Functional Characterization of Na⁺/H⁺ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The overall goal of the following experiment is to measure the activity of intracellularly expressed sodium proton exchangers. This is achieved by first selecting cells that express the intracellular sodium proton exchangers at the plasma membrane by proton killing selection. As a second step, the selected cells are acidified, and then the intracellular pH recovery is measured to confirm the sodium proton exchange activity of the cells.

The fast kinetics of the ion transport are then measured to determine the affinity constants for the transported cation and inhibitors. Ultimately, monitoring the changes in the intracellular pH and fast kinetics of the selected somatic cells allows the functional characterization of the intracellular protein exchange. The main advantage of this technique over existing methods like expressing mutant transporters or using per cells, is that it enables the expression of white type intracellular transporters at the plasma membrane making possible to directly measure their function.

Demonstrating the procedure will be Dr.Mallory poet, a research scientist from my laboratory To begin the proton killing procedure. First, incubate the cells expressing the intracellular sodium proton exchanger of interest in loading solution at 37 degrees Celsius and a carbon dioxide free incubator for one hour. Next, aspirate the loading solution and wash the cells twice with fresh medium.

After the second wash, aspirate the medium and incubate the cells in recovery solution in a carbon dioxide free incubator. After another hour, replace the recovery medium with regular culture medium and grow the cells under standard culture conditions. Repeating this cycle of selection twice a week until stable clones emerge to image the intracellular fluorescence pH of the cells.

First equipped an imaging set consisting of an inverted microscope coupled to a high sensitivity video camera with 450 nanometers and 490 nanometers narrow band interference filters paired with the appropriate quartz neutral density filters for excitation. If possible, use the appropriate set of filters and illumination conditions to set up comparable fluorescence values for channels one and two so that the ratio approaches one to measure the sodium proton exchanger forward activity. Next, acidify the cells with a one hour incubation in ammonium loading solution.

In the absence of carbon dioxide during the last five minutes of the incubation stain the cells with a ratio metric pH sensitive fluorescent die. At the end of the incubation period, rin the cells with ammonium loading solution to eliminate the extracellular probe and mount the dyed cells on the microscope. Select the regions of interest where the fluorescent values will be measured as appropriate.

Then record several images to establish a baseline and perfuse the cells with the RIN solution to initiate a drop in the fluorescence. After the pH stabilizes, peruse the cells with the solution of interest to measure the pH recovery rates as mediated by the lithium hydrogen, sodium hydrogen or potassium hydrogen exchange. Now peruse the cells with the solution of hippies supplemented with potassium chloride and gerin.

Then collect the fluorescence measurements from the images and export the data into a spreadsheet program in the text format to measure the initial rate of the sodium proton exchanger. Seven, by fast lithium uptake, acidify the cells and multi-well plates by the Imodium loading technique as just demonstrated. Next, rinse the plates twice rapidly as just demonstrated.

Then incubate the cells in uptake solutions containing one to 10 micromolar lithium and the desired concentrations of the cation or inhibitors of interest for about one minute or less at the end of the uptake period, carefully remove the uptake medium and rapidly wash the cells four times with ice cold PBS in less than 10 seconds total to prevent lithium efflux then lys the cells in 250 microliters of 25%nitric acid per well for at least one hour. Scraping each well with the end of the pipette tip at the end of the incubation and transferring the suspensions from each well into new micro centrifuge tubes. Finally, centrifuge's samples for five minutes at 15, 000 G at room temperature to remove the cellular debris and then measure the lithium content of the snat by atomic absorption spectroscopy.

According to the manufacturer's instructions, proton killing selection is based on the diffusion of the ammonium weak base. For example, cells that do not express a functional sodium proton exchanger at the plasma membrane cannot recover a neutral pH value and therefore exhibit a typical acidified aspect with a flat shape and a granular cytosol as seen in this representative image after repeated selection cycles, cells that fully and stably survive. The acidification, however, form individual cellular clones as seen here, as demonstrated the activity and ion selectivity of the intracellular sodium proton exchangers expressed at the plasma membrane can be characterized by measuring the changes in the intracellular pH induced under various experimental conditions as observed in these graphs following Imodium loading acidification.

As for the enzyme kinetics, the dose response for the initial uptake rates can be further used to derive the affinity constant values for the substrates and for the inhibition constant values of the inhibitors. Indeed, an interesting feature of the transporters is that different coupling cation will compete for the transport, yielding the possibility of measuring the affinity constant for sodium by competition with the lithium uptake as demonstrated in this figure Following this procedure. Other experiments like cell surface BioInnovation coupled to RNA silencing can be performed in order to verify that the transporter of interest is indeed expressed at the plasma membrane.