Generation of mutants
Constructs encoding mutant maxi-K channel forms were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Primers used are as follows:
Forward 5'-GGTATAATATGCTTTGTGCTGGAATTTACCGGCTGAGAGATGCG-3', Reverse 5' CGCATCTCTCAGCCGGTAAATTCCAGCACAAAGCATATTATAGG-3';
Forward 5'-GCA AAGCTCTGAAAACAGCTAATATGCTTTGTGCTGG-3',
Reverse 5'-CCAGCACAAAGCATATTAGCTGTTTTCAGAGCTTTGC-3'; and
Mutant Y1007A, F1015A, Y1015A,
For both patch-clamping and immunocytochemistry experiments, mutants were cloned into the pTracer expression vector, (Invitrogen, Carlsbad, CA) between restriction sites Not1 and Xba1.
Human myometrial tissue from the lower uterine segment was obtained from patients who, in the absence of spontaneous or induced labor contractions, underwent elective Cesarean section in late pregnancy (NL; 38-40 wk gestation) while under spinal anesthesia. All patients signed written consent forms approved by the University of Iowa's Internal Review Board (approval no. 199809066). Tissue was placed in Hanks' balanced salt solution or in phosphate-buffered saline (PBS) on ice, and was used to isolate hMSMCs within 1-3 h of collection.
Isolation, culture, transfection, and infection of cells
Human MSMCs were isolated and cultured as previously described , and were then utilized to study the effects of caveolins on endogenous cells by patch clamp analysis The adenoviral constructs containing caveolin-1 and caveolin-1-scrambled siRNAs were a gift from Dr. Debbie Thurmond (Indiana University School of Medicine), and their production was previously described  (Viraquest, North Liberty, IA). Cells were infected with 1 μl of 1.0 - 1.1 × 1012 particles/ml of the purified siRNA adenoviral constructs and incubated in the presence of the virus for an additional 72 hours before experiments were carried out. Transduction efficiency was gauged by EGFP fluorescence every 24 hours, which was typically greater than 90% by 72 hours.
The mouse Ltk- fibroblasts (Ltk-)  used for patch clamp, Western blot, immunoprecipitation and immunofluorescence studies were generated by transfecting cells of the Ltk- parent line, following growth to 50-80% confluency in T-75 flasks, 6-well or 12-well dishes, in DMEM/F12 (Sigma, St. Louis, MO) supplemented with 10% FBS (Gibco-BRL, Carlsbad, CA) and 50 μg/ml gentamicin (Sigma). Ltk- cells were transfected with 10 μg (T-75), 2 μg (6-well) or 1 μg (12-well) of plasmid DNA (LipofectAMINE PLUS Reagent Kit, Invitrogen), and incubated an additional 48 hours before the experiments were carried out. The Ltk- cells utilized for these experiments lack endogenous maxi-K channels but contain cav-1; this enabled us to study each caveolin-binding mutant without contamination from the endogenous channel.
Preparation of lipid rafts
Ltk- cells transfected with plasmid DNA (one T-75 flask) were trypsinized (0.25% TE/EDTA, Gibco-BRL) and spun down at 300 × g for 5 min. Cells were resuspended in 200 μl of Mes-buffered saline (MBS; 24 mM Mes, 150 mM NaCl, pH 6.5) plus 1% Triton X-100 and a Complete Protease Inhibitor tablet (Roche, Indianapolis, IN), incubated for 30 min on ice and dounce homogenized on ice for 1 min. A 100 μl aliquot was transferred to a microcentrifuge tube, and 300 μl of 53.33% sucrose/MBS was added and mixed in. 450 μl each of 30% and 5% sucrose/MBS were layered above this mixture, in this order, and the samples were centrifuged for 24 hours at 54,000 rpm, at 4°C. After this spin, 10 equal fractions were collected, starting at the top. We controlled for the presence of non-lipid membranes by monitoring the presence of the human transferrin receptor by Western blot, as this protein does not associate with lipid rafts.
Preparation of beads and immunoprecipitation
Protein G-Plus Agarose beads (Santa Cruz, Santa Cruz, CA) were incubated in 4 ml Dulbecco's Phosphate Buffered Saline, DPBS, (Gibco-BRL) with 1% BSA, 1% Normal Donkey Serum and 1% Normal Rabbit Serum, for 2 hours at 4°C. The beads were washed 1× with DPBS and resuspended in 1 ml DPBS with 0.025% Sodium Azide. 50 μl of each floating fraction were incubated in 1 ml Solubilization Buffer (10 mM Tris pH 8.0, 150 mM NaCl, 1% TX-100, 60 mM Octyl-glucopyranoside) plus a Complete Protease Inhibitor Tablet (Roche), for 45 min at 4°C, and were then mixed by nutation. These samples were spun down at 3500 × g for 5 min at 4°C, and the supernatants were transferred to new tubes. 50 μl of the prepared beads were added, and the samples were agitated overnight at 4°C. One μg of a maxi-K channel antibody (BD Biosciences, San Jose, CA) was then added, and immunoprecipitates were gently mixed overnight at 4°C. The samples were spun down at 3500 × g at 4°C, and washed 3× with 1 ml Buffer A (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% TX-100), after which the bead fractions (representing the immunoprecipitates) were resuspended in 40 μl of 2× sample loading buffer (4X: 10% Glycerol, 0.25 M Tris pH 7.0, 3% SDS, 5% 2-Mercaptoethanol, Bromophenol Blue).
The immunoprecipitates and mini lipid-raft fractions were separated by SDS-PAGE and immunoblotted as previously described . The primary antibodies used were one against the mouse maxi-K channel (1:250) (BD Biosciences), one against mouse cav-1 (1:1000) (BD Biosciences) and one against the mouse transferrin receptor (1:1000) (Zymed, South San Francisco, CA). The secondary antibody used in all cases was goat anti-mouse (1:3000) (Jackson Immunoresearch, West Grove, PA). All antibodies were diluted in PBST (1.37 mM NaCl, 81.01 mM Na2HPO4, 26.82 mM KCl, and 14.7 mM KH2PO4, 0.5% Tween, pH 7.2) containing 3% non-fat dry milk. Immunoreactivity was detected using HyGlo Western Blotting detection reagents (Denville Scientific Inc., Metuchen, NJ).
Ltk- cells transfected with plasmid DNA were fixed with 2% paraformaldehyde for 30 min at RT, quenched with 50 mM NH4Cl for 10 min at RT, permeabilized with 0.1% Triton X-100 for 10 min at RT, and blocked with 10% heat-inactivated FBS + 1% heat-inactivated donkey serum (blocking buffer) for 30 min at 37°C (all diluted in DPBS). For co-localization studies, Ltk- cells were incubated with rabbit maxi-K channel antibody (1:250; Millipore, Temecula, CA) for 1 hour at 37°C, and then with a Cy3-conjugated donkey anti-rabbit (1:1000; Jackson Immunoresearch) for 15 min, 37°C. They were then incubated for 15 min in blocking buffer for at 37°C, for 30 min in blocking buffer plus a mouse anti-caveolin antibody (1:500; BD Biosciences) for 30 min at 37°C, and finally for 15 min in blocking buffer plus a Cy5-conjugated donkey anti-mouse (1:1000; Jackson Immunoresearch) for 15 min at 37°C. Signals were visualized using a Zeiss 510 confocal scanning microscope (Zeiss, Oberkochen, Germany), and images were acquired with a Zeiss LSM 5 Image Browser (Zeiss). For controls, Ltk- cells were incubated with primary antibodies alone (30 min at 37°C) or secondary antibodies alone (15 min at 37°C). In general, Ltk- cells do not generate high background when an anti-mouse secondary antibody is used. Moreover, any background issues due to non-specific binding of the secondary antibody would be discovered by performing the experiments with the secondary antibody alone. Bleedthrough of fluorescent signals into the neighboring channels was controlled for by turning off all but one laser at a time, and recording images in all channels. Laser power, pinhole size and detector gain were then adjusted to ensure that the image appeared in its respective channel only. A nuclear counterstain was not performed since confocal microscopy of these cells showed sufficient morphological detail to identify individual cells.
All patch-clamping experiments were performed at RT. Whole-cell current was measured using an Axopatch 200-B amplifier (Molecular Devices, Sunnyvale, CA). Signals were filtered with a cutoff frequency of 5 kHz. Data acquisition was controlled using commercial pClamp 9.2 software (Molecular Devices), and data were digitized using a Digidata 1320 interface (Molecular Devices). Ltk- cells expressing wild-type and mutant channels (see Transfections), and hMSMC in which the caveolin-1 gene was inhibited (see siRNA) were placed in a pH 7.4 bath solution containing, in mM: 135 NaCl, 4.7 KCl, 1 MgCl2, 10 Glucose, 2 CaCl2 and 5 Hepes. Borosilicate glass pipettes of 2-5 MΩ were filled with a pH 7.2 solution containing, in mM: 140 KCl, 0.5 MgCl2, 1 EGTA, 5 ATP and 5 Hepes. Cells expressing GFP (the reporter for the plasmid and siRNA expression vector used) were clamped, currents were measured using a holding potential of -80 mV and prepulsing to -100 mV, and currents were elicited at step potentials from -80 to 160 mV in 20-mV intervals.
Each experiment was performed in a minimum of X independently transfected/infected samples. Statistical significance was calculated using Student's t-test for unpaired observations (siRNA and caveolin binding-site mutation experiments) and paired observations (for IbTX experiments; SigmaPlot software; SPSS, Chicago, IL). Differences were considered significant at P < 0.05.