Visualization and functional analysis of a maxi-K channel (mSlo) fused to green fluorescent protein (GFP)
Michael P. Myers *
Address of Correspondence:
Barium block , Calcium activated potassium
channel , Charybdotoxin , GPF fusion protein.
We have constructed a fusion protein between mSlo (a recombinant, high conductance, calcium - activated potassium channel or maxi-K), and GFP (green fluorescent protein). The GFP was fused in frame to the carboxy-terminus of the mSlo core protein (mSlo-GFP fusion protein). Expression of this fusion protein in COS-7 cells resulted in robust fluorescence localized near the cell membrane. Fluorescing cells that were patch clamped exhibited whole cell currents with a direction consistent with potassium currents. Conversely, non-fluorescing cells showed no significant currents.
Green Fluorescent Protein (GFP), cloned from the jellyfish Aequorea victoria is a powerful tool for studying the expression of various proteins. GFP is easily visualized when excited with UV light and its fluorescence does not depend on any exogenous compounds (Marshall et al., 1995). This protein, when expressed alone, appears cytosolic, with no cellular targeting mechanisms of its own, making it ideal to tag a protein of interest. Our fusion construct, called mSlo-GFP (for a review of GFP nomenclature see Gerdes and Kaether, (1996)), has the green fluorescent protein fused in frame to the c-terminus of the channel. The result is a functional tagged potassium channel. Our results demonstrate that the fusion protein can be expressed in COS-7 cells, harvested, and reconstituted into lipid bilayers allowing detailed single channel analysis.
Maxi-K channels belong to a class of calcium sensitive potassium channels which possess a large single channel conductance (greater than ~ 200 pS in symmetrical 150 mM KCl), thus they have been called BK (big potassium) or maxi-K channels (Latorre et al., 1989). These high conductance channels have been measured in numerous tissues and are associated with various physiological processes. Maxi-K channels have been cloned and expressed from the slowpoke locus (Adelman et al., 1992). This began the molecular characterization of the channel. The first maxi-K mammalian clone from the mouse brain (mSlo) appeared in 1993 (Butler et al., 1993). Human analogues (hSlo) of this channel have been cloned from a variety of sources.
For the expression of GFP alone, Green Fluorescent Protein cDNA (from the pGFP clone, Clontech, Palo Alto, CA) was subcloned into expression vector pMT3 (from the laboratory of Daniel D. Oprian, Brandeis University). The pMT3 vector is pMT2 (Sambrook et al., 1989) with the Pst I and Eco RI sites mutated to Kpn I and Not I, respectively. To express GFP as a fusion protein, the GFP was subcloned into an expression vector of pMT3 containing the a subunit of mSlo (mbr5) (Butler et al., 1993). The stop codon in mSlo was mutated to a lysine residue (TTG), which allowed for the translation of nine additional bases (CTCCCAGGA) at the c-terminus of mSlo before the sequence of GFP began. As a result of the subcloning steps, fourteen base pairs were eliminated from the 3' untranslated sequence of mSlo (CTATTTTTTTAAAG). A mismatch mutagenesis strategy (Kammann et al., 1989) using PCR (polymerase chain reaction) was employed to produce the mutations of the stop codon and subcloning. DNA fragments were separated by agarose gel electrophoresis and purified using QIAQUICK gel extraction kit (Qiagen, Chatsworth, CA). Oligonucleotides used in this study were custom made by the Oligonucleotide Core Facility at the University of Rochester.
To find the optimal conditions for transfection, the fusion protein was used to transfect COS-7 cells under differing conditions of cell density, concentrations of DNA to lipid reagent, and transfection times. When GFP was expressed alone in our cloning vehicle (plasmid vector GFP-pMT3), conventional fluorescent microscopy revealed cells labeled with intense fluorescence throughout the cell (figure 1, top panels). When GFP was fused to the maxi-K channel (plasmid vector mSlo-GFP-pMT3), the same microscopy revealed robust fluorescence localized near the cell membrane (figure 1, bottom panels). After transfection with mSlo-GFP-pMT3, fluorescent cells that were patch-clamped (4 out of 5), displayed typical outward going voltage gated maxi-K currents. COS-7 cells that were not fluorescing showed no currents (4 out of 4). The addition of green fluorescent protein to the mSlo channel had very little, if any effect on its functional characteristics.
Structural features of the mSlo-GFP fusion channel, namely the outer vestibule, were explored using CTX and Barium, respectively, as probes. CTX and Barium block of typical single channels revealed similar functioning of the two channels. To investigate the concentration dependence of CTX block of mSlo, the channel was assayed with the toxin at several different toxin concentrations. As seen with native maxi-K channels, the interaction of CTX with mSlo was bimolecular. A KD value of 7.4 nM for mSlo and 7.6 nM for mSlo-GFP was obtained for CTX block.
Using standard techniques of molecular biology, we constructed a functional reporter gene to monitor expression in various systems and conditions. This represents another tool for biotechnology. In this study, we have demonstrated that the mSlo channel expression and protein targeting, kinetics, and functional properties of the outer vestibule are all unchanged by GFP fusion at the carboxy-terminus. Our halo like location of the protein suggests that the channels are inserted into the plasma membrane. Therefore, sub-cellular localization of ion channels now appears possible. Thus this new construct represents a tag to not only monitor channel expression, but to locate the protein in living cells.
While attaching GFP to the carboxy-terminus of mSlo had no effect on channel function, results from an amino-terminus attachment to hSlo were quite different. Tagging the amino-terminus of hSlo with GFP apparently alters its calcium sensitivity (Meyer and Fromherz, 1999). No such affect on calcium sensitivity seen in our carboxy-terminus construct could be a clue that only the amino-terminus of the protein is involved in sensing calcium. A more detailed examination of the calcium sensitivity of our tagged protein will need to be done before a final conclusion can be made.
Another result of this study has been a single channel look at the functional inhibition of CTX on the a subunit of mSlo. Our KD value of 7.4 nM for mSlo and 7.6 nM for mSlo-GFP are on the order of the inhibition constant recently reported for the other CTX sensitive BK clone hSlo (Gribkoff et al., 1996). In addition, we demonstrate here that CTX block of mSlo is bimolecular, showing that the clone follows the interaction of CTX with native maxi-K channels.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an important reporter molecule for monitoring gene expression. Ion channels fused to GFP have limitless applications in studying the structure and function of these proteins. Our fluorescing protein represents a tagged maxi-K channel. The use of fluorescent mSlo-GFP will hopefully shed some light on the limited information we have about these critical proteins.
Adelman, J.P., Shen, K.Z., Kavanaugh, M.P., Warren, R.A., Wu, Y.N., Lagrutta, A., Bond, C.T.and North, R.A. (1992). Calcium-activated potassium channels expressed from cloned complementary DNAs. Neuron, 9:209-216.
Gribkoff, V.K., Lum-Ragan, J.T., Boissard, C.G., Post-Munson, D.J., Meanwell, N.A., Starrett, J.E., Jr., Kozlowski, E.S., Romine, J.L., Trojnacki, J.T., McKay, M.C., Zhong, J.and Dworetzky, S.I. (1996). Effects of channel modulators on cloned large-conductance calcium-activated potassium channels. Molecular Pharmacology, 50:206-217.
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