Journal of Neurochemistry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry
Rapid Communication
Expression of an Inwardly Rectifying Potassium Channel in Xenopus Oocytes Franqois Pkrier, Kathryn L. Coulter, Carolyn M. Radeke, and Carol A. Vandenberg Department of Biological Sciences and Neuroscience Research Institute, University of California, Santa Barbara, Cal$ornia, U.S.A.
Abstract: The murine macrophage-likecell line J774.1 was used as a source of mRNA for the expression of inwardly rectifying potassium channels in Xenopus oocytes. RNA was isolated, poly(A)+-selected, and size-fractionatedby sucrose density gradient centrifugation. Oocytes injected with 5774. l RNA expressed large-amplitude current (0.9 ? 0.07 PA; mean i SEM, n = 31) at -100 mV in 96 mM extracellular Kf showing prominent inward rectification. The inwardly rectifying currents were most strongly expressed by an mRNA size class of 4-5 kb. The expressed current displayed selectivity, conductance, and rectification properties of inwardly rectifying potassium channels in their native membranes. The current was potassium selective and was specifically blocked by Ba”. The conductance and the voltage dependence of the current rectification depended on the extracellular potassium concentration, with the midpoint in peak conductance following the potassium equilibrium potential. This high level of expression of inward rectifier current and the absence of other expressed currents suggest that 5774.1 mRNA represents an excellent starting material for expression cloning of the inward rectifier potassium channel cDNA. Key Words: Inward rectification-Potassium current-Xenopus oocyte -mRNA expression. P6rier F. et al. Expression of an inwardly rectifying potassium channel in Xenopus oocytes. J. Neurochern. 59, 1971-1974 (1992).
Inwardly rectifying potassium channels are characterized by asymmetry in their potassium conductance: They carry large inward currents at potentials negative to the potassium equilibrium potential and small currents at potentials positive to the potassium equilibrium potential. These channels are distinguished from voltage-activated potassium channels by their rectification, by the dependence of rectification on external K+ concentration, and by the square-root dependence of their conductance on external potassium concentration (reviewed by Hagiwara, 1983; Noma, 1987). Open-channel block by internal Mg2+is a major cause of both inward rectification (Hone and Irisawa, 1987; Matsuda et al., 1987; Vandenberg, 1987) and rectification dependence on external K+ (Hille and Schwarz, 1978). Inwardly rectifying potassium channels are found in high abundance in cardiac and skeletal muscle, glia, many neurons, macrophages, and invertebrate oocytes. Their pro-
posed functions include maintenance of long-duration action potentials, determination of the cell resting potential, modulation of membrane excitability by antagonizing membrane depolarization, involvement in action potential repolarization, and transport of K+ for buffering of extracellular ions by glial cells (Hagiwara, 1983; Brew et al., 1986; Shimoni et al., 1992). Several types of mammalian inwardly rectifying channels have been described electrophysiologically. For example, cardiac myocytes express the inward rectifier channel (Kl), which displays strong rectification; a G protein-coupled channel modulated by neurotransmitters (KACh),which shows moderate rectification; and an ATP-regulated channel (KATP), with modest rectification (Sakmann and Trube, 1984; Noma, 1987). Although these channels have been characterized extensively,little is known about their molecular nature. Cloning by functional expression in Xenopus oocytes (Kushner et al., 1989) or by complementation in yeast (Anderson et al., 1992; Sentenac et al., 1992)is an attractive method for the isolation of cDNA encoding these inwardly rectifying potassium channels. The success of these methods depends on the identification of an mRNA source that yields high levels of expression of these channels. Murine macrophage-like 5774.1 cells contain an inwardly rectifying K+ channel as their primary membrane conductance (McKinney and Gallin, 1988). Single-channel and macroscopic current measurements indicate that this inward rectifier is similar to the K, channel (McKinney and Gallin, 1988). In this report we show that size-fractionated poly(A)+RNA from the cell line 5774.1 expresses large-amplitude inwardly rectifying potassium currents when injected into Xenopus oocytes.
Resubmitted manuscript received July 29, 1992; accepted August 4. 1992. Address correspondence and reprint requests to Dr. C. A. Van-
MATERIALS AND METHODS RNA isolation J774.1 cells (American Type Culture Collection) were cultured as adherent cells in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (Gemini), 2 mMglutamine, 45 phfglutathione, and 50 U/ml of penicildenberg at Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, U.S.A.
1971
F. PERIER ET AL.
1972
lin/streptomycin at 37°C in an atmosphere containing 5% COz. Stock cultures were maintained under adherent conditions, and cells were harvested 2-4 days after plating because inward rectifier channels are up-regulated following adherence (McKinney and Gallin, 1990). Total RNA from 5774.1 cells was prepared as described by Chomczynski and Sacchi (1987). Poly(A)+RNA was isolated by two cycles of oligo(dT) cellulose chromatography (Sambrook et al., 1989). The poly(A)+ RNA (150-400 pg) was size-fractionated by centrifugation on a 10-3 1% (wt/wt) sucrose gradient [OS% sodium sarcosinate, 1 m M EDTA, and 10 mMTrisHCI (pH 7.5) (Frech and Joho, 1989) or 0.1% lithium dodecyl sulfate, 1 m M EDTA, and 10 mM HEPES (pH 7.5) (Sumikawa et al., 1982)]for 24 hat 25,000 rpm and 4°C in a Beckman SW 28.1 rotor.
Oocyte preparation and injection Oocytes were removed surgically from Xenopus laevis ovaries and treated with 2 mg/ml of collagenase (type IV; Sigma) for 2 h at 22°C before manual dissection to remove the follicular layer. The oocytes were microinjected 16-24 h postdissection with 46 nl of mRNA (1 p g / p l in water) and then incubated in Barth's solution at 16-18°C for 3-4 days before recording (Kushner et al., 1989).
Electrophysiological recording Oocyte currents were recorded by two-microelectrode voltage clamp techniques using either a Dagan TEV 200 or a Dagan 8500 voltage clamp at 22-24°C. Glass microelectrodes (
Rapid Communication
Expression of an Inwardly Rectifying Potassium Channel in Xenopus Oocytes Franqois Pkrier, Kathryn L. Coulter, Carolyn M. Radeke, and Carol A. Vandenberg Department of Biological Sciences and Neuroscience Research Institute, University of California, Santa Barbara, Cal$ornia, U.S.A.
Abstract: The murine macrophage-likecell line J774.1 was used as a source of mRNA for the expression of inwardly rectifying potassium channels in Xenopus oocytes. RNA was isolated, poly(A)+-selected, and size-fractionatedby sucrose density gradient centrifugation. Oocytes injected with 5774. l RNA expressed large-amplitude current (0.9 ? 0.07 PA; mean i SEM, n = 31) at -100 mV in 96 mM extracellular Kf showing prominent inward rectification. The inwardly rectifying currents were most strongly expressed by an mRNA size class of 4-5 kb. The expressed current displayed selectivity, conductance, and rectification properties of inwardly rectifying potassium channels in their native membranes. The current was potassium selective and was specifically blocked by Ba”. The conductance and the voltage dependence of the current rectification depended on the extracellular potassium concentration, with the midpoint in peak conductance following the potassium equilibrium potential. This high level of expression of inward rectifier current and the absence of other expressed currents suggest that 5774.1 mRNA represents an excellent starting material for expression cloning of the inward rectifier potassium channel cDNA. Key Words: Inward rectification-Potassium current-Xenopus oocyte -mRNA expression. P6rier F. et al. Expression of an inwardly rectifying potassium channel in Xenopus oocytes. J. Neurochern. 59, 1971-1974 (1992).
Inwardly rectifying potassium channels are characterized by asymmetry in their potassium conductance: They carry large inward currents at potentials negative to the potassium equilibrium potential and small currents at potentials positive to the potassium equilibrium potential. These channels are distinguished from voltage-activated potassium channels by their rectification, by the dependence of rectification on external K+ concentration, and by the square-root dependence of their conductance on external potassium concentration (reviewed by Hagiwara, 1983; Noma, 1987). Open-channel block by internal Mg2+is a major cause of both inward rectification (Hone and Irisawa, 1987; Matsuda et al., 1987; Vandenberg, 1987) and rectification dependence on external K+ (Hille and Schwarz, 1978). Inwardly rectifying potassium channels are found in high abundance in cardiac and skeletal muscle, glia, many neurons, macrophages, and invertebrate oocytes. Their pro-
posed functions include maintenance of long-duration action potentials, determination of the cell resting potential, modulation of membrane excitability by antagonizing membrane depolarization, involvement in action potential repolarization, and transport of K+ for buffering of extracellular ions by glial cells (Hagiwara, 1983; Brew et al., 1986; Shimoni et al., 1992). Several types of mammalian inwardly rectifying channels have been described electrophysiologically. For example, cardiac myocytes express the inward rectifier channel (Kl), which displays strong rectification; a G protein-coupled channel modulated by neurotransmitters (KACh),which shows moderate rectification; and an ATP-regulated channel (KATP), with modest rectification (Sakmann and Trube, 1984; Noma, 1987). Although these channels have been characterized extensively,little is known about their molecular nature. Cloning by functional expression in Xenopus oocytes (Kushner et al., 1989) or by complementation in yeast (Anderson et al., 1992; Sentenac et al., 1992)is an attractive method for the isolation of cDNA encoding these inwardly rectifying potassium channels. The success of these methods depends on the identification of an mRNA source that yields high levels of expression of these channels. Murine macrophage-like 5774.1 cells contain an inwardly rectifying K+ channel as their primary membrane conductance (McKinney and Gallin, 1988). Single-channel and macroscopic current measurements indicate that this inward rectifier is similar to the K, channel (McKinney and Gallin, 1988). In this report we show that size-fractionated poly(A)+RNA from the cell line 5774.1 expresses large-amplitude inwardly rectifying potassium currents when injected into Xenopus oocytes.
Resubmitted manuscript received July 29, 1992; accepted August 4. 1992. Address correspondence and reprint requests to Dr. C. A. Van-
MATERIALS AND METHODS RNA isolation J774.1 cells (American Type Culture Collection) were cultured as adherent cells in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (Gemini), 2 mMglutamine, 45 phfglutathione, and 50 U/ml of penicildenberg at Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, U.S.A.
1971
F. PERIER ET AL.
1972
lin/streptomycin at 37°C in an atmosphere containing 5% COz. Stock cultures were maintained under adherent conditions, and cells were harvested 2-4 days after plating because inward rectifier channels are up-regulated following adherence (McKinney and Gallin, 1990). Total RNA from 5774.1 cells was prepared as described by Chomczynski and Sacchi (1987). Poly(A)+RNA was isolated by two cycles of oligo(dT) cellulose chromatography (Sambrook et al., 1989). The poly(A)+ RNA (150-400 pg) was size-fractionated by centrifugation on a 10-3 1% (wt/wt) sucrose gradient [OS% sodium sarcosinate, 1 m M EDTA, and 10 mMTrisHCI (pH 7.5) (Frech and Joho, 1989) or 0.1% lithium dodecyl sulfate, 1 m M EDTA, and 10 mM HEPES (pH 7.5) (Sumikawa et al., 1982)]for 24 hat 25,000 rpm and 4°C in a Beckman SW 28.1 rotor.
Oocyte preparation and injection Oocytes were removed surgically from Xenopus laevis ovaries and treated with 2 mg/ml of collagenase (type IV; Sigma) for 2 h at 22°C before manual dissection to remove the follicular layer. The oocytes were microinjected 16-24 h postdissection with 46 nl of mRNA (1 p g / p l in water) and then incubated in Barth's solution at 16-18°C for 3-4 days before recording (Kushner et al., 1989).
Electrophysiological recording Oocyte currents were recorded by two-microelectrode voltage clamp techniques using either a Dagan TEV 200 or a Dagan 8500 voltage clamp at 22-24°C. Glass microelectrodes (
