The expert technical assistance of C&#233;line Gr&#252;ninger, Christina Lang, Sonja Mayer, and Ralf Rinke is gratefully acknowledged. We thank Dr. Morag K. Mansley for carefully reading the manuscript. This project was supported by a grant of the Deutsche Forschungsgemeinschaft (Grant SFB 423: Kidney Injury: Pathogenesis and Regenerative Mechanisms, to C. Korbmacher), grants of the Interdisziplin&#228;res Zentrum f&#252;r Klinische Forschung (to S. Haerteis and M. Krappitz), the ELAN program (to S. Haerteis) of the Friedrich-Alexander-Universit&#228;t Erlangen-N&#252;rnberg, and the University Library of Erlangen-N&#252;rnberg.

1. Isolation of Xenopus Oocytes and Microinjection of cRNA
<ol>
	<li>Obtain oocytes from adult female Xenopus laevis. Anaesthetize animals in 0.2% MS222, and resect ovarian lobes through a small abdominal incision.</li>
	<li>Isolate oocytes from the ovarian lobes by enzymatic digestion at 19 &#176;C for 3-4 hr with 600-700 U/ml of type 2 collagenase from Clostridium histolyticum dissolved in calcium free OR2 solution (recipe in Table 1).</li>
	<li>For selection, place the defolliculated oocytes in a Petri dish under a binocular microscope in a high sodium containing solution (ND96: recipe in Table 1).</li>
	<li>Select stage V-VI oocytes and place them in another Petri dish with a Pasteur pipette. NOTE: Blunt Pasteur pipette by flaming to prevent oocyte injury.</li>
	<li>Inject oocytes with cRNA (e.g. 0.2 ng per &#945;&#946;&#947;ENaC subunit). Dissolve cRNAs in RNase-free water. NOTE: Total volume injected into each oocyte is 46 nl.</li>
	<li>Store injected oocytes at 19 &#176;C in a low sodium solution to prevent sodium loading of the oocytes (ND9: recipe in Table 1). Supplement the solution with 100 U/ml sodium penicillin and 100 &#956;g/ml streptomycin sulphate to prevent bacterial growth. Carefully handle the oocytes to limit the amount of damaged or dead oocytes and maintain them in individual small groups in a 12-well-plate wells filled with bath solution during the two days after cRNA injection.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >OR2</td>
			<td >82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.4 with NaOH</td>
		</tr>
		<tr >
			<td >ND96</td>
			<td >96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.4 with Tris</td>
		</tr>
		<tr >
			<td >ND9</td>
			<td >9 mM NaCl, 87 mM NMDG-Cl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.4 with Tris</td>
		</tr>
		<tr >
			<td >biotinylation buffer</td>
			<td >10 mM triethanolamine, 150 mM NaCl, 2 mM CaCl2, adjusted to pH 9.5 with HCl</td>
		</tr>
		<tr >
			<td >quench buffer</td>
			<td >192 mM glycin, 25 mM Tris-Cl, adjusted to pH 7.5 with HCl</td>
		</tr>
		<tr >
			<td >lysis buffer</td>
			<td >500 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl, adjusted to pH 7.4 with HCl</td>
		</tr>
	</tbody>
</table>
Table 1. Solutions
2. Performing Two-electrode Voltage-clamp Experiments
<ol>
	<li>Measure the oocytes two days after injection.</li>
	<li>Fill one syringe of a gravity fed perfusion system with ND96 solution and another syringe with ND96 solution containing amiloride (2 &#181;M). Mount syringes 50 cm above the oocyte bath chamber. NOTE: The concentration of the ENaC inhibitor amiloride was chosen to be 20 times higher than its IC50 (100 nM).</li>
	<li>Turn on a 150 W halogen cold light source and adjust it to 10 cm above the oocyte bath chamber allowing good visualization with the binocular microscope. Then turn on suction and adjust the suction tube at the end of the oocyte bath chamber. Locate suction tube opposite to superfusion tubes&#8217; adapter entering the oocyte bath. NOTE: Suction power must be sufficient to support continuous flow of the solution superfusing the oocyte.</li>
	<li>Adjust superfusion speed of each solution to 3-5 ml/min by using the i.v. gravity flow control device. Connect the superfusion tubes with an adapter to the oocyte bath chamber.</li>
	<li>Pull glass capillaries with a micropipette puller to obtain tip diameters of &#60;1 &#181;m. Then fill capillaries to ~1/4 with 3 M KCl. NOTE: Make sure that the chlorinated part of the silver wire of the electrode holder is immersed in KCl solution. Check for air bubbles in the tip of the capillary. Air bubbles impair the measurement by increasing resistance stray capacitance.</li>
	<li>Insert the capillaries into the electrode holders of the current and the voltage electrode and place them into ND96 containing amiloride (2 &#181;M) solution using the micro-manipulators.</li>
</ol>
3. Measurement of Amiloride-sensitive Whole-cell Currents
<ol>
	<li>Zero the electrode potential of the voltage electrode (Vm) and the current electrode (Ve) by adjusting the Vm and Ve offsetbuttons. NOTE: The resistance should be 1-2 M&#937; for the electrode to measure Vm and 0.5-1 M&#937; for the current injection electrode.</li>
	<li>Place the oocyte into the bath chamber in close proximity to the voltage sensing electrode. NOTE: Do not damage the oocyte during any of these transfer steps. Use a Pasteur pipette to transfer the oocyte. To avoid damaging the oocyte the edges of the pipette should be blunted by flaming.</li>
	<li>Impale oocytes gently with both microelectrodes.</li>
	<li>Set the holding potential at the amplifier to -60 mV and turn on the chart recorder. Turn on the amiloride (2 &#181;M) containing solution. NOTE: The current should be about 0 &#177; 0.5 &#181;A. Larger leak currents indicate a leaky impalement. Therefore, these oocytes should be rejected. Moreover, the leak currents measured in the presence of amiloride (2 &#181;M) should be similar in &#945;&#946;&#947;-WT expressing oocytes to those measured in &#945;&#946;&#947;-mutant ENaC expressing oocytes. This indicates that the mutations do not affect the amiloride-sensitivity of the channel.</li>
	<li>Start recording. If necessary adjust the gain.</li>
	<li>After the measured current reaches a stable plateau, change to amiloride-free solution. NOTE: Downward current deflections in the current traces correspond to inward currents, i.e. movement of positive charge (Na+) from the extracellular side into the cell.</li>
	<li>After a current plateau is reached (after ~60 sec), switch the superfusion back to the amiloride containing solution. After the current of the oocyte reaches the initial baseline current, turn off the voltage clamp and gently withdraw the electrodes.</li>
	<li>To allow resealing of the plasma membrane at the sites of impalement, place the oocyte into one well of a 96-well plate containing 100-150 &#181;l of protease free ND96 solution.</li>
	<li>After 5 min, transfer oocyte to a protease containing solution or to a control solution without protease for a 30 min incubation time. NOTE: The incubation time depends on the protease and the studied channel.</li>
	<li>After the incubation step repeat the current measurement (see 3.2 and following). NOTE: It is possible to measure &#62;90% of the oocytes after incubation in protease solution.</li>
</ol>
4. Biotinylation Assay
<ol>
	<li>Select and discard defective oocytes under the binocular microscope. NOTE: Use injected oocytes from the same batch for the current measurements and for the biotinylation experiments.</li>
	<li>Keep biotin at RT for at least 20 min before its use in the experiment.</li>
	<li>Prepare the solutions: ND96 and ND96 containing the appropriate protease. Prepare Pasteur pipettes by labeling them and by briefly flaming their tips to avoid injury of the oocytes. NOTE: Here, the protease chymotrypsin 2 &#181;g/ml in ND96 is used. Treat each group with a separate pipette to avoid cross contamination of solutions.</li>
	<li>Fill each well of a 6-well plate with 2.5 ml control ND96 or ND96 containing a protease at RT. Then deposit 30 oocytes per well and incubate them for 30 min at RT. NOTE: For the subsequent procedures it is important to keep samples on ice at all times. All centrifugation steps are at performed at 4 &#176;C.</li>
	<li>Fill each well of a new 6-well plate with 2.5 ml ND96 (each group needs 3 wells for the washing steps) and weigh the biotin. NOTE: 2.5 mg biotin per well (1 mg/ml) is required. Dissolve the biotin in the biotinylation buffer (i.e. 25 mg biotin (for 10 groups) in 25 ml biotinylation buffer (recipe in Table 1).</li>
	<li>Transfer each group of oocytes to a well filled with 2.5 ml ND96. Transfer oocytes sequentially into two additional wells with ND96 to wash off any remaining protease. Incubate the oocytes for 5 min in ND96.</li>
	<li>Transfer the oocytes into a well containing 2.5 ml biotin solution and incubate them with gentle agitation (&#8216;shaker&#8217;) for 15 min. NOTE: Minimize the transferred ND96 with the pipette to avoid dilution of the biotin solution.</li>
	<li>Transfer each group of oocytes into a well containing 2.5 ml quench buffer (recipe in Table 1) to stop the biotinylation reaction. Then, transfer each group of oocytes into a second well also containing 2.5 ml quench buffer and incubate for 5 min with gentle agitation.</li>
	<li>Remove damaged or dead oocytes. NOTE: Choose the same number of oocytes per group for the following procedure.</li>
	<li>Transfer each group of oocytes to a 1.5 ml plastic microcentrifuge tube. NOTE: Minimize the quantity of quench buffer that is transferred.</li>
	<li>Subsequently, lyse the oocytes by passing them through a 27 G needle in 1 ml lysis buffer (recipe see Table 1) supplemented with protease inhibitors.</li>
	<li>Centrifuge the lysates for 10 min at 1,500 x g.</li>
	<li>Aspirate supernatant and transfer it to a 1.5 ml microcentrifuge tube containing 0.5% Triton-X-100 and 0.5 % NP40. Discard the remaining pellet. NOTE: The supernatant contains biotinylated plasma membrane proteins and non-biotinylated intracellular proteins.</li>
	<li>Incubate the microcentrifuge tubes for 20 min on ice. Repeatedly vortex the tubes during this period to completely dissolve the proteins in NP40 and Triton-X-100.</li>
	<li>Centrifuge 100 &#181;l of agarose beads per oocyte group for 3 min at 1,500 x g. After the centrifugation remove supernatant from the beads solution and wash three times with lysis buffer to equilibrate the beads with buffer.</li>
	<li>Pipette 100 &#181;l of the washed beads into each microcentrifuge tube containing the protein-detergent-solution prepared in 4.13 to allow binding of the biotinylated proteins to the beads.</li>
	<li>Incubate microcentrifuge tubes with overhead rotation O/N at 4 &#176;C.</li>
	<li>Centrifuge the microcentrifuge tubes for 3 min at 1,500 x g. Then transfer the supernatant into a new tube. NOTE: Intracellular proteins are not labeled with biotin. Supernatant can be stored at -20 &#176;C. Do not aspirate the beads.</li>
</ol>
5. Detection of ENaC Cleavage Fragments at the Cell Surface by Western Blot Analysis
<ol>
	<li>Wash the beads three times with lysis buffer and add 100 &#181;l of 2x SDS-PAGE sample buffer. NOTE: Samples can be stored at -20 &#176;C or immediately prepared for western blot analysis.</li>
	<li>Boil samples for 5 min at 95 &#176;C and then place tubes on ice.</li>
	<li>Centrifuge samples for 3 min at 20,000 x g and pipette the supernatant into a new microcentrifuge tube. NOTE: This supernatant contains the biotinylated plasma membrane proteins from the cell surface of the oocyte.</li>
	<li>Analyze 30 &#181;l of this supernatant by western blot to investigate cleavage fragments at the cell surface.</li>
	<li>Separate the biotinylated proteins by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) using an appropriate gel (8%, 10%, 12% depending on the molecular weight of the cleavage fragments investigated).</li>
	<li>Transfer the proteins to polyvinylidene difluoride (PVDF) membranes by semi-dry blotting.</li>
	<li>Probe the membrane with a specific antibody against human &#947;ENaC directed against an epitope in the C-terminus (see Figure 3 and 13).</li>
	<li>Use horseradish peroxidase-labeled goat anti-rabbit antibody as secondary antibody.</li>
	<li>Detect chemiluminescent signals.</li>
</ol>

<ol>
	<li>Kleyman, T. R., Carattino, M. D., Hughey, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ENaC+at+the+cutting+edge:+regulation+of+epithelial+sodium+channels+by+proteases.">ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.</a> The Journal of Biological Chemistry. 284, 20447-20451 (2009).</li><li>Rossier, B. C., Stutts, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+epithelial+sodium+channel+(ENaC)+by+serine+proteases.">Activation of the epithelial sodium channel (ENaC) by serine proteases.</a> Annu Rev Physiol. 71, 361-379 (2009).</li><li>Poirot, O., Vukicevic, M., Boesch, A., Kellenberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+regulation+of+acid-sensing+ion+channel+1+by+serine+proteases.">Selective regulation of acid-sensing ion channel 1 by serine proteases.</a> The Journal of Biological Chemistry. 279, 38448-38457 (2004).</li><li>Vukicevic, M., Weder, G., Boillat, A., Boesch, A., Kellenberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+cleaves+acid-sensing+ion+channel+1a+in+a+domain+that+is+critical+for+channel+gating.">Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating.</a> The Journal of Biological Chemistry. 281, 714-722 (2006).</li><li>Clark, E. B., Jovov, B., Rooj, A. K., Fuller, C. M., Benos, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+cleavage+of+human+acid-sensing+ion+channel+1+by+the+serine+protease+matriptase.">Proteolytic cleavage of human acid-sensing ion channel 1 by the serine protease matriptase.</a> The Journal of Biological Chemistry. 285, 27130-27143 (2010).</li><li>Ossovskaya, V. S., Bunnett, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-activated+receptors:+contribution+to+physiology+and+disease.">Protease-activated receptors: contribution to physiology and disease.</a> Physiological reviews. 84, 579-621 (2004).</li><li>Garcia-Caballero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+epithelial+sodium+channel+by+the+metalloprotease+meprin+&#946;-subunit.">Activation of the epithelial sodium channel by the metalloprotease meprin &#946;-subunit.</a> Channels (Austin. 5, 14-22 (2011).</li><li>Haerteis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+activation+of+the+epithelial+sodium+channel+(ENaC)+by+the+cysteine+protease+cathepsin-S.">Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S.</a> Pfl&#252;gers Archiv: European Journal of Physiology. 464, 353-365 (2012).</li><li>Harris, M., Firsov, D., Vuagniaux, G., Stutts, M. J., Rossier, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+neutrophil+elastase+inhibitor+prevents+elastase+activation+and+surface+cleavage+of+the+epithelial+sodium+channel+expressed+in+Xenopus+laevis+oocytes.">A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.</a> The Journal of Biological Chemistry. 282, 58-64 (2007).</li><li>Passero, C. J., Mueller, G. M., Rondon-Berrios, H., Tofovic, S. P., Hughey, R. P., Kleyman, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+activates+epithelial+Na++channels+by+cleaving+the+&#947;-subunit.">Plasmin activates epithelial Na+ channels by cleaving the &#947;-subunit.</a> The Journal of Biological Chemistry. 283, 36586-36591 (2008).</li><li>Svenningsen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+in+nephrotic+urine+activates+the+epithelial+sodium+channel.">Plasmin in nephrotic urine activates the epithelial sodium channel.</a> Journal of the American Society of Nephrology : JASN. 20, 299-310 (2009).</li><li>Patel, A. B., Chao, J., Palmer, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+kallikrein+activation+of+the+epithelial+Na+channel.">Tissue kallikrein activation of the epithelial Na channel.</a> American Journal of Physiology. Renal Physiology. 303, (2012).</li><li>Haerteis, S., Krappitz, M., Diakov, A., Krappitz, A., Rauh, R., Korbmacher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+and+chymotrypsin+have+distinct+preferences+for+channel+activating+cleavage+sites+in+the+&#947;-subunit+of+the+human+epithelial+sodium+channel.">Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the &#947;-subunit of the human epithelial sodium channel.</a> The Journal of General Physiology. 140, 375-389 (2012).</li><li>Chraibi, A., Vallet, V., Firsov, D., Hess, S. K., Horisberger, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease+modulation+of+the+activity+of+the+epithelial+sodium+channel+expressed+in+Xenopus+oocytes.">Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.</a> The Journal of General Physiology. 111, 127-138 (1998).</li><li>Volk, T., Konstas, A. A., Bassalay, P., Ehmke, H., Korbmacher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Na++removal+attenuates+rundown+of+the+epithelial+Na+-channel+(ENaC)+by+reducing+the+rate+of+channel+retrieval.">Extracellular Na+ removal attenuates rundown of the epithelial Na+-channel (ENaC) by reducing the rate of channel retrieval.</a> Pfl&#252;gers Archiv: European Journal of Physiology. 447, 884-894 (2004).</li></ol>

The authors have nothing to disclose.

In this manuscript a methodological approach which was successfully applied to study the mechanisms underlying the activation of ENaC by proteases is described8,13. The well established Xenopus laevis oocyte expression system was used to functionally express ENaC. ENaC function was assessed with the conventional two-electrode voltage-clamp technique. Site-directed mutagenesis was employed to identify functionally relevant protease cleavage sites. Biotinylation experiments performed in parallel with the electrophysiological measurements made it possible to correlate the appearance of ENaC cleavage products at the cell surface with proteolytic current activation. A correlation between the time course of current activation and the appearance of proteolytic cleavage fragments at the cell surface supports the concept of proteolytic channel activation.
Two-electrode voltage-clamp recordings require the impalement of an oocyte with two microelectrodes. This procedure is usually performed only once in an individual oocyte. However, it was feasible to remove the microelectrodes after an initial whole-cell current recording without apparent damage to the oocyte. Indeed, the plasma membrane at the sites of impalements appears to reseal within a few minutes. Thus, after completing a first two-electrode voltage-clamp measurement, it is possible to transfer the oocyte from the experimental flow chamber of the two-electrode voltage-clamp setup to a microfuge tube or a well of a 96-well plate filled with a small volume of test or control solution. Afterwards, the same oocyte can be transferred back to the flow chamber and can be impaled again to perform a second two-electrode voltage-clamp measurement. Remarkably, ENaC-mediated currents did not vary much between the first and second measurement when the oocyte was maintained in control solution. In contrast, incubation of the oocyte in a protease containing solution after the first measurement resulted in increased ENaC-mediated current in the second measurement (Figure 2). This finding indicates proteolytic channel activation.
Performing two separate current measurements in a single oocyte offers the advantage that the oocyte can be exposed to proteases or other pharmacological agents between the two measurements for a variable length of time in a small volume of test solution. This is important when using agents which are expensive and/or unavailable in large quantities, e.g. purified protease preparations. The limited availability of agents may make it impossible (or unaffordable) to use them in continuous two-electrode voltage-clamp recordings because of the large volumes of test solution required for continuously superfusing the oocytes with flow rates of several milliliters per minute. Moreover, continuous two-electrode voltage-clamp measurements are limited by the well-known phenomenon of spontaneous channel rundown also described for ENaC 15. In contrast, exposing oocytes to test solutions between two separate measurements for up to an hour or more does not generally pose a problem (see Figure 4A). Finally, two sequential measurements performed in the same oocyte allow paired observations of drug effects. This has an advantage over unpaired measurements from two separate groups of oocytes (protease-treated and vehicle-treated), because it reduces the problem of high variability between oocytes, usually observed in ion channel expression. With paired observations and the possibility to normalize the data to the first measurement, fewer oocytes are needed per experimental group to demonstrate a significant effect of a pharmacological agent. Normalization of the data also makes it easy to summarize data from different batches of oocytes with different ion channel expression levels and hence different baseline currents (Figure 2D). Obviously, control experiments are necessary for this approach to demonstrate that the ion channel activity of interest remains stable in vehicle-treated control oocytes from the first to the second measurement (see Figure 2).
To demonstrate that proteolytic current activation correlates with the appearance of ENaC cleavage products at the cell surface, a biotinylation approach originally described by Harris et al.9 can be used. This procedure (as detailed in the protocol section and shown in Figure 4) was adapted to demonstrate that exposure of channels to proteases and subsequent activation of ENaC-mediated currents is paralleled by the time-dependent appearance of cleavage fragments. The biotinylation method also allows the analysis of an overall increase or decrease of membrane proteins at the cell surface. Thus, this method is suitable to investigate the effect of proteases and other pharmacological agents upon channel insertion into the plasma membrane or upon channel retrieval. Moreover, western blot analysis of the biotinylated plasma membrane proteins allows detection of protein fragments (e.g. proteolytic ENaC fragments) or changes in the glycosylation pattern which may be functionally relevant.
In conclusion, the combination of methods used to investigate the stimulatory effect of proteases on ENaC-mediated whole-cell currents and to demonstrate a correlation with the occurrence of ENaC cleavage products at the cell surface may be useful for a broad range of applications. In particular, these methods may be suitable to address similar questions regarding the regulation of other ion channels, transporters or transmembrane receptors (e.g. protease-activated receptors PARs).

Proteases are enzymes that are involved in various physiological reactions ranging from the well-known proteolytic degradation of proteins, in the context of digestion, to highly sophisticated protease cascades involved in complex regulatory signaling pathways. Proteases are classified into seven groups according to their catalytic active site: aspartate, asparagine, cysteine, glutamic acid, metallo, serine, and threonine proteases. Different proteases target distinct cleavage sites which are not always easy to predict from the primary structure of a protein. The MEROPS database (<a href="http://merops.sanger.ac.uk/">http://merops.sanger.ac.uk/</a>) provides detailed information on a wide range of proteases and their preferential cleavage sites. Functionally relevant cleavage sites can be identified using site-directed mutagenesis.
It is well established that proteolytic processing of ENaC is an important mechanism of activation of this particular ion channel1,2. Interestingly, there is evidence that the function of the related acid-sensing ion channel 1a (ASIC1a) may also be modified by proteases3-5. At present it remains an open question whether proteolytic channel cleavage plays a relevant physiological role in regulating the activity of other ion channels or transporters. However, it is well established that proteolytic cleavage activates a group of G protein-coupled receptors, the protease-activated receptors (PARs)6. Several serine proteases (e.g. channel-activating proteases (CAP1-3), chymotrypsin, trypsin, furin, plasmin, neutrophil elastase, and kallikrein) have been shown to proteolytically activate ENaC2. In addition to serine proteases, other groups of proteases may be involved in proteolytic ENaC activation. Indeed, recent data shows that the metalloproteinase meprin-&#946;7 and the cysteine protease cathepsin-S8 can also activate ENaC. However, the (patho-)physiologically relevant proteases for ENaC activation remain to be determined and may differ from tissue to tissue.
Proteases are known to preferentially cleave at particular sites in the amino acid sequence. For instance, the serine protease chymotrypsin shows a specific cleavage pattern cleaving after the aromatic amino-acid residues phenylalanine and tyrosine. In contrast, the serine protease trypsin preferentially cleaves after the basic residues lysine or arginine. Using mutant human &#947;ENaC constructs generated by site-directed mutagenesis, functionally relevant cleavage sites in ENaC heterologously expressed in the oocyte expression system could be identified8-13.
By injecting cRNA for the three ENaC subunits (&#945;&#946;&#947;) into isolated oocytes, ENaC can be functionally expressed in these cells and the activity of channels present at the plasma membrane can be measured by using the two-electrode voltage-clamp technique. By using the diuretic amiloride, a specific ENaC inhibitor, the amiloride-sensitive ENaC-mediated whole-cell current component (&#916;Iami) can be separated from unspecific leak currents or from currents conducted by other ion channels. Thus, &#916;Iami values reflect overall ENaC activity and can be determined by subtracting whole-cell currents measured in the presence of amiloride from the corresponding whole-cell currents recorded in the absence of amiloride. To test whether a protease has a stimulatory effect on ENaC, &#916;Iami is measured twice in the same oocyte, i.e. before and after incubation of the oocyte in a protease containing solution. An increase of &#916;Iami from the first to the second measurement indicates proteolytic ENaC activation. Chymotrypsin or trypsin are known to maximally stimulate ENaC in the oocyte expression system2,14 and can be used to confirm that proteolytic ENaC activation is detectable in a given batch of oocytes.
In parallel to whole-cell current measurements, a biotinylation approach9 was used to investigate whether the increase in &#916;Iami detected upon exposure of the oocytes to proteases correlates with the appearance of ENaC cleavage fragments at the cell surface. Proteins at the cell surface are labeled with biotin and can be separated from intracellular proteins by binding the biotinylated proteins to Neutravidin-labeled agarose beads. The biotinylated proteins can be analyzed by western blot. &#947;ENaC cleavage fragments at the cell surface can be detected using a specific antibody directed against an epitope in the C-terminus of the &#947;ENaC. To identify functionally relevant cleavage site(s), predicted cleavage sites can be mutated using site-directed mutagenesis. Wildtype and mutant channels are compared in parallel experiments using oocytes from the same batch.
With this methodological approach it was demonstrated for the first time that proteolytic activation of ENaC-mediated whole-cell currents correlates with the time-dependent appearance of ENaC cleavage fragments at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, using site-directed mutagenesis of putative cleavage sites in combination with the two-electrode voltage-clamp technique, functionally relevant cleavage sites for plasmin, chymotrypsin13 and cathepsin-S8 were identified.

<table border="0" cellpadding="0" cellspacing="0" width="1446">
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			<td height="21" style="height:21px;">Bath Clamp Headstage for OC-725C-V</td>
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			<td>#SCOC150200EU</td>
			<td>brightness 4; mechanical aperture: D; color temperature: 3000 K</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">E Series Electrode Holder (Str, Vent, Ag Wire, 1.2 mm)</td>
			<td style="width:223px;">ADinstruments</td>
			<td>#ESW-F10v</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">left micromanipulator; MM-33L</td>
			<td style="width:223px;">Warner Instrument Corporation</td>
			<td>#64-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIH 1600 - computer interface</td>
			<td style="width:223px;">HEKA</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:644px;">magnetic valve system (ALA BPS-8) in combination with a TIB14 interface (HEKA)</td>
			<td style="width:223px;">ALA Scientific Instruments, HEKA</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OC-725C amplifier for two-electrode voltage-clamp recordings</td>
			<td style="width:223px;">Warner Instrument Corporation</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">P-97 FLAMING/BROWN Micropipette Puller</td>
			<td style="width:223px;">Sutter Instruments</td>
			<td style="width:119px;">-</td>
			<td>heat=550; velocity=22; time=200&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">right micromanipulator; MM-33R</td>
			<td style="width:223px;">Warner Instrument Corporation</td>
			<td>#64-0056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">Series Electrode Holder (45&#176;, Vent, Handle, Ag Wire, 1.2 mm)</td>
			<td style="width:223px;">ADinstruments</td>
			<td>#E45w-f10vh&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STAT 2 IV Gravity Flow Controller</td>
			<td style="width:223px;">Conmed</td>
			<td>#P-S2V-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">vacuum generator ejector SEG - for suction to remove bath solution</td>
			<td style="width:223px;">Schmalz</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1445px;">Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INFUJECT 60ml pump syringes for solutions</td>
			<td style="width:223px;">Braun</td>
			<td style="width:119px;">#22050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">Injekt-F for lysing the oocytes</td>
			<td style="width:223px;">Braun</td>
			<td style="width:119px;">#9166033V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">standard wall borosilicate tubing with filament&#160;</td>
			<td style="width:223px;">Sutter Instruments</td>
			<td>#BF150-86-10</td>
			<td>outside diameter: 1.50 mm; inside diameter: 0.86 mm; length: 10 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:223px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1445px;">Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">complete, Mini, EDTA-free protease inhibitor cocktail tablets</td>
			<td style="width:223px;">Roche Applied Science</td>
			<td style="width:119px;">#11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">EZ-Link Sulfo-NHS-Biotin</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">#21217</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horseradish peroxidase-labeled secondary goat anti-rabbit antibody&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>#sc-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">NeutrAvidin Agarose</td>
			<td>Thermo Scientific</td>
			<td>#29200</td>
			<td>Neutravidin-labeled agarose beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP40 (Nonidet P-40)</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">#I8896</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roti-Load 1 (2&#215; SDS-PAGE sample buffer)</td>
			<td>Carl Roth</td>
			<td>#K929.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperSignal West Femto Chemiluminescent Substrate for detection of chemiluminescent signals&#160;</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:119px;">#34095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:644px;">Triton-X-100</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td>#T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstration of proteolytic activation of the epithelial sodium channel (enac) by combining current measurements with detection of cleavage fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

To investigate whether the serine protease plasmin can activate ENaC-mediated currents, &#916;Iami of individual ENaC-expressing oocytes was determined before and after 30 min incubation of the oocytes in protease-free (control) (Figure 2A) or plasmin containing solution (Figure 2B) using the two-electrode voltage-clamp technique (see Figure 1). Exposure to plasmin increased &#916;Iami in every oocyte measured. In contrast, in control experiments, 30 min incubation of ENaC-expressing oocytes in protease-free solution had a negligible effect (Figure 2 C,D). Thus, by using this method a stimulation of ENaC-mediated current by plasmin can be detected.
To study the effects of mutating putative cleavage sites upon the activation of ENaC-mediated currents, as well as upon channel cleavage, the effect of chymotrypsin on WT-ENaC was compared with that on a mutant ENaC with mutated prostasin and plasmin cleavage sites (&#947;RKRK178AAAA;K189A). The time course of channel activation by chymotrypsin as well as the appearance of ENaC cleavage products at the cell surface was investigated by using different protease incubation times (Figure 4A). It was demonstrated that the mutant channel delays and reduces the activation of ENaC-mediated current by chymotrypsin. This is paralleled by a delayed appearance of a lower molecular weight &#947;ENaC cleavage fragment of 67 kDa corresponding to the fully cleaved subunit. Cleavage fragments were detected using a &#947;ENaC antibody directed against an epitope in the C-terminus (Figure 3). This methodological approach demonstrates that the time course of proteolytic activation of ENaC-mediated currents correlates with the appearance of a 67 kDa &#947;ENaC cleavage product at the cell surface (Figure 4 B,C). This supports the concept of a causal link between proteolytic channel cleavage and channel activation13. Moreover, by combining current measurements and the detection of &#947;ENaC fragments at the cell surface it was demonstrated that the mutated cleavage sites are functionally relevant for proteolytic channel activation.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51582/51582fig1highres.jpg" width="500" /> 
Figure 1. Procedure of determining the stimulatory effect of a protease on ENaC heterologously expressed in Xenopus laevis oocytes. ENaC activity is estimated by measuring the amiloride-sensitive whole-cell current component &#916;Iami.
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/51582/51582fig2highres.jpg" width="400" /> 
Figure 2. Plasmin stimulates ENaC-mediated currents in oocytes expressing ENaC. (A-D) Oocytes expressing human ENaC were incubated for 30 min in protease-free solution (control) or in solution containing plasmin (10 &#956;g/ml). To determine &#916;Iami before (-) and after (+) incubation, oocytes were clamped at a holding potential of -60 mV. (A,B) Four representative whole-cell current traces from one batch of oocytes. Amiloride (ami) was present in the bath solution to specifically inhibit ENaC as indicated by black bars. (C) Data points obtained from an individual oocyte are connected by a line. (D) Summary of similar experiments as shown in C. Columns represent relative stimulatory effect on &#916;Iami calculated as the ratio of &#916;Iami measured after a 30 min incubation (&#916;Iami 30 min) to the initial &#916;Iami (&#916;Iami initial) measured before incubation. Numbers inside the columns indicate the number of individual oocytes measured. N indicates the number of different batches of oocytes. (This figure has been modified from [Haerteis et al. 2012 J Gen Physiol 140, 375-389, doi:10.1085/jgp.201110763])
<img alt="Figure 3" fo:content-width="4in" src="/files/ftp_upload/51582/51582fig3highres.jpg" width="400" /> 
Figure 3. Model of the &#947;ENaC subunit showing cleavage sites for proteolytic activation and the binding site of the antibody used. Proteolytic cleavage by the Golgi-associated convertase furin is important for ENaC maturation in the biosynthetic pathway before the channel reaches the plasma membrane. After cleavage by furin a 76 kDa fragment can be detected at the cell surface using a biotinylation approach and an antibody against an epitope in the C-terminus of the &#947;-subunit. The pivotal final step in proteolytic ENaC activation probably takes place at the plasma membrane where &#947;ENaC is cleaved by extracellular proteases (e.g. plasmin or chymotrypsin) in a region distal to the furin site resulting in a 67 kDa cleavage fragment. (This figure has been modified from [Haerteis et al. 2012 J Gen Physiol 140, 375-389, doi:10.1085/jgp.201110763])
<img alt="Figure 4" fo:content-width="3in" src="/files/ftp_upload/51582/51582fig4highres.jpg" width="300" /> 
Figure 4: Mutating both the plasmin (K189) and the prostasin cleavage site (RKRK178) delays the activation of ENaC-mediated currents and the appearance of a 67 kDa cleavage product of the channel&#8217;s &#947;-subunit. Oocytes expressing WT (open symbols) and &#947;RKRK178AAAA;K189AENaC mutant channel (closed symbols) were incubated for 30 min in protease-free solution (control) or for 5, 30, or 60 min in a solution containing chymotrypsin (2 &#956;g/ml). (A) To determine &#916;Iami before and after incubation, oocytes were clamped at a holding potential of -60 mV. Circles represent the ratio of &#916;Iami measured after 5, 30, or 60 min incubation (&#916;Iami min) to the initial &#916;Iami (&#916;Iami initial) measured before incubation. Each data point represents the mean &#916;Iami measured in 22-24 individual oocytes of four different batches. (B-D) In parallel to the detection of &#916;Iami, expression of biotinylated &#947;ENaC at the cell surface was analyzed by SDS-PAGE. &#947;ENaC was detected with an antibody against an epitope in the C-terminus of human &#947;ENaC. Representative western blots from one batch of oocytes are shown. (C-E) Densitometric analysis of three western blots similar to those shown in B or D. For each lane, the signals detected in the regions of 76 kD (open columns) and 67 kD (gray columns) were determined and normalized to the sum of the total signal detected. N indicates the number of different batches of oocytes. <a href="https://www.jove.com/files/ftp_upload/51582/51582fig4highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments at JoVE.c

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

demonstration-proteolytic-activation-epithelial-sodium-channel-enac

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12.8K Views.  Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU). Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

Video: Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...