Mutations of Kv4.2 C-terminal phosphorylation site at S552 (Kv4.2S552A and Kv4.2S552D) were performed using the Quick-Change site-directed mutagenesis kit (Stratagene, San Diego, CA). We mutagenized the serine (S) to Alanine (A) to remove the phosphorylation of PKA site or to Aspartic acid (D) to mimic constitutive phosphorylation at site 552 on construct Kv4.2-GFP (Kv4.2, GFP tagged at C-terminus). Mouse KChIP4a was produced by PCR amplifying mouse cDNA using 5’-GGGAATTCGATGAACTTGGAGGGGCTTG and 5’-GGTCTAGAGATCACATTTTCAAAGAG primers (GenBank accession number AF453243) and cloned into p3xFlag-CMV vector (Sigma, St. Louis, MO). Since there is no satisfactory commercially available antibody against KChIP4a, we used a Flag tag for detection. KChIP1, KChIP2a and human KChIP4a constructs were kindly provided by Dr. Pfaffinger, Baylor College of Medicine. Dr. Covarrubias, Thomas Jefferson University, kindly provided the Kv4.3 construct and the pcDNA-AKAP79 construct was kindly provided by Dr. Dell'Acqua at University of Colorado. All constructs were confirmed by sequencing analysis.
Cell Western Assays
Assays were performed as described by the manufacturer (LI-COR Biosciences application note (2004)). Briefly, 0.2×106 of COS7 cells were plated into 24-well tissue culture plates. The cells were co-transfected with wild type Kv4.2 or Kv4.2S552A with KChIP4a-Flag for 24-48 hr using Fugene 6. COS7 cells were fixed with PBS containing 4% paraformaldehyde. After blocking with Odyssey blocking solution for 1-1.5 hr at RT, cells were incubated with mouse anti-Kv4.2 antibody (1:200, NeuroMab, Davis, CA) in Li-COR blocking buffer at 4°C for over night. Cells were washed and incubated with the secondary antibody IRDye 800 Goat anti-mouse (1:1000, Rockland, Gilbertsville, PA) at 37 °C for 1 hr. After a wash in PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. We used rabbit anti-beta actin antibody (1:1000, sigma) and goat anti-rabbit secondary antibody IRDye 680 (1:800, Invitrogen) to detect actin. The intensity of the 700-nm and 800-nm infrared signal for each well was quantified using the Odyssey infrared imaging system software (LI-COR Biosciences, Lincoln, NE).
Co-immunoprecipitation and Western Blotting
COS7 cells were co-transfected with wild type Kv4.2 or Kv4.2S552A with KChIP4a-flag. To confirm an interaction between AKAP79 and Kv4.2 or KChIP4a, COS7 cells were transfected with Kv4.2 alone as control or co-transfected with AKAP79 and either KChIP4a or Kv4.2. After 24-48 h transfection, cells were lysed in lysis buffer: 150 mM NaCl, 20 mM Tris-HCl, 1% NP40, 0.5% SDS and protease inhibitor mixture (Roche, Indianapolis, IN). Anti-Flag (2μg/500μg protein, Sigma, St. Louis, MO), nonspecific IgG (Invitrogen) or anti-AKAP79 antibody (2ug/500ug protein, Millipore, Bedford, MA) was then added to the centrifuged detergent lysate. The mixture was then incubated and rotated at 4°C for overnight. The antibody-antigen complex was immobilized by adsorption onto 50 μl of immobilized protein A (Pierce, Rockford, IL) and incubated for 2 h at RT. The protein-bead mixtures were washed six times with lysis buffer. The beads were resuspended in reducing SDS sample buffer and analyzed on 10% SDS polyacrylamide gels. The separated proteins were immuonoblotted using Kv4.2 (1:2000, NeuroMab, Davis, CA) or Flag antibody (1: 6000, Sigma) for KChIP4a and AKAP79 antibody (1:1000) and visualized by Alexa Fluor 800 secondary antibody (1:10,000, Rockland, Gilbertsville, PA) and Alexa Fluor 680 secondary antibody (1:10,000, Invitrogen). Immunoreactivity was detected with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska). Quantification of results was performed using Odyssey software (LI-COR Biosciences, Lincoln, Nebraska).
Biotinylation assays were performed as previously described (Kim et al., 2007
). Briefly, transfected COS7 cells were rinsed with ice-cold PBS, surface protein were biotinylated with 1.5mg/ml sulfo-NHS-SS-biotin reagent (Pierce, Rockford, IL) in PBS for 30 min on ice. Unbound biotin was quenched with cold 50 mM glycine in PBS. Cells were lysed with ice-cold lysis buffer: 150 mM NaCl, 20 mM Tris-HCl, 1% NP40 and protease inhibitor mixture (Roche, Indianapolis, IN), sonicated and centrifuged at 12,000 g for 10 min. Cell lysates were incubated overnight at 4°C with immobilized-Streptavidin agarose beads (Pierce, Rockford, IL), unbound protein was removed from the beads with 5 washes in lysis buffer. The bound proteins were eluted with 2×SDS sample buffer. Surface expressed proteins were separated by electrophoresis on 10% Tris-bis SDS-PAGE (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes. Western blots were probed with the following antibodies: mouse anti-Kv4.2 (1:2000, NeuroMab, Davis, CA), mouse anti-GAPDH (1:1000, Calbiochem, San Diego, CA). Secondary antibodies conjugated to infrared dyes (Rockland Immunochemicals, Gilbertsville, PA) were detected using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Quantification of results was performed using Odyssey software.
To study the effect of KChIP4a on the stabilization of Kv4.2 protein complexes, we transfected COS7 cells with Kv4.2 or Kv4.2S552A along with KChIP4a, KChIP1 or control (empty vector) for 24 hr, then treated with 10 μM of forskolin (Sigma) for 24 hr at 37°C. After washing with ice-cold PBS, the cells were solubilized in 500 μl of lysis buffer (described above). Lysates were centrifuged for 10 min at 20,000 g rpm at 4°C after shaking for 2 h at 4°C. 20 μl of the supernatant was analyzed by 10% SDS-PAGE. Kv4.2, KChIP4a and KChIP1 were immuonoblotted using Kv4.2 antibody (1: 2000, NeuroMab, Davis, CA), Flag antibody (1: 6000, Sigma) and KChIP1 antibody (1:1000, NeuroMab, Davis, CA), then visualized by anti-rabbit Alexa Fluor 800 secondary antibody (1:10,000) and anti-mouse Alexa Fluor 680 secondary antibody (1:10,000). Detection and quantification was performed using the Odyssey infrared imaging system and its software as described above.
Rat hippocampal neuron culture and immnofluorescence staining
Primary hippocampal neurons cultures were prepared as previously described (Kim et al., 2007
). Neurons were infected by a modified sindbis virus expression system (Kim et al., 2004
). Briefly, hippocampi from E18-19 rat embryos were triturated after trypsinization and suspension with MEM plus 10% FBS plating media following by plating on Poly-DLysine and Laminin coated glass coverslips (BD, San Jose, CA) in a 24-well plate. After 5-6 hr, the media was replaced with Neurobasal media plus B27 supplements (Invitrogen). Cultures were maintained at 37°C with 10% CO2
. Neurons (DIV18-21) were infected with Kv4.2g (Kv4.2-GFP, see (Kim et al., 2005
) ) sindbis virus for 1 hr. The next day, neurons were fixed with PBS containing 4% paraformaldehyde, and 0.12 M sucrose for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min, endogenous AKAP150 was immunostained with anti-AKAP150 antibody (1:100, Millipore, Bedford, MA) for 1 h at 37°C, washed with PBS and then incubated with Alexa Fluor 555-conjugated goat anti-rabbit (1:500, Invitrogen) in DPBS with 5% NGS, 0.05% triton X-100 for 1 h at 37°C. Coverslips were mounted onto glass slides with Hard Set mounting medium (Vector Laboratories, Burlingame, CA). Images were captured with Zeiss LSM510 confocal microscope (Carl Zeiss, Germany) using a 63X objective with 1.4 NA.
HEK 293 cells were plated on poly-D-lysine/laminin-coated glass coverslips (BD BioCoat) in 12-well dish at a density of 22-24 × 103 cells/well and transfected using the Fugene 6 system (Roche). Cells were transfected with Kv4.2 , Kv4.2S552A or Kv4.2S552D constructs, along with either mouse KChIP4a, KChIP1, KChIP2, AKAP79 or control plasmids. Recordings were made 24-48 hr after transfection. Coverslips containing transfected HEK 293 or COS7 cells were submerged in the recording chamber and exposed to a continuous flow of ACSF consisting of the following (mM): 145 NaCl, 5 KCl, 2 CaCl2, 1.3 MgCl2, 10 glucose, 10 HEPES. ACSF was perfused with 95% O2-5% CO2, and TTX (~0.5 μM, Sigma) was included to block any endogenous sodium current. An infrared differential interference contrast (IR-DIC) videomicroscopy system (Zeiss Instruments, Diagnostic Instruments) was used to visualize cells. Patch pipettes were pulled from thick-walled borosilicate glass (Warner Instruments) to achieve a tip resistance of 2-5 MΩ. Pipettes were filled with an internal solution containing (mM): 20 potassium chloride, 125 potassium gluconate, 10 HEPES, 4 NaCl, 0.5 EGTA, 10 phosphocreatine, 4 ATP, 0.3 TrisGTP. For the PKA inhibition and activation experiments, H89 (10 μM) and FSK (10 μM) was applied to HEK 293 cells in culture 1 h and 24h before and during recordings, respectively. Ht31(25 μM, Promega) inhibitor peptide was applied to cultures 1h before and during recordings in some experiments.
Whole-cell voltage clamp recordings were made using a Multiclamp 700B amplifier (Molecular Devices) and Clampex 10.1 software (Molecular Devices). Signals were digitized at 10 kHz with a Digidata 1440A (Molecular Devices) and filtered at 4 kHz. A-type K+ currents were elicited by delivering a 400 ms prepulse from the holding potential of -60 mV to -120 mV followed by a 500 ms step to +120 mV. This protocol was repeated 20-45 times for each cell to determine the average peak A-current. Leak currents were subtracted online by a P/6 procedure. Only cells with a holding current greater than -100 pA were selected for recordings. Series resistance ranged from 4-11 MΩ, and was monitored throughout the experiments. All recordings were performed at room temperature. Recordings in which the series resistance increased by more than 25% of the initial value were excluded from analysis. No series resistance compensation was employed. Recordings were analyzed using Clampfit 10.1 (Molecular Devices) and Microsoft Excel. To determine A-current density, peak currents were normalized to whole-cell capacitance. To estimate the time constant of channel inactivation (inactivation rate), current traces were fitted with a single exponential function. Statistical significance was evaluated using Student's t-test (unpaired, two tails).