Protein: A New Tool for Studying ... fluorescent protein (GFP) from the jellyfish Aequorea victo- ..... experiments, the total amount of DNA added was -20 I~g.
Neuron, Vol. 14, 211-215, February, 1995, Copyright © 1995 by Cell Press
The Jellyfish Green Fluorescent Protein: A New Tool for Studying Ion Channel Expression and Function 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 cotransfection 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 mammalian cells is identifying the cells that express the channel 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. Although powerful in certain applications, each of these approaches has limitations. It has been shown recently that expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (Prasher et al., 1992) in Escherichia coil or Caenorhabditis elegans produces a protein that is strongly fluorescent, 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 describe expression of GFP in human embryonic kidney cells (HEK 293; Graham et al., 1977). It produces a robust signal, 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 fluorescence when viewed with conventional epifluorescence optics for fluorescein isothiocyanate. This signal, which is
Neurotechnique
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 adversely, 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 GFPassociated 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 fluorescent 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 corresponding 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÷ channels in membrane patches isolated from intensely or weakly fluorescent cells from the same transfection are shown in Figures 2C and 2D, respectively. The singlechannel activity recorded at +20 mV is 3-4 times higher in the patch from the intensely fluorescent cell. It was consistently 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-Daspartate (NMDA) receptor subunit NMDAR1, a strongly
Neuron 212
A
C
¢,
Figure 1. GFP Expression in HEK 293 Cells Cotransfection with plasmids encoding GFP and the Ca2+-activated K÷ channel bSIo produces cells with a strong green fluorescence (A and B). This signal is present in only a fraction of the cells, as seen in the double exposures (B and C), and is distributed throughout the cell. There is heterogeneity in the intensity of the signal, producing bright (C, hollow arrow) and dim (C, solid arrows) cells. The chimeric NMDAR1-GFP protein (D and E) produces labeling in the membrane compartments of the cells and, in cases of particularly strong expression, is concentrated in the perinuclear organelles (D, hollow arrow). Bar in (A), 16 p.m.
GFP as a Reporter in Mammalian Cells 213
A
C 9 --
L•A
8-7--
350 300 ~ 250
200ms
~ ~ p A
6-200
5--
150 100 50 0
4-3-2--
los
1-O--
200rra B
10pA
D 3 -120pA
•
12--__ ~
10S 0--
Figure 2. The Intensity of the Fluorescent Signal Correlates with the Level of Functional Channel Expression (A and S) Whole-cell patch-clamp recordings from H EK 293 cells cotransfected with cDNAs encoding GluR6(R) and GFP. Each cell was voltage clamped at -86 mV, and inward currents were evoked by superfusing the cell with a solution containing 10 pM kainate (duration indicated by the bar above each record). The recording in (A) is from a cell that showed very bright GFP-associated fluorescence, whereas the recording in (B) is from a cell that gave only a very weak fluorescent signal. Note that the calibrations in (A) and (B) are different. The records were low pass-filtered at 200 Hz (-3 dB), (C and D) Recordings of Ca2+-activated K+ currents in inside-out patches excised from HEK 293 cells that were cotransfected with cDNAs encoding a Ca2+-activated K+ channel (bSIo) and GFP. The currents shown in (C) were recorded in a patch taken from an intensely fluorescent cell, and the currents in (D) were recorded in a patch from a cell that was only dimly fluorescent. Each patch was voltage clamped at +20 mV in symmetrical KCI solutions. The free [Ca2+]of the solution in contact with the cytoplasmic face of the patches was -0.3 #M. Single-channel currents are outward and have a unitary amplitude of -5.5 pA. Current levels indicative of the number of channels open simultaneously are shown at the left of each record. The records were low pass-filtered at 500 Hz (-3 dB).
fluorescent protein is produced in transfected cells. In contrast to the signal seen with GFP alone, the fluorescent chimeric protein shows a more restricted distribution within the cell (Figures 1D and 1E). The signal in weakly labeled cells appears to be largely at the plasmalemma, whereas in brighter cells there are also large signals associated with intracellular organelles. Cotransfection of the NMDAR1-GFP chimera with cDNAs encoding either the NMDAR2-A or NMDAR2-C subunit results in the expression of functional NMDA-type ion channels. The macroscopic properties of heterooligomeric channels that contain the NMDAR1-GFP chimera are similar to the corresponding properties of heterooligomeric channels that contain authentic NMDAR1, It is known that NMDARI/NMDAR2-C heteromeric channels show substantially slower decay kinetics (Monyer et al., 1992) and substantially greater sensitivity to glycine (Kutsuwada et al., 1992) than heteromeric NMDARI/NM DAR2-A channels. These differences are conserved in heteromeric channels that contain the chimeric construct. Figure 3 shows a concentration-response curve obtained for glycine modulation of glutamate-activated currents from a
GlycinConcent e ratio(~M) n Figure 3. Heteromeric Channels Containing the NMDARI-GFP Chimera Show Normal Sensitivity to the Coagonist Glycine Concentration-response data obtained from a HEK 293 cell that was cotransfected with cDNAs that encode the NMDAR1-GFP chimeric protein and the NMDAR2-C subunit. The cell was voltage clamped in the whole-cell patch-clamp configuration, and inward currents were evoked at-80 mV by the application of I ~M glutamate in the presence of different glycine concentrations. The steady-state amplitude of the currents is plotted. The smooth curve is the best fit to the data according to a Hill-type equation: I = Im,,([glycine]lEC~o)",l([glycine]l ECso)°H + 1), which gave ECs0 and n, values of 0.64 I~M and 1.1, respectively.
cell expressing NMDAR1-GFP and NMDAR2-C. The fit gives an apparent ECs0 value of 0.64 I~M, which is similar to the value of 0.7 ~M determined for dimeric NMDARI/ NMDAR2-C channels by Kutsuwada et al. (1992). Further evidence for the functional integrity of the N M D A R 1 - G F P chimera was obtained from analysis of single-channel currents. Figure 4A shows examples of unitary currents activated by 1 I~M glutamate in an outside-out patch excised from a HEK 293 cell that coexpressed the N M D A R 1 - G F P chimera and NMDAR2-A. Varying the membrane potential of the patch showed that the singlechannel currents reversed near 0 mV. The amplitude of the currents at - 8 0 mV (Figure 4B) is consistent with a main conductance level of about 50 pS, although a small number of openings to a conductance level of about 40 pS were also seen. These results are similar to those of Stern et al. (1992, 1994) for recombinant N M D A R I / NMDAR2-A channels expressed in either oocytes or HEK 293 cells. The gating kinetics of channels containing the chimeric subunit also appear to be largely unaltered. Dwell-time histograms for channel openings and closings are shown in Figures 4C and 4D, respectively. The distribution of apparent open durations has been fitted with three exponential components with time constants of 37 ~s, 1.8 ms, and 4.7 ms, respectively. Four exponential components were detected in the distribution of shut times. The three briefest components (which likely are bounded by openings of the same channel molecule) had respective time constants of 30 ~s, 0.21 ms, and 3.9 ms. These results are similar to the corresponding results reported for the heteromeric channels expressed after cotransfection of the cDNAs encoding NMDAR2-A and the authentic NMDAR1 subunit (Stern et al., 1993).
Neuron 214
appr oach might of f er significant a d v a n t a g e s over immunocytochemical visualization.
Experimental Procedures GFP Constructs lO ~
lo4 10~ lo`2 A p p a r e n t O p e n T i m e (see)
Inward Current (pA)
10-~
lo~
lo~ lo" lo" A p p a r e n t S h u t T i m e (sec)
lo*
Figure 4. Unitary Properties of Heteromeric Channels Containing the NMDAR1-GFP Chimera (A) Examples of single-channel currents evoked by 1 ~.M glutamate in an outside-out patch excised from a HEK 293 cell that was cotransfected with cDNAs encoding the NMDAR1-GFP chimera and NMDAR2-A. The patch was voltage clamped at -80 mV, and channel openings are downward. The lower trace shows an expanded portion of the upper trace taken from above the 100 ms time bar. The records were low pass-filtered at 1 kHz (-3 dB). (B) Histogram of the amplitude of individual sample points obtained from a longer stretch of record from the same patch at -80 mV. Gaussian functions (smooth curves) have been fitted to the peaks corresponding to the closed level (left) and the main open level (right). The fit to the open sample points gives a mean inward current amplitude of 4.0 pA, which corresponds to a single-channel conductance of 50 pS. (C and D) Dwell-time histograms for channel openings (C) and closings (D) in the same patch at -80 mV. For the kinetic analysis, the records were low pass-filtered (digital Gaussian) at 3 kHz (-3 dB) and sampled at 20 kHz. Note that log durations are plotted. The distributions of open times were fitted with three exponential components with time constants (and relative areas) of 0.037 ms (0.62), 1.8 ms (0.33), and 4.7 ms (0.05). The distributions of closed times were fitted with four exponential components with time constants (and relative areas) of 0.030 ms (0.52), 0.21 ms (0.18), 3.89 ms (0.22), and 12.5 ms (0.08).
Discussion T h e e x p r e s s i o n of GFP m a k e s it possible to identify rapidly cells that have been successfully transfected, and there is a correlation between the intensity of the o b s e r v e d fluorescence and the relative level of functional ion channel expression. The c o e x p r e s s i o n of GFP d o e s not a p p e a r to be deleterious to the cells or to alter, in any obvious way, the functional properties of either bSIo or GluR6. Thus, GFP promises to be a useful tool for investigations on recombinant ion channels. The chimeric N M D A R 1 - G F P subunit is c a p a b l e of forming hetero-oligomeric receptor complexes, and these complexes exhibit both macroscopic and unitary properties that are indistinguishable from the c o r r e s p o n d i n g properties of hetero-oligomers containing N M D A R I . This chimeric subunit could be used to visualize the receptor subunit in transfected cell lines (or p e r h a p s primary neurons) in o r d e r to a d d r e s s questions related to subunit assembly or clustering, or to the targeting of subunits to specific membrane compartments. Because the subunit-associated fluorescence can be imaged repeatedly in live cells, this
To express GFP, the KpnI-EcoRI fragment of plasmid TU #65 (Chalfie et al., 1994) was inserted into the CMV expression vector pcDNA3 (Invitrogen). For the NMDAR1 chimera, polymerase chain reaction amplification with Vent polymerase (NEB) was used to introduce Nhel sites into the 3' end of the NMDAR1 subunit (Moriyoshi et al., 1991) and the 5' end of the GFP cDNA. These sites were then used to create a chimera in which the last 13 amino acids of the NMDAR1 subunit were replaced with an alanine followed by the GFP sequence coding for amino acids 2-238. The chimera was then inserted into pcDNA3. GluR6(R) was in pBK-CMV (Stratagene), NMDAR2-A was in pcDNAAMP (Invitrogen), and NMDAR2-C was in a modified version of the pcB6 vector (supplied by Dr. M. Caplan, Yale University). bSIo was in the pcDNA3 vector.
Cell Culture and Transfection Human embryonic kidney cells (HEK 293, ATCC CRL 1573) were raised in MEM-E supplemented with 10% fetal bovine serum. Exponentially growing cells were plated in 35 mm dishes, and calcium phosphate transfection (Chen and Okayama, 1987) was carried out with commercially prepared reagents (Stratagene). For cotransfection experiments, the total amount of DNA added was - 2 0 I~g.
Microscopy and Electrophysiology Standard epifluoresence optics (Zeiss cube #10, Olympus cube B) were used to visualize GFP. Whole-cell recordings and recordings from membrane patches were made as described by Hamill et al. (1981) with an EPC-9 patch clamp. The standard extracellular solution (pH 7.2) contained 150 mM NaCI, 2.5 mM KCI, 1 mM CaCI2,and 10 mM Na-HEPES. For whole-cell recording, the standard intracellular solution (pH 7.2) contained 140 mM CsCI, 0.5 mM CaCI2, 5 mM Na-EGTA, and 10 mM Na-HEPES. The experiments on the Ca2÷-activated K+ channel clone were made in symmetrical solutions containing 150 mM KCI, 5 mM Na-EGTA, and 3.8 mM CaCI2buffered with 10 mM Na-HEPES (pH 7.2). Solutions were applied by local superfusion via a wide-mouthed pipette positioned within - 1 0 0 t~m of the cell or patch. For the experiments on homomeric GluR6(R) channels, the cells were pretreated with concanavalin A (10-25 p.M) for 10-15 rain to remove desensitization. Whole-cell and single-channel currents were low pass-filtered (Bessel-type) at 10 kHz (-3 dB) and stored on videotape with a VCR-PCM recorder at a sampling rate of 94 kHz. For the analysis of singlechannel currents, the data were digitized at 20 kHz and low passfiltered (digital Gaussian) at 2-4 kHz (-3 dB). A half-amplitude threshold-crossing routine was used to detect and measure the duration of channel openings and closings (Colquhoun and Sigworth, 1983). Histograms of dwell-time distributions were constructed as described by Sigworth and Sine (1987) and were fitted with two to four exponential components to estimate the time constant and relative area of each component.
Acknowledgments We are indebted to Jim Eberwine for his suggestion to use GFP and Martin Chalfie for generously sharing GFP. We thank Whit Tingley and Rick Huganir for NMDAR2-A, Steve Heinemann for GluR6(R), S. Nakanishi for NMDAR1, Peter Seeburg for NMDAR2-C, and MicroTech Optical, Inc. for the loan of Olympus epifluorescence optics. This work was supported by the Research to Prevent Blindness Foundation and NEI R01 EY08362 (-I. E. H.) and NIH R01 NS30996 (J. R. H.). Fellowship support was provided by The Connecticut American Heart Association (J. M.) and Donaghue Medical Research Foundation (G. W. J. M.). J. M. is now at Brown University. Received November 11, 1994; revised November 23, 1994.
GFP as a Reporter in Mammalian Cells 215
References
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-805. Chen, C., and Okayama, H. (1987). High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745-2752. Colquhoun, D., and Sigworth, F. J. (1983). Fitting and statistical analysis of single-channel records. In Single-Channel Recording, B. Sakmann and E. Neher, eds. (New York: Plenum Press), pp. 191-263. Egebjerg, J., Bettler, B., Hermans-Borgmeyer, I., and Heinemann, S. (1991). Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature 351,745-748. Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59-74. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. PflLigers Arch. 391, 85-100. Kutsuwada, T., Kashiwabuchi, N., Mori, H., Sakimura, K., Kushiya, E., Araki, K., Meguro, H., Masaki, H., Kumanishi, T., Arakawa, M., and Mishina, M. (1992). Molecular diversity of the NMDA receptor channel. Nature 358, 36-41. Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M., Lomeli, H., Burnashev, N., Sakmann, B., and Seeburg, P. H. (1992). Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256, 1217-1221. Moriyoshi, K., Masu, M., Ishii, T., Shigemoto, R., Mizuno, N., and Nakanishi, S. (1991). Molecular cloning and characterization of the rat NMDA receptor. Nature 354, 31-37. Moss, G. W. J., Marshall, J., Morabito, M., Moczydlowski, E. G., and Howe, J. R. (1995). Cloning of a maxi-Ca-activated K channel from bovine aortic muscle: sensitivity to a Kunitz inhibitor. Biophys. J., in press. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111,229-233. Sigworth, F. J., and Sine, S. M. (1987). Data transformations for improved display and fitting of single channel dwell time histograms. Biophys. J. 52, 1047-1054. Stern, P., B~h~, P., $choepfer, R., and Colquhoun, D. (1992). Singlechannel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors. Prec. R. Soc. Lond. (B) 250, 217-277. Stern, P., B~he, P., Schoepfer, R., and Colquhoun, D. (1993). Singlechannel kinetics of recombinant NM DA receptors. J. Physiol. 473, 48P. Stern, P., Cik, M., Colquhoun, D., and Stephenson, F. (1994). Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes. J. Physiol. 476, 391397. Wang, S., and Hazelrigg, T. (1994). Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369, 400-403.
