Expressing and Characterizing Mechanosensitive Channels in Xenopus Oocytes

Abstract

The oocytes of the African clawed frog (Xenopus laevis) comprise one of the most widely used membrane protein expression systems. While frequently used for studies of transporters and ion channels, the application of this system to the study of mechanosensitive ion channels has been overlooked, perhaps due to a relative abundance of native expression systems. Recent advances, however, have illustrated the advantages of the oocyte system for studying plant and bacterial mechanosensitive channels. Here we describe in detail the methods used for heterologous expression and characterization of bacterial and plant mechanosensitive channels in Xenopus oocytes.

1 Introduction

Oocytes of the African clawed frog (Xenopus laevis) have been used for several decades for the expression of transporters and receptors from bacteria [1], plants [2, 3], and mammals [4–6] due to the simplicity and reliability of this experimental system [7]. Relatively low background ion channel activity [8], large size (1–1.5 mm), low cost, and ease of handling make these cells a favored system for the expression of human, plant and bacterial channels. Xenopus oocytes have also been used as a model system for the study of processes unrelated to ion transport such as apoptosis [9, 10], gene expression [11], the structure and function of the nuclear envelope [12] and many others [13].

While most mechanosensitive channels (MSCs) have been characterized under native conditions [14–16], we have recently expressed and characterized two MSCs in Xenopus oocytes, MscS from Escherichia coli and MSL10 from Arabidopsis thaliana [17, 18]. Our analyses were made possible by previous studies showing that the endogenous MSCs known to be present in the oocyte membrane [8, 19, 20] can be effectively blocked or inactivated [21, 22]. A number of useful papers on frog maintenance [23], oocyte expression and transport systems [3, 24–28], oocyte isolation, and the methods used for characterization of ligand- and voltage-gated channels in the Xenopus oocyte system [29–33] have been published to date. Here we focus on the methods we used for the heterologous expression and characterization of MscS and MSL10 at the single-channel level. We describe in detail the techniques for surgery, oocyte isolation and preparation, in vitro RNA production and injection into oocytes, and basics of MSC electrophysiology, including patch-clamp and pressure-clamp.

2 Materials

Special attention must be paid to water quality at several stages in this procedure. The water used for surgery solutions should be the same water in which the frog is maintained in the animal facility. Water used in buffers for oocyte isolation and patch-clamp should be distilled and filter-sterilized (0.2 μm) or autoclaved. Water used for complimentary RNA (cRNA) production must be DNase- and RNase-free. All solutions (apart from those made right before the experiment or requiring freezer storage) should be kept in a refrigerator. Gentamycin-containing buffers should be stored together with oocytes at 16–18°C.

2.1 Frog Surgery

1. Anesthetic solution: 0.5-1 L of 1.5 g/L MS-222 (ethyl-3-aminobenzoate methanesulfonate) buffered with 840 mg/L NaHCO₃ and dissolved in pretreated water from the animal facility.

2. Complete N96 buffer: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM Hepes, pH 7.38.

3. Penicillin streptomycin (Pen Strep) solution (100× solution).

4. Sterile autoclaved forceps and scissors.

5. Sutures (2 pieces each of Monocryl undyed monofilament (Ethicon) and Ethilon black monofilament (Ethicon)).

6. Parafilm.

7. 50-ml conical tube with 20 ml of ND96 buffer.

8. Lab coat, gloves and eye protection.

9. 70 % ethanol or Lysol for benchtop sterilization.

2.2 Oocyte Isolation

1. Ca²⁺ -free N96 buffer: 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM Hepes, pH 7.38.

2. 0.1 g collagenase A type I, in 20 ml Ca²⁺ -free ND96.

3. 250 ml of 0.1 % BSA in Ca²⁺ -free ND96.

4. 30 ml of 50 mM KH₂PO₄/50 mM K₂HPO₄ with 0.01 % BSA in water, pH 6.5 (made in advance and stored as 45 mL aliquots at −20°C).

5. Complete ND96 buffer supplemented with 50 mg/ml gentamycin.

6. 50-ml conical tubes.

7. 100 mm × 15 mm petri dishes.

8. Plastic transfer pipettes (62 μl aperture, large bulb).

9. Incubator capable of maintaining 16–18°C (such as a wine cooler).

2.3 RNA Preparation

1. mMessage mMachine kit (Ambion).

2. RNase-free water.

3. RNase-free pipette tips and tubes.

4. Spectrophotometer suitable for measuring the concentration and purity of nucleic acids (e.g., NanoPhotometer, Implen).

2.4 Oocyte Injection

1. RNase-free water.

2. Mineral oil.

3. Gridded petri dish (a regular 35 mm petri dish with a polypropylene mesh glued to its bottom, see Fig. 1).

4. Capillary glass for an injection pipette. We use a thin-walled 8 in.-long pipette.

5. Micropipette or automatic injection system (see Fig. 2).

6. Pipette puller (P97, Sutter Instrument Co) (see ^{Note 1}).

7. RNase-free pipette tips.

8. 6-well tissue culture plates.

Fig. 1.

Image of a custom-made gridded petri dish. Plastic mesh is glued to the bottom of a 35-mm dish. Each cell of the grid accommodates a single oocyte

Fig. 2.

Setup for oocyte injection. A manually driven micropipettor (on the right) should be capable of reproducibly and accurately injecting 50 nl of solution into a single oocyte. Inset: RNAse-free water is sucked into the pipette from a drop in a petri dish

2.5 Patch-Clamp

1. 10 ml of hypertonic buffer: 200 mM K⁺ -aspartate, 20 mM KCl, 1 mM MgCl₂, 10 mM EGTA, 10 mM Hepes, pH 7.4.

2. 10 ml of ND96 with 10 mM of MgCl₂.

3. 10 ml of TEA-Cl buffer: 98 mM TEA-Cl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM Hepes, pH 7.38 adjusted with TEA-OH, 0.2 μm filtered.

4. Patch-clamp rig: amplifier (Axopatch 200B, Molecular Devices), digitizer (Digidata 1440A, Molecular Devices), micromanipulator (Patchstar, Scientifica) and (preferably inverted) microscope with 20× lens (see Figs. 3a and 4).

5. Pressure-clamp machine (ALA Scientific Instruments HSPC-1) (see ^{Note 2}).

6. Anti-vibration table (Technical Manufacturing Corporation, system 63–533) with a Faraday cage.

7. Noise filter/surge suppressor (Isobar 12 Ultra, Tripp-Lite).

8. Pipette puller (P97, Sutter Instrument Co).

9. Capillary glass for a patch-pipette. We use a 1.5 mm inner diameter, 1.8 mm outer diameter, 100 mm long borosilicate pipette (see ^{Note 3}).

Fig. 3.

Diagram of the experimental setup and typical results. (a) Schematic representation of the patch-clamp setup. (b) Illustration of a membrane patch with applied suction and a single MSC opening in response to increased membrane tension. (c) Representative trace showing five opening and closing MSL10 channels. A 50 mV command potential and a −120 mmHg symmetric pressure ramp were used. The trace was sampled at 20 kHz with a 5 kHz low-pass Bessel filter (Axopatch 200B), then filtered at 1 kHz and exported as graphics via Clampfit software (Molecular Devices)

Fig. 4.

Experimental setup, shown from two angles. The glass patch pipette (1) is located above the ground electrode (2) immersed into a petri dish filled with bath buffer (3). The microscope objective is located underneath. Also shown are the pressure-clamp headstage (4), the pipette holder (5), the common ground point (6), and the inverted microscope condenser (7)

3 Methods

Surgery is not performed until cRNA has been made and quantified (see Subheading 3.3). This prevents unnecessary surgeries. The surgery and oocyte isolation occur on the same day and injections are done the following day. Our protocol for surgery and oocyte isolation is based on [34].

3.1 Frog Surgery

1. Immerse a frog in the MS-222/NaHCO₃ solution for 10–15 min, until she does not respond to pinching of the skin between the toes. 13 min is optimal in our hands.

2. Rinse the frog with pretreated animal facility water to remove the anesthetic from its skin.

3. Put the frog on a wet paper towel and perform the surgery with sterile instruments, on a benchtop sterilized by spraying with 70 % ethanol, using sterile gloves. Keep the frog moist with sterile ND96 solution (see ^{Note 4}).

4. Make a small incision through the skin of the abdomen, using sharp scissors, followed by an incision of about 1 cm in the muscle layer. Rinse with ND96. (If the skin is slippery, grasp it with two fingers through a piece of clean paper towel). Place a sheet of Parafilm with a square cut in the middle over the incision, so that oocytes extracted from the abdomen will not come in contact with the surface of the frog’s skin.

5. Pull 2–4 ml of oocytes out of the body cavity with dull forceps and place in 25 ml of ND96 in a 50 ml conical tube.

6. Repair the incision with two sets of sutures, one for the muscle tissue (undyed) and one for skin (black). Each stitch is made with two loops of single knots and a third loop of double knots (three stitches) per 1–1.5 cm incision. The last knot should be pulled tight, and the ends trimmed closely.

7. Wash the frog with 0.5 L of clean water taken from the facility in order to get rid of any blood and anesthetic on her skin.

8. Allow the frog to recover in 0.5 L of Pen Strep solution (5 ml of 100× Pen Strep stock in 0.5 L of treated water from the frog facility) for 40–60 min. Tilt the plastic bath 45° from the vertical so that most of the frog’s body is submerged in the buffer. Care must be taken that the frog does not drown (keep her nostrils above the water level) but her skin stays wet. When she starts to move in response to pinching, she’s almost recovered. Once the frog recovers, return her to a clean cage with fresh water.

9. Depending on institutional rules, a frog could be operated on up to 5 times, with the last operation terminal. The operations should be 4–6 weeks apart and alternating sides of the abdomen used. After the last operation the frog may be euthanized according to the rules of your institution (see ^{Note 5}).

3.2 Oocyte Isolation

1. Separate the ovarian tissue into clumps of 10–20 oocytes each in a petri dish, using forceps and working under a stereomicroscope. Transfer the clumps into Ca²⁺ -free ND96. Try to avoid damage to the oocytes.

2. Incubate the oocytes in a petri dish with slow shaking for 25–40 min at room temperature in 20 ml of Ca²⁺ -free ND96 with collagenase. Ca²⁺ -free medium is used to slow down the collagenase reaction as Ca²⁺ strongly activates collagenase [35]. Stop the process as soon as single oocytes are released.

3. Wash the oocytes 5 times with 25 ml ND96 + 0.1 % BSA in a 50 ml conical tube.

4. Add 25 ml 0.1 M potassium phosphate pH 6.5 buffer and incubate for 40–50 min on a slow shaker. At 15-min intervals, draw the oocytes into a wide-bore plastic pipette (made by trimming the tip) and pipette up and down to facilitate dispersal.

5. Wash the oocytes 5 times with 25 ml ND96 + 0.1 % BSA in a 50 ml conical tube.

6. Sort the oocytes in a petri dish under the microscope, selecting only mature oocytes (Dumont stage V-VI, [36]) with clear animal–vegetal pole divisions and without any spots, markings or residual ovarian tissue on the surface. Manipulate single oocytes with plastic transfer pipettes (see Subheading 2). Store the selected oocytes in complete ND96 with 50 mg/ml gentamycin at 16–18°C.

3.3 RNA Preparation

The sequence of the RNA of interest should first be cloned into an expression vector appropriate for cRNA production and its translation in oocytes. We use pOO2, a vector that allows cloning of a cDNA in between the 5′- and 3′-untranslated β-globin sequences from Xenopus [37].

1. Linearize approximately 5 μg of template DNA in a 70 μl reaction. Use a restriction enzyme that recognizes a single site in the vector downstream of the sequence to be transcribed (see ^{Note 6}).

2. Run 4 μl of the sample on a gel to ensure linearity.

3. Add 5 μl 0.5 M EDTA, 4 μl 5 M NaCl, 25 μl RNase-free water and mix well.

4. Add 200 μl 100 % ethanol.

5. Mix well and place at −20°C for at least 20 min.

6. Spin for 15 min on a benchtop centrifuge at top speed.

7. Carefully remove supernatant and dry the pellet.

8. Resuspend the pellet in 25 μl RNase-free water and calculate the concentration of DNA by measuring the OD₂₆₀.

9. Follow the protocol supplied with Ambion mMessage mMachine kit to produce the cRNA of interest (see ^{Note 7}).

10. Aliquot the cRNA solution (it should be approximately 1 μg/μl) into 3 μl aliquots and store at −20°C.

3.4 Oocyte Injection

1. Place 10–40 oocytes into a gridded petri dish (see Fig. 1) filled with complete ND96. Place each oocyte into a square of the plastic grid with the light side (vegetal pole) up.

2. Thaw RNase-free water and cRNA on ice.

3. Pull long needles from 8 in. capillary glass on a puller. For a Sutter P-97 the following program may be a good starting point: heat = 560, pull = none, velocity = 120, time = 100. Trim the end of each needle with scissors 5–6 mm before the point where the pulled portion of the pipette becomes flexible.

4. Backfill the needle with 13–15 μl of mineral oil using a micropipette, then apply the needle to the pipette or injector apparatus. Make sure there are no bubbles in the oil, then pull approximately 500 nl of RNase-free water into the tip from a drop placed on a petri dish, followed by all 3 μl of your cRNA sample (see ^{Note 8}).

5. Using a microinjector or a micropipette attached to the glass needle (see Fig. 2), inject 50 nl of the cRNA solution into each oocyte, preferably into the light side and close to the animal/vegetative border (see ^{Note 9}).

6. Repeat steps 4 and 5 above with fresh oocytes and RNase-free water as a negative control.

7. Place the oocytes into a 6-well plate filled with complete ND96 with gentamycin.

8. Change the solution and remove defective oocytes on a daily basis until analysis (see ^{Notes 10 and 11}).

3.5 Patch-Clamp

The microscope stage setup, including a dish with bath medium, command and ground electrodes, patch pipette and a pressure clamp headstage, is presented in Figs. 3a (schematic view) and 4 (actual setup).

1. Conventional single-channel patch-clamp is carried out using pipettes with BN (“bubble number”, see ref. 38) of 4–7 in symmetric and asymmetric buffers.

2. Prior to patching, the transparent vitelline membrane of each oocyte should be removed with a pair of sharp forceps after 5–10 min incubation in hypertonic buffer, which makes the cell shrink and the vitelline membrane more visible (see ^{Note 12}). After removal of the vitelline membrane, the oocyte becomes very fragile, so it should not be exposed to the surface of the buffer and care should be taken when moving it. We suggest removing the vitelline membrane in the petri dish to be used for patch-clamp, already filled with patching bath buffer.

3. A patch-pipette should be fabricated using a pipette puller (see appropriate manual for measuring glass melting temperature and initial proxy settings that can be adjusted to get the pipette of desired size and shape). Typically, a pipette with tip size between 0.5 and 2 µm is required, which corresponds to BN 4–6 and a resistance of several MΩ, depending on the tips’ length and geometry.

4. The command (or pipette) electrode is a small piece of Ag wire that has been chlorinated either by immersion into 6 % bleach for 15–25 min or by subjecting it to proper potential in KCl buffer [ 39 ].

5. The ground electrode is an Ag/AgCl pellet, connected to the ground input of the amplifier’s headstage through the common ground (such as a Faraday cage or anti-vibration table) via Ag wire (see ^{Note 13}, Figs. 3a and 4). It does not matter where the electrode is grounded, only that everything metal be grounded at a single common point, with a stable electrical contact between the wires and grounding point. This can be tested with a standard multimeter. The ground electrode could be immersed into the bath solution directly or via an agar salt bridge (see ^{Note 14}). The Ag/AgCl pellet should be washed with distilled water after experiments are completed.

6. Back-fill the patch-pipette with buffer, making sure it is free of air bubbles. If bubbles form, they can sometimes be eliminated by tapping the pipette. Bubbles can be prevented in the first place by pulling some buffer into the empty pipette with a syringe before back-filling the rest of the pipette.

7. Before and during immersion of the pipette into the bath buffer, a positive pressure of 20–30 mmHg should be applied to it. After the pipette tip is immersed, the pressure may be lowered to 5–10 mmHg (see ^{Note 15}).

8. Bring the pipette tip close to the oocyte membrane. The state of the pipette tip should be monitored by periodic application of low amplitude square voltage pulses of approximately 5 mV to the command electrode. In Axopatch amplifiers, use the “seal test mode”, where steps are gated by the amplifier’s internal line frequency oscillator. If the tip resistance grows (the current decreases) before the tip touches the oocyte membrane, replace the pipette, as its tip is likely obstructed with debris.

9. The moment of touching the oocyte membrane is observed electrically as a decrease of the measured current. When it decreases to 2/3–1/2 of its initial value, stop pipette motion and immediately decrease the pressure in the pipette from slightly positive to about −10 mmHg.

10. Observe the gradual formation of the “gigaseal” between the membrane and the pipette as the resistance of the pipettemembrane contact increases to 1 GΩ or more (see ^{Note 16}). After a gigaseal is reached you have obtained the cell-attached configuration (see ^{Note 17}). Next, bring the pipette pressure to between −1 and −3 mm Hg.

11. Excise the patch by rapid removal of the pipette away from the oocyte membrane. If the patch is not stable, this may result in patch rupture. Alternatively a membrane vesicle may form in the pipette tip and block it (usually when the pipette is not moved away from the cell quickly enough, see ^{Note 18}).

12. Once the patch is in the “excised” (in this particular case, “inside-out”) patch configuration (see ^{Notes 19 and 20} and Fig. 3b), one can proceed to stimulation of MSCs. We recommend using a fixed transmembrane voltage of −20 to −40 mV and applying triangular 4- or 5-second-long symmetric ramps of negative pressure to the patch pipette, allowing for the gradual opening and closing of individual MSCs (see ^{Note 21}). A typical trace obtained from the membrane patch of an oocyte expressing MSL10 from A. thaliana [18] is presented in Fig. 3c for reference.

13. Once a tension-induced channel activity is detected and recorded, be sure to repeat patch excision and current measurement on water-injected oocytes to ensure the absence of artifacts or unusually high endogenous channel activity.

14. We have observed that the localization of the channel usually is not uniform within the oocyte membrane and several patches from the same cell may be necessary to locate channel activity, especially if a small pipette diameter is used.

15. In our hands, the endogenous cation-selective MSCs that are often observed in the cell-attached configuration inactivate upon patch excision, especially in the presence of elevated MgCl₂ or in TEA-Cl buffer (see ^{Notes 22–24}).

16. The rare appearance of an endogenous, large-conductance Ca²⁺ -activated chloride channel (see ref. 8) should also be taken into account (see ^{Note 25}). However, it usually does not present a serious problem as long as small patch-pipettes are used, due to its relatively low expression.

17. A good starting point for recording settings would include the following: (1) acquisition at 20 kHz with the amplifier’s low pass filter set to 5–10 kHz (depending on the available settings); (2) a 4–5 s pressure ramp of −20 to −80 mmHg (depending on the pipette size, see ^{Note 26}), which in most cases eliminates artifacts caused by a too rapid increase in tension; and (3) a holding potential of 20 mV to 40 mV (pipette voltage) for the inside-out configuration, which typically provides clean and artifact-free traces (see ^{Notes 26 and 27}).

18. Once several channels are activated in the patch, one may study the channel’s voltage dependence. To do so, repeat the measurement with the same transmembrane pressure but at various potentials (see ^{Note 28}).

19. Pressure may be gradually increased by increments of −10 to −40 mmHg in order to activate more channels in the patch and obtain a dose-response curve (see ^{Note 29}).

20. For additional recommendations on data collection and statistical analysis, see ^{Notes 30 and 31}.

Fig. 5.

A laser-scanning confocal micrograph of the fluorescent signal from the periphery of an oocyte RNA 7 days after injection with MscS-GFP cRNA. An oocyte in ND96 buffer was placed onto a glass slide with concaved bottom and covered with a coverslip. Under bright field the lens was focused on the edge of the oocyte, then the confocal scan with GFP filters (488/510 nm) setup was performed. Inset: oocyte image in bright field