The E.coli MscL and MscS coding sequences were a generous gift from Boris Martinac (Victor Chang Cardiac Research Institute, Australia). Both cDNAs were sub-cloned into pIRES2-eGFP (Clontech). The MscL-FLAG construct in pIRES2-eGFP was made by insertion of the FLAG-tag sequence (DYKDDDDK) after position I68 of E.coli MscL. MscL G26C mutagenesis was performed using overlap-extension PCR with synthetic complementary oligonucleotides. All cDNA constructs were verified by DNA sequencing.
Cell lines and transfection
CHO, HEK-293, HeLa and COS-7 cells were transfected using Lipofectamine 2000 (Invitrogen), plated onto glass cover slips, and used for electrophysiological recordings and/or delivery experiments 18 - 48 h after transfection. CHO cells stably expressing the E.coli MscL wild type construct (CHO-MscL-WT) or MscL G26C (CHO-MscL-G26C) in pIRES2-eGFP were obtained after transfection with the respective cDNA, selection of stable cells with G-418 (Invitrogen), and further selection by fluorescence-activated cell sorting (FACS) of GFP positive cells. To obtain a monoclonal MscL G26C CHO cell line, FACS sorted cells were diluted and selected in 96-well plates for single colony growth. Colonies were subsequently picked and screened for uniform dye uptake.
The channel activities of recombinantly expressed E.coli
MscL and MscS were examined in excised patch recordings in symmetric potassium (in mM; 150 KCl, 20 HEPES, 1 EGTA, pH 7.4). The bath solution in outside-out patch recordings was equivalent to the solution used for delivery experiments (in mM; 140 KOH, 100 aspartate, 12 NaOH, 4 HCl, 1 MgCl2
, 2 Mg-ATP, 10 HEPES, pH 7.3). Electrodes with a resistance of 1.5 to 2.5 MΩ and a pipette bubble number of 5.5 to 6.537
were pulled from borosilicate glass (WPI) using a Flaming/Brown pipette puller (Sutter Instruments). Pressure steps and ramps were applied using a HSPC-1 high speed pressure system (ALA Scientific)38
controlled by pClamp software (Molecular Devices). A P-V pump unit was used as a pressure/vacuum source (ALA Scientific). Bath temperature was regulated using a bipolar temperature controller and an in-line heater/cooler with continuous perfusion (Warner Instruments). The bath temperature was monitored using a thermistor placed in close proximity to the recording electrode. Membrane voltage was controlled and currents recorded using an Axopatch 200B amplifier (Molecular Devices). All recordings were low-pass filtered at 5 kHz and acquired at 10 or 20 kHz. Data analysis was performed off-line using Clampfit 9 (Molecular Devices) and Origin 7 (OriginLab) and currents further digitally filtered off-line for display purposes. Data are presented as mean ± SEM. Statistical comparison was made using Student's unpaired t-
value <0.05 was considered statistically significant). The normalized current - pressure relation for MscL and MscS was fitted to a Boltzmann function (I = Imax
)/(1 + exp ((P – P0.5
)), where Imax
are the maximum and minimum current values, P is the test pressure, P0.5
is the pressure activation midpoint, and k
is the slope factor). Single channel conductance was calculated from the slope of linear regression fits and the open probability (NPOPEN
) after LPC treatment was computed by the equation NPOPEN
) using Clampfit 9 (Molecular Devices).
For a live-cell staining of CHO cells transfected with the MscL-FLAG construct, cells were incubated for 30 min at 4°C with monoclonal anti-FLAG M2 IgG (2 μg/ml, Sigma). Non-specific binding was blocked by addition of 2% goat serum and 2% BSA (Sigma). Cells were washed 3× in blocking buffer followed by incubation for 30 min at room temperature (RT) with an Alexa Fluor 546 conjugated anti-mouse IgG secondary antibody (2 μg/ml, Invitrogen). For staining of fixed cells (4% paraformaldehyde, 45 min, RT), MscL-FLAG transfected cells were incubated in 0.1% Triton X-100 in PBS for 45 min at RT and nonspecific binding blocked by subsequent incubation in 2% goat serum and 2% BSA for 45 min at RT. Labeling was carried out by incubation with anti-FLAG IgG (2 μg/ml, 1h, RT) followed by anti-mouse IgG secondary antibody (2 μg/ml, 30 min, RT). Images were acquired with a FluoView-1000 laser scanning confocal microscope (Olympus).
In a typical delivery experiment, stably or transiently transfected cells were plated on glass cover slips 24 - 48 h prior to the experiment. After removal from the 37°C, 5% CO2 incubator, cover slips were placed in Ringer solution (in mM; 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4) and kept at RT for the entire length of the experiment. The cells grown on a cover slip were first briefly rinsed in the K-aspartate based delivery solution (in mM; 140 KOH, 100 aspartate, 12 NaOH, 4 HCl, 1 MgCl2, 2 Mg-ATP, 10 HEPES, pH 7.3) and subsequently incubated with MTSET (1 mM) to activate MscL G26C and allow for delivery of the desired molecule. Channel closure was achieved after a brief rinse in K-aspartate based solution by incubation with DTT (1 mM) for 10 min. MTSET was diluted from a 10 mM stock, which was freshly prepared before each use and kept on ice. After delivery of the respective molecule (dye, dye-dextran conjugate, phalloidin), cover slips were transferred to a live imaging chamber in Ringer solution and imaged by laser scanning fluorescence confocal microscopy (Fluoview-1000, Olympus). Confocal images were analyzed using Image-J software (NIH). The delivery efficiency of different size molecules at discrete MscL activation times was measured by analyzing the average pixel intensity in cells per field. First, images were background-subtracted. For each field of view analyzed, a mask of the areas corresponding to cells expressing MscL was created by thresholding of the GFP channel (the construct being expressed from a bicistronic IRES-GFP vector). This mask was subsequently applied to the fluorescence image of the delivered molecule (typically a Texas Red or Alexa Fluor 594 conjugate), followed by calculation of the mean fluorescence pixel intensity. The mean fluorescence intensity within cells for the whole field was calculated for five independent fields of view and averaged. To avoid detector saturation, acquisition parameters (e.g., PMT voltage, confocal aperture) were established first for the conditions giving maximum dye fluorescence (8 min activation), and kept constant across all experiments.
The metabolic integrity of cells after activation of E.coli MscL for different durations was assessed using MTT following the manufacturer's instructions (Cayman Chemical). Briefly, cells were seeded at 0.5 × 105/ml per well in 96-well plates and cultured in a CO2 incubator at 37°C for 24 h. Following the typical delivery protocol, cells were treated for different durations with MTSET (0 - 20 min) to induce channel opening in MscL G26C expressing cells, but not MscL WT expressing control cells. To further control for side effects of the delivery solution or the reducing agent, both cell lines were treated with DTT alone (1 mM, 10 min) or incubated in the K-aspartate based delivery solution for 20 min followed by a 10 min DTT treatment. Cells were subsequently placed in fresh growth media (DMEM/F12) and incubated at 37°C, 5% CO2 for 24 h. MTT absorbance was measured using a microplate reader (PerkinElmer). Cell survival data were collected 2-3 independent times in quadruplicate, background-subtracted and normalized to untreated control cells.
MTSET was purchased from Toronto Research Chemicals, DTT and lysophosphatidylcholine (LPC) from Sigma and Alexa Fluor 594 hydrazide, Texas Red dextran (3,000 and 10,000 Da) and Alexa Fluor 568 phalloidin were obtained from Invitrogen.