The use of animals in the present studies was in accordance with the National Institutes of Health guidelines for the humane care and use of animals in research, and was approved by the Institutional Animal Care and Use Committee of the University of Vermont.
Cell Culture and Transient Transfection
Postganglionic sympathetic neurons were obtained from superior cervical ganglia of neonatal rats. The ganglia (SCG) were dissociated with collagenase/hyaluronidase digest followed by a trypsin digest. Cells were plated onto type I rat tail collagen coated dishes (RT-PCR and western analyses) or coverslips (immunohistochemistry). The day after plating, non-neuronal cells were growth arrested with mitomycin C (1 hour, 10 μg/ml). All neuronal cultures were grown in neuronal growth medium (DMEM/F12 supplemented with 10% NuSerum, 5% FBS, 100 U/ml penicillin/streptomycin, and 50 ng/ml NGF). Vascular smooth muscle cells (VSM) were isolated from explants of adult postpartum Sprague Dawley rat tail arteries. VSM were grown in DMEM supplemented with 10% FBS, and 100 μ/ml penicillin/streptomycin. VSM were used at passage 2. Clonal human embryonic kidney 293 (HEK293) cells were maintained in DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum, 10 U/ml penicillin and streptomycin, and 2 mM L-glutamine. Cells were transiently transfected with 3.0 μg of GFP-Kv1.3/-Flag or GFP-Kv1.3-ΔTDV-Flag, using the Lipofectamine LTX transfection reagent (Invitrogen). Confluent transfected cultures were plated to a low density (25,000 cells/cm2) onto poly-D-lysine-coated tissue culture plates (Corning Glass Works) and subsequently placed in serum-free medium overnight for experimentation the following day.
All mutagenesis reactions were performed using the Quickchange Lightning Site-Directed Mutagenesis Kit (Strategene), following the manufacturer’s protocol. The GFP-Kv1.3-ΔTDV truncation was created by inserting two premature stop codons into the wild-type channel using the following primer sequence and its reverse complement: 5′-GTCAACATCATCAAAAAGATATTCTGATGAACTGATGTCTAATAGGGATCCACC-3′. Similar to previous reports [18
], the Flag epitope was created by inserting the amino acid sequence, DYKDDDDK, into the first extracellular loop of GFP-Kv1.3/-ΔTDV and incorporating D222 as the first aspartic acid residue in the Flag epitope using the following primer sequence and its reverse complement: 5′-TCTCCGTCGCAGGACTACAAGGACGACGACGACAAGGTGTTTGAGGCTGCC-3′.
Cultures of postganglionic sympathetic neurons and vascular smooth muscle cells were each rinsed in PBS and fixed in 4% formaldehyde in PBS for 12 min. Cultures were permeabilized with 0.2% Triton X-100 in PBS for 5 min and subsequently rinsed in PBS. Cells were blocked for 1 hour at room temperature in 3% goat serum, 0.1% fish skin gelatin in PBS. Cells were co-stained with anti-Kv1.3 mouse monoclonal antibody (0.84 μg/mL; NeuroMab) and anti-GM130 rabbit polyclonal antibody (0.7 μg/mL; Calbiochem) overnight at 4°C, followed by three 5 min washes in PBS. Alexa Fluor Goat anti-mouse (488 nm) and goat anti-rabbit (647) secondary antibodies (4 μg/ml; Invitrogen) were applied for 1 hour at room temperature.
To detect total Kv1.3, transiently transfected HEK293 cells were rinsed in PBS, fixed in 4% formaldehyde in PBS for 20 min, and subsequently permeabilized with acetone for 5 min and rinsed in PBS. Cells were then blocked for 20 min at 37°C in 3% goat serum, 0.1% fish skin gelatin in PBS. Cells were incubated 20 min at 37°C in a rabbit polyclonal GM130 antibody, raised against recombinant protein containing amino acids 371–990 of human GM130 (Lot # D00004465), to identify the Golgi apparatus and subsequently rinsed 3 × 5 min in PBS. Alexa Fluor goat anti-rabbit (647 nm) secondary antibody (4.0 μg/ml; Invitrogen) was applied for 20 min at 37°C.
To detect Kv1.3 on the cell surface, transiently transfected HEK293 cells were live labeled with anti-Flag M2 antibody (1μg/mL; Sigma) at 37°C for 15 min. Cells were then rinsed 3 × 5 min in PBS, and fixed, permeabilized and labeled as described above. Alexa Fluor goat anti-mouse (647 nm) and goat anti-rabbit (568 nm) secondary antibodies (4.0 μg/ml; Invitrogen) were used to detect Kv1.3 surface expression and GM130 localization, respectively. All cells were mounted using ProLong Gold antifade reagent (Invitrogen). All images were taken using the Olympus IX70 microscope and DeltaVision Restoration Imaging System (Applied Precision, LLC) and background subtracted with the appropriate IgG isotype controls (R&D Systems).
Golgi Localization was quantified as follows: Golgi region of interest (ROI) was defined using the GM130 marker and the amount of GFP-Kv1.3-Flag signal intensity present in the Golgi ROI was expressed as a percent of the total Kv1.3 signal for the entire cell (Kv1.3 in Golgi region/total Kv1.3) × 100). Surface expression was quantified as follows: the intensity of the live labeled anti-Flag M2 antibody was normalized to the total GFP-Kv1.3-Flag intensity of the cell (surface Kv1.3/total Kv1.3).
Cells were washed with ice-cold phosphate-buffered saline and lysed in radioimmune precipitation assay buffer (50 mM Tris, 150 mM NaCl, 11 mM EDTA, 0.25% deoxycholate, 1% Nonidet P-40, 10% glycerol, 1 mM NaF, 1 mM Na3VO4, 1 mM Na4BAPTA, 1mM dithiothreitol, protease inhibitors (Sigma, catalog no. P8340), phosphatase inhibitors (Calbiochem, catalog nos. 524624 and 524625), pH 8.0). Lysates were centrifuged at 20,000 × g for 5 min, and resulting supernatants were combined with sample buffer and separated by SDS-PAGE. Western blotting detection of Kv1.3 was done with anti-Kv1.3 monoclonal antibody (0.42 μg/mL; NeuroMab). A monoclonal anti-GAPDH antibody (1.0 μg/mL; Millipore) was used to control for loading efficiency. Blots were imaged and quantified with the Odyssey infrared imaging system (Li-Cor Biosciences).
Electrophysiological recordings were performed at room temperature, utilizing the whole cell patch clamp technique. Data acquisition and analysis were obtained using the Axopatch 200B (Axon Instruments) patch clamp amplifier and pCLAMP 9.2 (Axon Instruments) software. Electrodes were pulled in two stages from thin wall filament glass capillary tubing (Warner Instruments) and fire polished to a resistance ranging from 1 – 2 MΩ. Voltage clamp recording solutions were as follows (in mM): external (bath) solution 100 NaCl, 5.4 KCl, 1.8 CaCl2. 0.8 MgCl2, 23 glucose, 5 Na Hepes, pH 7.4; internal (pipette) solution: 120 KCl, 3.69 CaCl2, 0.094 MgCl2, 5 BAPTA, 5 EDTA, 5 Na HEPES, 5 glucose, pH 7.2. Cells were held at −60 mV, followed by a 20 ms hyperpolarization to −90 mV, and stepped from −70 mV to +50 mV in 10 mV increments. Leak currents (P/8) were subtracted from all traces.
pMAL-Kv1.3-C-term, pMAL-Kv1.3ΔTDV-C-term, and the empty pMAL vector were transformed into BL21 Gold bacterial cells (Strategene). Cultures were grown in LB supplemented with Glucose/Ampicillin and protein expression was induced by adding 0.3mM IPTG for 2 hours. Cells were harvested at 4000× g for 10 min and resuspended in Column Buffer (20mM Tris-HCl, 200mM NaCl, 1mM EDTA, 1mM sodium azide, 1mM DTT). Cells were lysed and the protein extracts (MBP-Kv1.3, MBP-ΔTDV or MBP) were coated onto Amylose resin (New England Biolabs). HEK293 cells transfected with pGW1-CMV-myc-PSD-95 were lysed in RIPA and precleared with MBP coated Amylose resin by rocking for 30 min at 4°C. Precleared myc-PSD-95 lysates were then rocked with either MBP-Kv1.3, MBP-ΔTDV or MBP coated Amylose resin for 30 min at 4 °C, washed 3x in RIPA, needle aspirated, combined with sample buffer and separated by SDS-PAGE. Western blotting detection of myc-PSD-95 was done with an anti-Myc monoclonal antibody (0.1 μg/mL; Invitrogen) and Kv1.3 was detected using an anti-Kv1.3 monoclonal antibody (0.42 μg/mL; NeuroMab). Blots were imaged and quantified with the Odyssey infrared imaging system (Li-Cor Biosciences).
HEK293 cells were cotransfected with soluble GFP, used as a marker of live cells with intact plasma membranes, and FLAG-Kv1.3 or FLAG-Kv1.3ΔTDV. Transfected HEK cells were treated with 0.5% sodium azide for 30 min at 37 °C to block endocytosis. Surface Kv1.3 was labeled with a monoclonal anti-Flag M2 antibody (1ug/mL; Sigma) directed against an extracellular epitope inserted within the channel. Secondary labeling was done with a fluorescently conjugated goat anti-mouse IgG (0.25ug/mL; Southern Biotech). Kv1.3 surface levels were quantified as the number of live cells (GFP positive) emitting at 667 nm with fluorescence intensity above a threshold value determined using cells labeled with a mouse IgG2a isotype control (R&D Systems). Flow cytometry was done with the Easycyte single laser flow cytometer (Guava Technologies). Analysis of cell populations and histograms was done with FCS Express and WinMDI softwares, respectively.
Cell Surface Biotinylation
HEK cells transiently transfected with either GFP-Kv1.3-Flag or GFP-Kv1.3-Flag-ΔTDV were rinsed 2 × 5 min in Hank’s Buffered Saline Solution (HBSS) at room temperature (RT). Cells were incubated with a 25mM solution of the cell impermeant EZ-Link Sulfo-NHS-SS-Biotin (Thermo Scientific) for 30 min at RT. Cells were then washed 4 × 5 min in HBSS containing 50mM Tris pH 7.4, lysed in RIPA buffer not containing dithiothreitol, and allowed to rock at 4°C for 10 minutes. Lysates were centrifuged at 20,000 × g for 5 min, and a portion of the supernatant was retained for SDS-PAGE analysis. The remaining supernatant was combined with prewashed NeutrAvidin Agarose Resin (Thermo Scientific) and allowed to rock at 4°C for 4 hours. The resin was centrifuged at 2,000 × g for 2 min, washed 3 x in RIPA buffer an needle aspirated. Bound proteins were eluted from resin, separated by SDS-PAGE, and processed according to the Immunoblot Analysis methods listed previously.
Data are presented as means ± standard errors. As noted, either nonparametric Mann-Whitney U tests, unpaired Student’s t-tests assuming unequal variances, or one-way ANOVA with post hoc Bonferroni test were used to determine statistical differences. Differences were considered significant if p < 0.05.