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[24] E x p r e s s i o n o f G a p J u n c t i o n a l P r o t e i n s in X e n o p u s Oocyte Pairs B y LISA EBIHARA

Introduction The study of gap junctional channels poses a unique problem not encountered with other ionic channels in that the formation of functional gap junctional channels requires the close apposition of two cell membranes. To overcome this difficulty, Dahl and associates 1,2 studied electrical coupling between pairs of "stripped" Xenopus oocytes which had been injected with mRNA isolated from estrogen-induced rat myometrium or mRNA synthesized in vitro from gap junction cDNA. The main advantage of this system is that it provides a rapid method for assessing the function of normal and altered junctional proteins. In addition, it is possible to visualize the distribution of the junctional protein within the cell as a function of time following mRNA injection using antibodies for the protein. 3 One disadvantage is that it does not allow the study of single gap junctional channels. For such studies, it would be advantageous to stably integrate the gene into a mammalian cell line which has few or no endogenous gap junctional channels such as the human cell line SKHepl.4 Alternatively, the channel protein could be overexpressed in bacteria and reconstituted into planar bilayers. Methods

In VitroSynthesis of Messenger RNA The connexin cDNAs were separately subeloned into the transcription vector SP64T. 5 This vector contains a cloning site between the 5' and 3' noncoding of regions of p-hemoglobin and has been reported to increase the efficiency of translation of some cloned cDNAs for gap junctional proteins? The DNA is linearized using an appropriate restriction enzyme

G. Dahl, T. Miller, D. Paul, R. Voellmy, and R. Werner, Science 236, 1290 (1987). 2 R. Werner, T. Miller, R. Azarnia, and G. Dahl, J. Membr. Biol. 87, 253 (1985). 3 K. I. Swenson, J. R. Jordan, E. C. Beyer, and D. L. Paul, Cell (Cambridge, Mass.) 53, 145 (1989). 4 A. P. Moreno, B. Eghabali, and D. C. Spray, Biophys. J. 236, 243a (1990). 5 p. A. Krieg and D. A. Melton, Nucleic Acids Res. 12, 7057 (1984).

METHODS IN ENZYMOLOGY. VOL. 207

Copyrisht© 1992by AcademicPress,Inc. All fishtsof ~.im3ductionin any form reserved.

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and transcribed with SP6 RNA polyrnerase.6 The methylated cap 7mGppNP is added to the transcription buffer to produce capped mRNAs. 7

Preparation of Oocytes Female Xenopus laevis frogs are anesthetized with tricaine methyl sulfonate and small pieces of ovary removed through an incision in the abdomen. The pieces of ovary are stored in sterile modified Barth's solution (MB) containing (in mM) 88 NaC1, 1 KC1, 2.4 NaHCO3, 15 Hepes, 0.3 CAN03, 0.41 CaC12, 0.82 MgSO4, 50 rag/1 Gentamicin, pH 7.4. Only stage V - V I oocytes are used. The follicular cell layer is removed by incubating small clumps of oocytes in MB without calcium or magnesium and containing 20 mg/ml collagenase (Worthington Freehold, NJ) for 30-45 min. The oocytes are then transferred to another dish containing MB without calcium or magnesium, and the follicular cell envelope is manually removed. After defolliculation, the oocytes are washed 3 times in MB with calcium and injected with 40-50 nl of mRNA dissolved in autoclaved water (20 ng/ml). The oocytes are then incubated overnight in MB solution at 18 ° in order to allow time for protein synthesis. The time course of protein accumulation has been measured by Swenson et aL,3 who were able to detect induced junctional proteins above the background of endogenous proteins within 6 hr after injection. The following day the oocytes are devitellinized by first incubating the oocytes in a hypertonic stripping solution (200 m M potassium aspartate, 20 m M KC1, 1 m M MgC12, 10 m M EGTA, 10 m M HEPES, pH 7.4) for 5- 30 rain until the vitelline membrane comes away from plasma membrane, s The vitelline membrane is then removed with a pair of fine forceps. The devitellinized oocytes are fragile and will lyse if exposed to an airwater interface. They are gently transferred to another dish and manipulated together into pairs. The transfer pipettes consist of either a Pipetman with a large orifice tip (USA Scientific, Ocala, FL) or a glass capillary tube with an inner diameter just larger than an oocyte in a pipetting device. After pairing, the oocytes tend to roll apart unless they are mechanically held in contact with each other. To keep them together, oocyte pairs are wedged between two strips of Parafilm separated by a 2 mm space, which are affixed onto the bottom of the dish. Gap junctions will typically form 6 D. A. Melton, P. A. Krieg, M. R. Rebagliatti, T. Maniatis, K. 7inn, and M. R. Green, Nucleic Acids Res. 12, 7035 (1984). 7 M. M. Konarska, R. A. Padgett, and P. A. Sharp, Cell (Cambridge, Mass.) 38, 731 (1984). s C. Methfessel, V. Witzemann, T. Takahashi, M. Mishina, S. Numa, and B. Sakmann, Pfluegers Arch. 407, 577 (1986).

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after several hours of incubation in MB at 18 °. The oocyte pairs can be stored at 18 ° in MB solution for several days. The membrane potential of the devitellinized oocyte pairs measured 1 day after devitellinization ranges between - 4 0 and - 9 0 mV. Oocytes which have sustained minor, initially undetectable degrees of damage during this procedure will have lower membrane potentials and usually lyse within 1 - 2 days. Electrophysiological experiments are performed using a dual, twomicroelectrode voltage clamp technique. The microelectrodes are filled with 3 M KCI and have resistances between 1 and 4 MQ. Each oocyte is impaled with two microelectrodes, one passing current and one for measuring voltage. Both oocytes are then voltage clamped and a voltage clamp step applied to cell 1 while cell 2 is maintained at the holding potential. Under these conditions, the change in current observed in cell 2 in response to a voltage clamp step applied to cell 1 will be entirely due to current flowing through the gap junction. Results We chose to study initially the properties of a gap junctional protein isolated from rat heart called connexin 43. 9 Injection of connexin 43 mRNA induced the formation of gap junctional channels. The junctional conductance in the mRNA-injected cell pairs was approximately 2 orders of magnitude larger than that observed in noninjected or water-injected controls. Figure 1 shows a family of junctional current traces recorded in response to a series of depolarizing and hyperpolarizing voltage clamp steps. The junctional conductance was time and voltage independent for potentials between - 6 0 and +60 mV. For larger voltage clamp steps, a small, slowly decaying current component was sometimes observed. Potential Problems One problem with this system is that the oocyte pairs have low levels of background coupling which has been attributed to the presence of an endogenous gap junctional protein called Xenopus connexin 38 (alpha 2). ~°,H The level of background coupling varies among frogs but is usually similar in oocytes obtained from the same frog. Factors that increase the level of endogenous coupling include incubating the cell pairs at tempera9 E. C. Beyer, D. L. Paul, and D. A. Goodenough, J. CellBiol. 105, 2621 (1987). ~oL. Ebihara, E. C. Beyer, K. I. Swenson, D. L. Paul, and D. A. Goodenough, Science 243, 1194 (1989). H R. L. Gimlich, N. M. Kumar, and N. B. Gilula, J. Cell Biol. 110, 597 (1990).

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GAP JUNCTIONALPROTEINSIN OOCYTEPAIRS

B

200

nA

500

ms'~ec

60mv

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°

00 nA 500 msec

FIG. 1. Junctional currents recorded in a connexin 43/43 pair. The cellswere initially held at a holding potential of -60 mV, and a series of hyperpolarizing (A) or depolarizing (B) voltage clamp steps were applied to cell 1. Junctional current was recorded in cell 2.

tures above 25 ° and allowing the oocytes to remain in contact with each other for more than 24 hr. T o control for endogenous coupling we always perform similar experiments on water-injected or noninjected control pairs which are matched with respect to ovary and stage o f development. The endogenous junctional channels can also be distinguished from mRNA-induced channels because o f their striking dependence on transjunctional voltage. Figure 2 shows a family o f junctional current traces recorded from a control oocyte pair which displayed unusually high levels of endogenous coupling.

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10 nA 500 msee

FIG. 2. Endogenous junctional currents recorded from a control noninjected cell pair during a series of depolarizing transjunctional voltage clamp steps between 0 and +50 mV in 10-mV increments. The holding potential was -33 inV.

Conclusions Expression o f gap junctional proteins in Xenopus oocyte pairs allows the direct m e a s u r e m e n t ofcell-to-ceU coupling. This technique can be used to confirm that a cloned gene encodes a gap junction protein and to study the electrophysiological properties o f the different connexin subtypes. It should also be a valuable tool in studying the relationship between structure and function. Acknowledgments This work was supported by National Institutes of Health Grants HIA5377 and HL28958, a New York Heart Grant-in-Aid, and an Irma Hirschl Career Scientist Award.

Expression of gap junctional proteins in Xenopus oocyte pairs.

376 EXPRESSION OF ION CHANNELS [24] [24] E x p r e s s i o n o f G a p J u n c t i o n a l P r o t e i n s in X e n o p u s Oocyte Pairs B y LISA E...
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