Microtransplantation of Neurotransmitter Receptors From Cells to Xenopus Oocyte Membranes

New Procedure for Ion Channel Studies Ricardo Miledi, Eleonora Palma, and Fabrizio Eusebi

Summary

The Xenopus oocyte is largely used as a cell expression system for studying both structure and function of transmitter receptors and ion channels. Messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional ion channels. A new method was developed further to transplant neurotransmitter receptors from human brain or cultured cell lines to the membrane of Xenopus oocytes. This method represents a modification of the method used many years ago of injecting into oocytes membrane vesicles from Torpedo electroplaques, yielding the expression of functional Torpedo acetylcholine receptors. We describe this approach by extracting membrane vesicles from human hippocampus or temporal neocortex and from mammalian cell lines stably expressing glutamate or neuronal nicotinic receptors. Because the human neurotransmitter receptors are "microtransplanted" with their native cell membranes, this method extends the usefulness of Xenopus oocytes as an expression system for addressing issues in many fields, including channelopathies.

Key Words: Epilepsy; human brain tissues; transfected cell lines; voltage clamp; Xenopus oocytes.

1. Introduction

The heterologous expression of ligand-gated and voltage-gated ion channels in the oolemma of Xenopus oocytes is regarded as one of the most powerful and convenient tools available to study the properties of receptors and ion channels. This experimental approach is particularly useful when the native cells (i.e., human nerve cells) are not easily accessible for extensive investigations.

Two methods with similar efficacy may be used to express ion channels in the oocyte membrane. The first method consists of either cytoplasmic injections into Xenopus oocytes with poly(A+) messenger RNAs (mRNAs) extracted from native tissues (1-4) or intranuclear injections of complementary DNAs (cDNAs) encoding for

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

ion channels (5-7). The second method consists of injecting into the oocyte membrane vesicles extracted from the native tissue (8-11). A detailed description of the first method is beyond the scope of this chapter because many reviews have already addressed this issue (1,3-6,12-14).

This chapter describes the method of membrane transplantation from cells to oocytes, as used particularly for cultured cells. This procedure is relatively new and overcomes some of the problems associated with oocyte injections of mRNAs or cDNAs. For example, with the latter procedure, the proteins are synthesized and posttranslationally processed by the oocyte's own machinery, and the receptors are assembled in oocyte membranes. In contrast, when the foreign membranes are injected into the oocyte, the original ion channels, still embedded in their native cell membrane, are transplanted to the oocyte plasma membrane (see Note 1).

2. Materials

1. Anesthetic (2% 3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO).

2. Xenopus laevis frogs.

3. Oocytes ringer (OR): 82.5 mM NaCl, 2.5 mM KCl, 5 mM HEPES, pH 7.4. To prepare 1 L of 10-fold concentrated OR (OR 10X) without Ca2+ and Mg2+, weigh 48.2 g NaCl, 1.86 g KCl, 11.9 g HEPES, pH 7.2 with 5M NaOH, add 5 mL phenol red. Store at 4°C and use within 3 to 4 wk. To make 1 L of the final OR, take 100 mL OR 10X, add 1 mL 1 M MgCl2 and 2.5 mL 1 M CaCl2, complete to 1 L with water, check pH, and store at 18°C.

4. Sterile filter system for oocyte Ringer (Corning, NY).

5. Collagenase type I (Sigma).

6. Stereomicroscope.

7. Spinner flask (100 mL; Bellco Glass, NJ).

8. Barth's modified saline solution: 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2. Prepare 500 mL of Barth's 10X stock solution. Weigh 25.67 g NaCl, 0.37 g KCl, 1 g NaHCO3, 11.9 g HEPES, 1 g MgSO4-7 H2O, 0.39 g Ca(NO3)2, 0.3 g CaClr2 H2O at pH 7.2 with 5 MNaOH. Filter and store at 4°C in sterilized glass bottle. To prepare 500 mL Barth's medium for experiments, take 50 mL Barth's 10X, add 5 mL penicillin/streptomycin and 1 mL kanamycin solution (100 U/100 |lg penicillin/streptomycin and 100 |lg of kanamycin solution), complete to 500 mL with water, and store at 18°C.

9. Antibiotics: 100 U/100 |g penicillin/streptomycin; 100 |g kanamycin solution (Gibco-BRL).

10. Glycine buffer: 200 mM glycine, 150 mM NaCl, 50 mM ethyleneglycolbistetraacetic acid (EGTA), 50 mM ethylenediaminetetraacetic acid (EDTA), 300 mM sucrose plus protease inhibitor solution (100 |L/10 mL homogenized tissue). Prepare 20 mL glycine buffer: solution made with 4 mL 1 M glycine, 0.6 mL 5 M NaCl, 5 mL 0.2 M EGTA, 2 mL 0.5 M EDTA, 6 mL 1 M sucrose, 2.4 mL water at pH 9.0 with NaOH. Keep in 5-mL aliquots at 4°C.

11. Protease inhibitor: for stock solution, protease inhibitor cocktail (Sigma P2714) in 100 mL water; keep 5-mL aliquots at -20°C.

12. Homogenizer (Ultra-Turrex T8; metal tip S8N-5G and S8N-8G; IKA, Staufen, Germany).

13. Ultracentrifuge (Beckmann centrifuge with F 241.5 rotor, CA).

14. Assay buffer: 20 mL 5 mM glycine; keep 5-mL aliquots at 4°C.

15. BCA Protein Assay Reagents Kit (Pierce, Rockford, IL).

16. Vertical micropipet puller.

17. Microinjection apparatus (Harvard Apparatus, www.harvardapparatus.com; 6.66-|lL glass micropipets, Drummond, www.drummondsci.com; micromanipulator, Singer Instruments, Roadwater, UK).

18. Frog ringer: 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM HEPES. To prepare 1 L of solution, weigh 6.72 g NaCl, 0.149 g KCl, 0.265 g CaClr2 H2O at pH 7.2 with 1 M NaOH and store at 18 °C.

19. Microelectrode solution (3 M KCl) stored in a sterile bottle at 4°C.

20. 1 M CaCl2 and 1 M MgCl2 stock solutions (stored at 4°C in sterile glass bottles).

21. Micromanipulators for voltage clamp recording (Leica, Wetzlar, Germany).

22. Voltage clamp amplifier (GeneClamp 500B; Axon Instruments).

23. Recording chamber (Automate Scientific, CA).

24. Perfusion system (Biologique RSC-200; Claix, France) (see Note 2).

3. Methods

The methods described here outline the procedures for: (1) oocyte preparation and the expression of ligand-gated and voltage-gated channels in Xenopus oocytes following the extraction of membranes from either (2) native brain tissue or (3) cell lines; (4) membrane cytoplasmatic injections into oocytes; and (5) voltage clamp recordings from oocytes.

3.1. Preparation of Xenopus Oocytes

Preparation of oocytes are detailed elsewhere (7,15,16) and are summarized here. Adult female frogs can be obtained from commercial suppliers. The steps for preparing oocytes are as follows:

1. Anesthetize the frog with 0.2% 3-aminobenzoic acid ethyl ester and sacrifice it by decapitation.

2. Cut part of the ovary into small pieces using a razor blade and thin forceps and place the segments in OR solution without CaCl2.

3. Weigh 0.1 g of collagenase type I, and dissolve in 50 mL OR solution.

4. Add the collagenase solution and place the pieces of ovary in 100-mL spinner flask under slow agitation at 18°C for 2 to 3 h.

5. Check visually if the oocytes are isolated and defolliculated and wash three or four times with OR plus 2.5 mM CaCl2 or frog Ringer.

6. Transfer the oocytes in Barth's modified saline solution plus antibiotics and incubate at 18°C.

7. Inject oocytes with membrane preparation 12 to 24 h after collagenase treatment (procedures for injections are described in Subheading 3.4.).

3.2. Membrane Preparation From Mammalian Brain Tissues

The general method of membrane preparation has been described and used to extract membranes from the electric organ of Torpedo and the rat and human brains (8-10,17). The following protocol describes the procedures we have used for isolating membranes from, with minor modifications, (1) human temporal lobe (TL) and hippocampus of medically intractable epileptic patients who underwent surgical TL resection; (2) TL of patients affected by oligodendroglioma; (3) TL of P60 DBA mice; and (4) the cortex of P0-P12 BDF1 mice. Figure 1 is a diagram illustrating the main steps used to obtain nervous tissue membranes for injection into oocytes. Figure 2

Fig. 1. Neurotransmitter receptors from the human brain microtransplanted to the Xenopus oocyte plasma membrane to study their functional properties. Two alternative procedures can be used to incorporate receptors in the oolemma: (1) injecting the oocytes with brain cell membranes (as shown) or (2) by injecting mRNA extracted from surgically removed brain tissues.

Fig. 1. Neurotransmitter receptors from the human brain microtransplanted to the Xenopus oocyte plasma membrane to study their functional properties. Two alternative procedures can be used to incorporate receptors in the oolemma: (1) injecting the oocytes with brain cell membranes (as shown) or (2) by injecting mRNA extracted from surgically removed brain tissues.

shows examples of responses obtained with this method. The steps for isolating membranes are as follows:

1. Prepare 4 mL glycine buffer by adding 40 ||L protease inhibitor stock solution immediately before use.

2. Clean the metal tip (S8N-8G; IKA, Germany) of the homogenizer by rinsing twice with ethanol (EtOH) 70% and water.

3. Homogenize the tissue in a 50-mL Falcon tube with 4 mL glycine buffer. Take care to keep the Falcon tube on ice during the homogenization (see Note 3).

4. Transfer the filtrate to 1.5-mL vials and centrifuge at 9500g for 15 min at 4°C.

5. Centrifuge the supernatant at 100,000g for 2 h at 4°C in a Beckmann centrifuge TL-100 (rotor TLA-100).

6. Withdraw the supernatant.

7. Wash the pellet twice with water.

8. Resuspend the pellet in 400 |L assay buffer (5 mM glycine). Use when necessary an Eppendorf micropipet with a 1-mL plastic tip cut to enlarge three or four times the original opening to facilitate resuspension. Take 1 |L sample and measure the amount of total proteins using a standard BCA Protein Assay Reagent Kit.

9. Prepare 10- to 50-|L aliquots to use directly and to store frozen at -80°C for later use.

3.3. Membrane Preparation From Cell Lines

The procedure described next has been used to prepare membranes from the following cell lines: (1) human HEK (human embryonic kidney) stably expressing functional rat homomeric aminohydroxymethyl-isoxazole-proprionic acid (AMPA)-type glutamate receptor type 1 (GluR1); (2) human HEK stably expressing functional human neuronal nicotinic acetylcholine receptor a4p2 (AChR-a4p2); (3) rat GH4-C1

Fig. 2. Examples of currents evoked by different neurotransmitters (as indicated) in Xeno-pus oocytes injected with membranes (A), or mRNA (B) extracted from human mesial temporal lobe epilepsy (MTLE), or with membranes extracted from human oligondendroglioma (C). The responses to AMPA were evoked in the presence of 50 |lM cyclothiazide. GABA, y-amino-butyric acid. KAI, kainic acid.

Fig. 2. Examples of currents evoked by different neurotransmitters (as indicated) in Xeno-pus oocytes injected with membranes (A), or mRNA (B) extracted from human mesial temporal lobe epilepsy (MTLE), or with membranes extracted from human oligondendroglioma (C). The responses to AMPA were evoked in the presence of 50 |lM cyclothiazide. GABA, y-amino-butyric acid. KAI, kainic acid.

Fig. 3. Examples of currents evoked by acetylcholine (ACh) and AMPA as indicated, from Xenopus oocytes injected with membranes extracted from left, GH(4)C1 cells stably trans-fected with human a7-nAChR subunit; middle, HEK293 cells stably transfected with human a4- and P2-nAChR subunits; right, HEK293 cells stably transfected with rat flip variant of glutamate type 1 receptor subunit. The AMPA response was evoked as in Fig. 2.

Fig. 3. Examples of currents evoked by acetylcholine (ACh) and AMPA as indicated, from Xenopus oocytes injected with membranes extracted from left, GH(4)C1 cells stably trans-fected with human a7-nAChR subunit; middle, HEK293 cells stably transfected with human a4- and P2-nAChR subunits; right, HEK293 cells stably transfected with rat flip variant of glutamate type 1 receptor subunit. The AMPA response was evoked as in Fig. 2.

pituitary cells expressing functional human neuronal a7-AChR (see Fig. 3 and ref. 11). The steps for extracting membranes are as follows:

1. Collect 1 to 8 x 108 cells in 2 mL glycine buffer by scraping the cells.

2. Homogenize the cells in a 15-mL Falcon tube using the Ultra-Turrex T8 homogenizer with a small metal tip (S8N-5G; IKA, Germany) to match the small amount of cells (see Note 4).

3. Follow the protocol described in Subheading 3.2., steps 4 to 7.

4. Resuspend the pellet in 200 ||L assay buffer and, after measuring the protein concentration (10-15 mg/mL), use directly or store 5-|L aliquots at -80°C for subsequent use.

3.4. Cytoplasmatic Injection Into Oocytes

For membrane injection, it is convenient to use only oocytes at stages V-VI of development, with a 1- to 1.5-mm diameter, and with a tough membrane. The steps for cytoplasmatic injection are as follows:

1. Pull the glass micropipets (6.66 |L) for the injection using a standard puller.

2. Break slightly the micropipet tip under a stereomicroscope (20x; Zeiss, Germany) and insert the micropipet in a standard holder connected to a pressure microinjector.

3. Fill the glass micropipet with 4 to 5 | L membrane samples placed on a clean piece of parafilm.

4. Calibrate the injector according to the diameter of the glass micropipet, setting both duration and injecting pressure.

5. Inject each oocyte with about 100 nL membrane preparation (1-2 mg proteins/mL; see Notes 5 and 6).

6. After the injection, maintain the oocytes in Barth's solution at 18°C until the electro-physiological recordings are performed.

3.5. Voltage Clamp Recordings From Oocytes

Full-size currents are recorded about 12 h after the membrane injections. Membrane currents are recorded using two conventional voltage clamp electrodes as previously reviewed (5,6,14,15,18). One electrode is used to record the transmembrane potential of the oocyte. The amplifier compares the resting potential recorded by the voltage electrode to the desired holding potential, and the current is injected into the oocytes from the second electrode to keep the potential stable at this value. Both the electrodes are filled with 3 M KCl (19). The steps for voltage clamp recordings are as follows:

1. Prepare glass microelectrodes with filament, pulling them with a single-step vertical pipet puller. Bend the electrodes with a Bunsen flame to get electrodes more stably into the oocyte.

2. Transfer the oocyte to the recording chamber using a blunt and fire-polished Pasteur pipet.

3. Plunge the voltage and current electrode tips in the solution of the recording chamber and check their resistance (see Note 7).

4. Insert the voltage-recording electrode into the oocyte to measure its resting potential, then insert the passing current electrode. The resting potential of healthy membrane-injected oocytes ranges from -25 to -70 mV. After inserting the microelectrodes, there is a drop in membrane potential, which usually recovers after superfusing with the standard solution (OR) for a few minutes.

5. Superfuse with the standard solution.

6. Shift to voltage clamp configuration and hold the oocyte at -60 mV. Measure the transmembrane resistance of the oocyte (best conditions at 1- to 5-MQ resistance).

7. Start recording membrane currents (Fig. 2; see Note 8).

4. Notes

1. The method of transplantation of receptors by injecting membranes is very powerful and has some advantages over the expression of receptors by mRNA injections, such as: (1) faster appearance of functional receptors in the oocyte plasma membrane; (2) problems with ribonuclease contamination are avoided; (3) small amounts of tissue are used to obtain abundant membrane preparations; (4) the same aliquots of membrane preparation can be used after thawing and freezing without much precaution. However, the main advantage is that the receptors are the original ones already assembled and in their original cell membranes.

2. Solution exchange is conveniently achieved using electromagnetic valves and a computer-controlled perfusion system consisting of 18 syringes (50 mL) connected with polythene tubing to 18 valves mounted directly on the Faraday cage and grounded. The aspiration of the solution from the recording chamber is achieved using a small plastic tube connected to a vacuum through an air pump for an aquarium in which the flux of the air has been inverted.

3. The amount of tissue to be used for membrane extraction can be variable. Its wet weight may be as low as 0.2 g to produce a membrane pellet suitable for many experiments.

4. Convenient membrane preparations from cultured cells are obtained using more than 108 cells.

5. It is easier and faster to inject oocytes using a dark background, such as a black plastic chamber.

6. If the membrane preparation is too viscous or dishomogeneous at visual inspection, it is necessary to sonicate the sample for 10 s before the injection.

7. Optimal electrode resistance is between 0.5 and 2 MQ.

8. There is usually large variability in the number of receptors transplanted and consequently in the values of the recorded transmembrane currents. To reduce this variability, we recommend sonicating the membrane preparation just before injection and to inject in the equatorial region.

Acknowledgments

We thank Zulma Duenas and Flavia Trettel for help and discussion. This work was supported in part by FIRB and COFIN grants from MIUR (to F. E.) and NSF (Neuronal and Glial Mechanisms; to R. M.).

References

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