Generation of functional fluorescent BK channels by random insertion of GFP variants.

Target Plasmids, Transposons, and Transposition Reactions The full-length human slo cDNA isolated from uterine smooth muscle (hslo; GenBank/EMBL/DDBJ accession no. U11058 ; Wallner et al., 1995 ) was modified by Dr. A. Tinker in the 5′ end to carry a six-histidine tag followed by the FLAG epitope DYKDDDDK. This construct was provided to us by G. Moss (University College London, London, UK). A HindIII/NotI fragment carrying this modified hslo cDNA was cloned into a pCMVSport/βgal-derived vector. The CFP transposon was as previously reported ( Sheridan et al., 2002 ). The transposon encoding YFP was made by replacing the AscI-flanked EGFP fragment in ( Sheridan et al., 2002 ) with the corresponding YFP fragment (Venus variant; Nagai et al., 2002 ). The two transposons use different reading frames to increase the chance of insertions yielding functional proteins ( Sheridan et al., 2002 ). In view of the low probability of transposition in an in vitro reaction, a selectable marker was introduced into each transposon in the form of a Kanamicin resistance cassette (Kan r ) flanked by SrfI restriction sites. Immediately before the Kan r there is a stop codon; therefore, each clone containing an in-frame insertion should encode a truncated protein with YFP or CFP at the COOH terminus. The transposon can be inserted anywhere in the target plasmid; in our case, the probability that the insertion occurs into the hslo cDNA, in the correct orientation and reading frame, is expected to be ∼9%. Thus our screening strategy consisted of several steps. First, we performed two separate in vitro transposition reactions, one for each transposon, following the protocol described by Sheridan et al. (2002) . The resulting DNA was transformed into Escherichia coli . Single colonies resistant to ampicillin (transformed clones) and kanamycin (marker for transposition) were picked and grown in 96-well plates. Transposed plasmids were then isolated in a 96-well format with Eppendorf PerfectPREP-96 Vac Direct Bind miniprep kits (Eppendorf) on a PerkinElmer MultiPROBE II HT liquid handling robot. Approximately 10,000 transposed clones were obtained; we purified DNA from 672 clones of each reaction (1,344 clones in total). We identified correctly oriented in-frame insertions by transiently transfecting each truncated construct into HEK293 cells, which were screened for fluorescence. The probability of transposition is not uniform ( Goryshin et al., 1998 ), so we expected some clones to have identical insertion sites. The 5′ boundaries of the insertions were determined for all fluorescent clones by DNA sequencing (HHMI Biopolymer/Keck Foundation Biotechnology Laboratory, Yale University School of Medicine) to identify unique insertions and localize the exact insertion site. These clones were digested with SrfI to remove the Kan r cassette and obtain the full-length fusion constructs. Screening of Transposed Clones HEK-293 cells were seeded in 96-well glass bottom tissue culture plates (NalgeNUNC) and grown in DMEM (GIBCO BRL) supplemented with 10% FBS at 37°C in an atmosphere containing 5% CO 2 . Cells were transfected with plasmid DNA from individual CFP- and YFP-transposed clones and Lipofectamine 2000 (GIBCO BRL) following standard protocols. The cells were screened for YFP and CFP fluorescence ∼24 h after transfection with a 20× objective on a Carl Zeiss MicroImaging, Inc. inverted fluorescence microscope. Cell-surface Immunostaining CHO cells were plated in Falcon culture slides (Becton Dickinson Labware) and grown in α-MEM (GIBCO BRL) with 10% FBS at 37°C in 5% CO 2 . Cells were transfected 18–24 h before experiments with plasmid DNA of each full-length fusion protein and Lipofectamine 2000 (GIBCO BRL). All staining procedures were performed at 4°C. Cells were washed twice with PBS 2+ (PBS with 2 mM CaCl 2 and 0.5 mM MgCl 2 ), blocked with 1% BSA and 10% goat serum for 1 h and further incubated with 5 μg/ml mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) for 45 min. Cells were then fixed with 2% formaldehyde in PBS 2+ for 10 min, washed extensively with PBS and incubated with 10 μg/ml of an Alexa Fluor 594–conjugated goat anti–mouse IgG antibody (Molecular Probes) for 45 min. After several washes with PBS, the cells were mounted with Aquamount (Lerner Laboratories). Images of the slides were collected with a Carl Zeiss MicroImaging, Inc. laser scanning microscope equipped with a 40× oil-immersion objective. YFP and CFP were excited using the Argon laser 488-nm line, whereas Alexa 594 was excited using the He-Ne laser 568-nm line. The emission signals were filtered with a Carl Zeiss MicroImaging, Inc. 515–565-nm filter (YFP/CFP emission) or with a 590–640 filter (Alexa 594 signal).

Triton X-100 Solubilization and Western Blot Analysis

A protocol modified from Zarei et al. (2004) was used. Transfected CHO cells were permeabilized in lysis buffer (150 mM NaCl, protease inhibitors, 1 μg/ml DNaseI, 20 mM Tris-HCl, pH 7.4) containing various concentrations of Triton X-100 (0, 0.1, 0.4, 1, 2%). After a 10-min incubation on ice, lysates were centrifuged for 5 min at 15,000 g . Solubilized fractions were set aside while insoluble fractions were washed twice with PBS and resuspended in equal volume as soluble fractions (60 μL). SDS loading buffer was added to both soluble and insoluble fractions and 30-μl aliquots were separated in 7.5% SDS-PAGE gels. We did not boil the samples, since that consistently produced aggregates of hslo α subunits that would run as high molecular weight complexes. Proteins were transferred to Immun-blot PDVF membranes (Bio-Rad Laboratories), and hslo was detected by Western blotting with 10 μg/ml anti-FLAG monoclonal antibodies (Sigma-Aldrich). Anti–mouse IgG secondary antibodies conjugated to peroxidase (Sigma-Aldrich) were used at 1:10,000 dilution and the signal was developed with the ECL+ system (Amersham Pharmacia Biotech).

Electrophysiological Recordings

CHO cells were grown on 12-mm glass coverslips (Fisherbrand) in α-MEM (GIBCO BRL) with 10% FBS and transfected with plasmid DNA and Lipofectamine 2000 (GIBCO BRL). 18–24 h after transfection, fluorescent cells expressing hslo-YFP or hslo-CFP were assayed for hslo function. Recordings were done in the cell-attached or inside-out patch clamp configurations ( Hamill et al., 1981 ) at 22–24°C. Patch pipettes were made of borosilicate glass (Kimax) and had resistances of 1–3 MΩ. Data were acquired using an EPC-9 amplifier and Pulse acquisition software (HEKA Electronik). Records were digitized at 50-μs intervals. Capacitance and leak currents were subtracted using a P /5 leak subtraction protocol. Conductance–voltage ( G-V ) curves were obtained by measuring the amplitude of tail currents 500 μs after repolarization to −70 mV from the various test voltages. Current levels were highly variable between patches; however, for all functional constructs, the maximal currents at 120 mV were consistently in the range of 500 pA to 5 nA, ∼60% the amplitude obtained with wild-type hslo channels. Currents obtained at all concentrations within a patch were normalized to the maximum peak current at 100 μM Ca 2+ and G-V curves were fitted to a Boltzmann function using Igor Pro software (Wavemetrics Inc.). Values in the text are given as mean ± SEM. Recording solutions contained (in mM): pipette, 80 KMeSO 3 , 60 N -methylglucamine-MeSO 3 , 20 HEPES, 2 KCl, 2 MgCl 2 (pH 7.4); bath solution, 80 KMeSO 3 , 60 N -methylglucamine-MeSO 3 , 20 HEPES, 2 KCl, 1 HEDTA, and CaCl 2 to give the appropriate free Ca 2+ concentration (pH 7.4). No Ca 2+ chelator was used in solutions containing 100 μM free Ca 2+ or higher. To prevent Ba 2+ block at high voltages ( Diaz et al., 1996 ), 50 μM (+)-18-crown-6-tetracarboxylic acid (18C6TA) was added to all bath solutions. The amount of total CaCl 2 needed to obtain the desired free Ca 2+ concentration was calculated using the program Max Chelator ( Bers et al., 1994 ), which was downloaded from www.stanford.edu/~patton/maxc.html . Free Ca 2+ was measured with a Ca 2+ -sensitive electrode (Orion electrode, Thermo Labsystems). Solutions were exchanged using the DAD-VC superfusion system from ALA Scientific Instruments.