The overall goal of the following experiment is to investigate the effect of proteases on the epithelial sodium channel or ENaC heterologous expressed in zap lava cytes. This is achieved by first measuring the amorite sensitive whole cell current in an oocyte. After allowing the oocyte to recover, it is exposed to protease and the current is measured again.
Then in parallel oocytes are biotinylated and subjected to western blotting to monitor the appearance of gamma ENaC cleavage fragments at the cell surface results are obtained that show a causal link between channel cleavage and channel activation based on a combination of the two electrode voltage clamp technique and the biotin approach. The main advantage of this technique over existing methods like unplanned measurement, is that the cyte can be exposed to protease or other pharmacological agents between the two measurements for a variety length of time in a small volume of test solution. Two days after isolating zap lava cytes and injecting with CN according to the text protocol, use ND 96 to fill one syringe of a gravity fed perfusion system and ND 96 containing amide to fill a second syringe.
After mounting the syringes 50 centimeters above the bath chamber, turn on a 150 watt halogen cold light source and adjust it to 10 centimeters above the cyte bath chamber, allowing good visualization with the binocular microscope turn on suction and place the suction tube opposite the superfusion tube's adapter in the oocyte bath chamber. Next, using the IV gravity flow control device, adjust the superfusion speed of each solution to three to five milliliters per minute. Then with an adapter, connect the superfusion tubes to the oocyte bath chamber using a micro pipette polar pull glass capillaries to obtain tip diameters of less than one micrometer.
Then use three molar potassium chloride to fill them about one quarter full. Insert the capillaries into the electrode holders of the current and the voltage electrode and with the use of the micro manipulators, place them into ND 96 containing two micromolar Amide solution. To measure amide sensitive whole cell currents begin by adjusting the VM offset button to zero the electrode potential of the voltage electrode and the VE offset button to zero the current electrode.
Place an UO site into the bath chamber and close proximity to the voltage sensing electrode. Then using both micro electrodes, gently impale the UO site, set the amplifier holding potential to negative 60 millivolts and turn on the chart recorder. Then turn on the amide containing solution.
Next, start recording, and if necessary, adjust the gain After the measured current reaches a stable plateau, switch to amide free solution. Once a current plateau is reached, switch the perfusion back to the amide containing solution. Then after the current of the UO site, reaches the initial baseline current, turn off the voltage clamp and gently withdraw the electrodes to allow resealing of the plasma membrane at the sites of Impalment.
Place the Uoc site into one well of a 96 well plate containing 100 to 150 microliters of protease free ND 96 solution for five minutes before transferring the UOC site to a protease containing solution or to a control solution without protease. For 30 minutes after the incubation, repeat the current measurement, discard defective oocytes under the stereo microscope and prepare anti 96 with and without appropriate protease. Prepare past pipettes by labeling them and briefly flame their tips to avoid injury of the oocytes.
Fill each well of a six Well plate with 2.5 milliliters of room to temperature ND 96 or ND 96 containing a protease. Then add 30 cytes per well and incubate them for 30 minutes at room temperature. After adding 2.5 milliliters of ND 96 to the wells of a new six well plate transfer each group of oocytes to a well filled with ND 96.
Move the cytes sequentially into two additional wells with ND 96 to wash off any remaining protease and incubate them in the final well for five minutes, prepare biotin in biotin elation buffer according to the text protocol. After placing each group of oocytes into a well containing 2.5 milliliters of biotin elation, buffer incubate for 15 minutes with gentle agitation. Then after placing each group of oocytes into a well containing 2.5 milliliters of quench buffer, to stop the biotin reaction, transfer them to a second dwell of quench buffer and incubate for five minutes.
With gentle agitation, transfer each group of oocytes to a 1.5 milliliter micro centrifuge tube and using one milliliter of lysis buffer, supplemented with protease inhibitors, lys the cytes by passing them through a 27 gauge needle. Centrifuge the lysates for 10 minutes at 1500 times G.Transfer the S natin to a new 1.5 milliliter tube containing TRITTON X 100 and MP 40, and incubate the tubes on ice for 20 minutes. Repeatedly vortexing the tubes to dissolve the proteins after AC equilibrating 100 microliters of vagaro beads per oocyte group, add them to the protein detergent solutions and incubate the samples with overhead rotation at four degrees Celsius overnight.
The following day, centrifuge the samples for three minutes at 1500 times G.Then transfer the supernatant into a new tube to detecting act cleavage fragments at the cell surface. Repair gel samples by using lysis buffer to wash the beads three times before adding two XSDS page sample buffer. After centrifuging the samples for three minutes at 20, 000 times, G, pipette the supernatant into new micro fuge tubes.
Analyze the samples by western blot according to the text protocol to investigate whether the serine, protease plasmin can activate ENaC mediated currents. The delta IE of individual ENaC expressing cytes was determined before and after a 30 minute incubation of the cytes in protease free or plasmin containing solution. Using the two electrode voltage clamp technique as shown here, exposure to plasmin increased delta IE in every cyte measured.
In contrast, incubation in protease free solution had a negligible effect. This figure compares the effect of chi trypsin on wild type ENaC and on ENaC with mutated prostasin and plasmin cleavage sites. As seen here, the mutant channel delays and reduces the activation of ENaC mediated current by chio trypsin.
In addition, it generates a cleavage fragment at a reduced rate compared to the wild type ENaC. After watching this video, you should have a good understanding of how a combination of the described methods can be used to correlate the stimulatory effect of proteases on inner occurrence with the occurrence of cleavage products at the cell surface. This may be useful for a broad range of applications, for example, to address similar questions regarding the regulation of other iron channels, transporters or transmembrane receptors.
