids. Interestingly, the extent of rubidium uptake inhibition by 10 micromolar SC 2 8 0 8 0 is markedly reduced for the delta Y construct, whereas delta YY and delta Q-E-L-Y-Y are no longer sensitive to 10 micromolar S CH 2 8 0 8 0 at all SDS page analyses indicate that the total amount of proteins in membrane fractions for the various deletion constructs were similar.
However, the relative amount of protein in the plasma membrane fractions decreased with C terminal deletion taken together with drug inhibition results. These data have implications on the steady state distribution of reaction cycle intermediates as discussed in the accompanying text, rubidium fluxes can also be utilized to measure thermodynamic parameters like activation energies. Rubidium uptake by proton potassium atpa is expressing oocytes is strongly dependent on temperature.
An aous plot yields a good linear correlation of the data resulting in an activation energy of 96 kilojoules per mole. First results with lithium uptake showed that unspecific lithium uptake in cytes is low, whereas uptake by proton potassium atpa increases in a dose-dependent manner and appears resistant to SC 2 8 0 8 0. These data shed light on enzyme states during the stationary proton lithium cycling.
While attempting this procedure, it's important to remember to select the cytes for homogenous size and to currently stick to incubation times and temperature for each flux experiment. Following this a S procedure. Other methods like pharmacological screening can be performed in order to identify novel drugs for therapy against human diseases such as gas osteopath or hypertension.
