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• W2604401907 abstract "John Marshall,* Raymond Molloy,t Guy W. J. Moss,* James R. Howe,* and Thomas E. Hughestt *Deparment of Pharmacology tSection of Neurobiology SDepartment of Ophthalmology and Visual Science Yale University School of Medicine New Haven, Connecticut 06520 Summary Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotrans- fection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate studies of ion channel expression, localization, and processing. Introduction A problem in transfecting cloned ion channels into mam- malian cells is identifying the cells that express the chan- nel and/or localizing the channel within a cell. A variety of strategies have been employed to overcome this problem, including generating stable cell lines and using antibodies, fluorescent enzyme substrates, or the luciferase gene. Al- though powerful in certain applications, each of these ap- proaches has limitations. It has been shown recently that expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victo- ria (Prasher et al., 1992) in Escherichia coil or Caenorhab- ditis elegans produces a protein that is strongly fluores- cent, and that this fluorescence does not appear to depend upon exogenous substrates or coenzymes (Chalfie et al., 1994). Subsequently, GFP was used to construct chimeric proteins that were functional and fluorescent in Drosophila melanogaster (Wang and Hazelrigg, 1994). Here we de- scribe expression of GFP in human embryonic kidney cells (HEK 293; Graham et al., 1977). It produces a robust sig- nal, making it possible to identify cells for patch-clamp studies or to follow chimeric receptor proteins. Results Cotransfection of GFP with Cloned Receptors Identifies the Transfected Cells GFP expressed under the control of a cytomegalovirus (CMV) promoter is readily detected in HEK 293 cells 2 days after the transfection (Figures 1A-1C). When GFP is expressed alone, or in pairwise combination with an ion channel, the signal produced is a bright green fluores- cence when viewed with conventional epifluorescence op- tics for fluorescein isothiocyanate. This signal, which is resistant to photobleaching, makes it possible to combine both bright-field and epifluorescence illumination so that the transfected and untransfected cells are visible. The green fluorescence is apparent in both living cells and those fixed with paraformaldehyde. Although adjacent HEK 293 cells are known to form gap junctions, there was no evidence that GFP spread to surrounding cells. The expression of GFP does not appear to affect the cells ad- versely, and strong signals can be found in living cells at least 5 days after the transfection. Whole-cell recordings from cotransfected, fluorescent cells reveal that they invariably express the introduced ion channels (n = 42). Such expression is absent from cells that do not exhibit detectable fluorescence. In addition, there is cell to cell variability in the intensity of the GFP- associated signal (Figure 1C), and there is a correlation between the intensity of the fluorescence and the level of expression of functional ion channels. Figures 2A and 2B show whole-cell recordings from two cells cotransfected with cDNAs encoding GFP and the fully edited (R) version of glutamate receptor 6 (GluR6; Egebjerg et al., 1991). The recording shown in (A) was made from an intensely fluorescent cell, whereas the recording in (B) was obtained from a nearby cell that exhibited a much weaker fluores- cent signal. Both cells were voltage clamped at -80 mV, and inward currents were evoked by the application of 10 I~M kainate. The current recorded from the intensely fluorescent cell is about 40 times larger than the corre- sponding current in the weakly fluorescent cell. A similar correlation between the intensity of the fluorescent signal and functional channel expression was observed for cells that coexpressed GFP and a Ca2+-activated K + channel that was cloned from bovine aorta (bSIo; Moss et al., 1995). Single-channel currents through Ca2÷-activated K ÷ chan- nels in membrane patches isolated from intensely or weakly fluorescent cells from the same transfection are shown in Figures 2C and 2D, respectively. The single- channel activity recorded at +20 mV is 3-4 times higher in the patch from the intensely fluorescent cell. It was con- sistently true that patches from intensely fluorescent cells showed substantially higher levels of channel activity than patches isolated from weakly fluorescent cells. GFP did not appear to alter the macroscopic properties of currents through GluR6(R) channels, or the properties of recombinant Ca2÷-activated K ÷ channels studied in patches from GFP-identified cells. These latter channels were very similar in their properties to the corresponding native channels expressed in smooth muscle cells from which the cDNA clone was isolated. We therefore found no indication that GFP alters the properties of ion channels with which it is coexpressed. A NMDAR1-GFP Chimeric Protein Makes It Possible to Follow Functional NMDA Receptor Proteins in Living Cells When GFP is fused to the 3' end of the N-methyi-D- aspartate (NMDA) receptor subunit NMDAR1, a strongly" @default.
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• W2604401907 date "1995-01-01" @default.
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• W2604401907 title "The Jellyfish Green Fluorescent Protein: A New Tool for Studying Ion Channel Expression and Function Neurotechnique" @default.
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