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.
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’s γ-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 (ΔIami) 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 γENaC 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 γENaC 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 γENaC 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.
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 (http://merops.sanger.ac.uk/) 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-β7 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 γENaC 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 (αβγ) 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 (ΔIami) can be separated from unspecific leak currents or from currents conducted by other ion channels. Thus, ΔIami 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, ΔIami is measured twice in the same oocyte, i.e. before and after incubation of the oocyte in a protease containing solution. An increase of ΔIami 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 ΔIami 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. γENaC cleavage fragments at the cell surface can be detected using a specific antibody directed against an epitope in the C-terminus of the γENaC. 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.
1. Isolation of Xenopus Oocytes and Microinjection of cRNA
OR2 | 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.4 with NaOH |
ND96 | 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.4 with Tris |
ND9 | 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 |
biotinylation buffer | 10 mM triethanolamine, 150 mM NaCl, 2 mM CaCl2, adjusted to pH 9.5 with HCl |
quench buffer | 192 mM glycin, 25 mM Tris-Cl, adjusted to pH 7.5 with HCl |
lysis buffer | 500 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl, adjusted to pH 7.4 with HCl |
Table 1. Solutions
2. Performing Two-electrode Voltage-clamp Experiments
3. Measurement of Amiloride-sensitive Whole-cell Currents
4. Biotinylation Assay
5. Detection of ENaC Cleavage Fragments at the Cell Surface by Western Blot Analysis
To investigate whether the serine protease plasmin can activate ENaC-mediated currents, ΔIami 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 ΔIami 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 (γRKRK178AAAA;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 γENaC cleavage fragment of 67 kDa corresponding to the fully cleaved subunit. Cleavage fragments were detected using a γENaC 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 γENaC 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 γENaC fragments at the cell surface it was demonstrated that the mutated cleavage sites are functionally relevant for proteolytic channel activation.
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 ΔIami.
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 μg/ml). To determine ΔIami 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 ΔIami calculated as the ratio of ΔIami measured after a 30 min incubation (ΔIami 30 min) to the initial ΔIami (ΔIami 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])
Figure 3. Model of the γENaC 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 γ-subunit. The pivotal final step in proteolytic ENaC activation probably takes place at the plasma membrane where γENaC 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])
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’s γ-subunit. Oocytes expressing WT (open symbols) and γRKRK178AAAA;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 μg/ml). (A) To determine ΔIami before and after incubation, oocytes were clamped at a holding potential of -60 mV. Circles represent the ratio of ΔIami measured after 5, 30, or 60 min incubation (ΔIami min) to the initial ΔIami (ΔIami initial) measured before incubation. Each data point represents the mean ΔIami measured in 22-24 individual oocytes of four different batches. (B-D) In parallel to the detection of ΔIami, expression of biotinylated γENaC at the cell surface was analyzed by SDS-PAGE. γENaC was detected with an antibody against an epitope in the C-terminus of human γENaC. 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. Click here to view larger image.
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).
The authors have nothing to disclose.
The expert technical assistance of Céline Grüninger, 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 Interdisziplinäres Zentrum für Klinische Forschung (to S. Haerteis and M. Krappitz), the ELAN program (to S. Haerteis) of the Friedrich-Alexander-Universität Erlangen-Nürnberg, and the University Library of Erlangen-Nürnberg.
Bath Clamp Headstage for OC-725C-V | Warner Instrument Corporation | – | |
Cold light source – Schott KL 1500 LCD | Schott | #SCOC150200EU | brightness 4; mechanical aperture: D; color temperature: 3000 K |
E Series Electrode Holder (Str, Vent, Ag Wire, 1.2 mm) | ADinstruments | #ESW-F10v | |
left micromanipulator; MM-33L | Warner Instrument Corporation | #64-0055 | |
LIH 1600 – computer interface | HEKA | – | |
magnetic valve system (ALA BPS-8) in combination with a TIB14 interface (HEKA) | ALA Scientific Instruments, HEKA | – | |
OC-725C amplifier for two-electrode voltage-clamp recordings | Warner Instrument Corporation | – | |
P-97 FLAMING/BROWN Micropipette Puller | Sutter Instruments | – | heat=550; velocity=22; time=200 |
right micromanipulator; MM-33R | Warner Instrument Corporation | #64-0056 | |
Series Electrode Holder (45°, Vent, Handle, Ag Wire, 1.2 mm) | ADinstruments | #E45w-f10vh | |
STAT 2 IV Gravity Flow Controller | Conmed | #P-S2V-60 | |
vacuum generator ejector SEG – for suction to remove bath solution | Schmalz | – | |
Material | |||
INFUJECT 60ml pump syringes for solutions | Braun | #22050 | |
Injekt-F for lysing the oocytes | Braun | #9166033V | |
standard wall borosilicate tubing with filament | Sutter Instruments | #BF150-86-10 | outside diameter: 1.50 mm; inside diameter: 0.86 mm; length: 10 cm |
Reagent | |||
complete, Mini, EDTA-free protease inhibitor cocktail tablets | Roche Applied Science | #11836170001 | |
EZ-Link Sulfo-NHS-Biotin | Thermo Scientific | #21217 | |
Horseradish peroxidase-labeled secondary goat anti-rabbit antibody | Santa Cruz Biotechnology | #sc-2004 | |
NeutrAvidin Agarose | Thermo Scientific | #29200 | Neutravidin-labeled agarose beads |
NP40 (Nonidet P-40) | Sigma-Aldrich | #I8896 | |
Roti-Load 1 (2× SDS-PAGE sample buffer) | Carl Roth | #K929.2 | |
SuperSignal West Femto Chemiluminescent Substrate for detection of chemiluminescent signals | Thermo Scientific | #34095 | |
Triton-X-100 | Sigma-Aldrich | #T8787 |