Kv4.2 deletion mutant (Kv4.2ΔPDZ
) lacking the C-terminal PDZ domain, which includes the four amino acids, VSAL, was prepared by PCR using the primers 5′GGAGGAAATATCGTCAGGCCTCCGTCGACGGTACCGC (forward) and 5′-GCGGTACCGTCGACGGAGGCCTGACGATATTTCCTCC (reverse) on construct Kv4.2-GFP. The truncated C-terminal deletions of Kv4.2 were generated by PCR using the forward primer 5′-CGAATTCTGGGGTACACCCTGAAGAGC and reverse primers F (1-560) 5′-GTCGACGGCTGAATTGTACTGAGTTCTTG; F (1-490) 5′-GTCGACGGGTTCGTGGTTTTCTCCAGGCAGTG; F (1-417) 5′-GTCGACGGTTGGTTTTGGTGGTAGATCCG and F (1-406) 5′-GTCGACGGCACGATCACAGGCACGGGTAG and subcloned into the EcoR1-SalI sites of the pEGFP vector. Kv4.2S552A
mutation of Kv4.2 C-terminal phosphorylation site at S552 was described in Lin et al. (2010)
. Briefly, we mutagenized the serine (S) to Alanine (A) to remove the phosphorylation of PKA site at site 552 on construct Kv4.2-GFP. Both mutants were performed using the Quick-Change site-directed mutagenesis kit (Stratagene, San Diego, CA) and confirmed by sequencing analysis. pcDNA3 or pEGFPN1 vectors encoding AKAP79 Wild-type and mutations AKAP79ΔPKA, AKAP79ΔCaN, AKAP79ΔPIX(deletion of the 7 residues composing the PXIXIT-like motif in CaN binding region), AKAP79ΔMAGUK, AKAP79 fragments 1-153, 150-427 and AKAP150shRNAi constructs were as previously described and characterized (Dell’Acqua et al., 2002
; Gomez et al., 2002
; Oliveria et al., 2003
; Hoshi et al., 2005
; Robertson et al., 2009
Rat hippocampal neuron culture and expression
Primary hippocampal neurons cultures were prepared as previously described (Kim et al., 2007
). Neurons were infected with Kv4.2Myc Sindbis virus (Hammond et al., 2008
) using a modified Sindbis virus expression system (Kim et al., 2004
). Briefly, cultured DIV14 hippocampal neurons were infected with Kv4.2Myc virus for 1h at 37 °C. Neurons were transfected by using the Nucleofector System (Amaxa, Gaithersburg, MD). Cultured hippocampal neurons (6×106
) were resuspended in 100 μl of rat neuron Nucleofectorsolution with 10 μg of DNAs, electroporated using the O-03 program, then plated on poly-D-lysine and laminin coated glass coverslips (BD, San Jose, CA) in a 24-well or 100mm culture plate in Minimum Essential Medium (MEM) (Invitrogen) supplemented with 10% FBS, penicillin and streptomycin. After 5–6 h, the medium was replaced with Neurobasal medium plus B27 supplements (Invitrogen). Cultures were maintained at 37°Cwith 10% CO2
Co-immunoprecipitation and Western Blotting
To confirm an interaction between Kv4.2 and AKAP79/150, we performed co-IP experiments either in native, detergent-solubilized rat brain extracts or COS7 cells co-transfected with various WT and mutant Kv4.2 and AKAP79 constructs for 24–48 h. Rat brain or 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-Kv4.2 (2μg/500μg protein, NeuroMab, Davis, CA), nonspecific IgG (Invitrogen) or anti-AKAP79 antibody (2ug/500ug protein, Millipore, Bedford, MA) was then added to the 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) and AKAP79 antibody (1:1000) and visualized by Alexa Fluor 680 secondary antibody (1:10,000, Invitrogen) and Alexa Fluor 800 secondary antibody (1:10,000, Rockland, Gilbertsville, PA). 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).
On cell Western assays
Assays were performed as described by Lin et al (2010)
. Briefly, 0.1×106
of rat or mouse (for Kv4.2−/−
experiments) hippocampal neurons were plated into 24-well tissue culture plates. DIV14 cultured hippocampal neurons were treated with either 10μM Ht31 control or Ht31 peptide for 15 min at 37 °C and then were fixed with PBS containing 4% paraformaldehyde. After blocking with Odyssey blocking solution for 1–1.5 hr at RT, neurons were incubated with mouse anti-Kv4.2 antibody (1:200, NeuroMab, Davis, CA) or anti-Myc antibody (1:100, Sigma) in Li-COR blocking buffer at 4°C for overnight. 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).
Biotinylation assays were performed as previously described (Kim et al., 2007
). Briefly, either DIV14 culture hippocampal neurons or 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 100 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 15 min. Cell lysates were incubated overnight at 4°C with immobilized-Streptavidin agarose beads (Pierce, Rockford, IL), after washed 5 times in lysis buffer; the bound proteins were eluted with 2×SDS sample buffer. Surface expressed Kv4.2 was separated 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), rabbit anti-GAPDH (1:1000, Calbiochem, San Diego, CA) and rabbit anti-beta-actin (1:1000, Abcam, Cambridge, MA). 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.
For patch-clamp recordings, coverslips containing DIV14 hippocampal primary culture neurons were transferred to a submerged recording chamber with a continuous flow of extracellular solution containing (mM): 145 NaCl, 3 KCl, 10 HEPES, 2 CaCl2, 8 glucose, 2 MgCl2 (pH 7.2 with NaOH). Hippocampal primary culture neurons were visualized by using infrared differential interference contrast (IR-DIC) videomicroscopy system (Carl Zeiss, Germany). The patch pipettes (2~5 MΩ) were filled with an internal solution containing (mM): 20 KCl, 125 K-gluconate, 10 HEPES, 4 NaCl, 0.5 EGTA, 10 phosphocreatine, 4 ATP, 0.3 TrisGTP (pH 7.2 with NaOH). For some experiments, 50μM Tris-HCl (no peptide control), 10 μM Ht31 control peptide, 50 nM rapamycin (FK506 control, Sigma), Ht31peptide (Promega, Madison, WI) or 4μM FK506 (Tocris Bioscience, Ellisville, Missouri) were included in the internal solution to treat neurons for ~ 20min before recording. All patch-clamp recordings were made by using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) and Clampex 10.1 software (Molecular Devices). Signals were digitized at 10kHz with a Digidata 1440A (Molecular Devices) and filtered at 4 kHz.
Nucleated patch recordings of voltage-gated K+ currents were made in voltage-clamp mode at room temperature. TTX (0.5 μM, Tocris Bioscience) was included in the bath solution. Only neurons with holding currents greater than −100 pA were selected for recording or analysis. Membrane potential holding potential was −60 mV while peak current was measured at +60 mV. Current ensemble averages were constructed from 15 to 30 sweeps. Leakage currents were subtracted digitally by using P/6 protocol. Transient current was isolated from sustained current using a 150 ms prepulse step to −20mV to inactivate transient channels. To determine A-current density in nucleated patches, peak currents were normalized to membrane capacitance.
Neuronal firing properties were recorded in current-clamp mode at 31~33°C. Series resistance was 5~12 MΩ. Neurons in which resting membrane potential changed by more than ±5mV of the initial value were excluded from analysis. Current injection series were repeated 10 times and the results reported as the average of the 10 individual measurements. For current clamp analyses, the numeric values (AP onset, threshold etc.) were measured using a +200pA current injection. The data were taken from the first AP initiated upon current injection, which was determined using the first derivative of the voltage trace with respect to time. Peak and steady-state potentials were measured from the maximal hyperpolarization and the voltage at the end of a 1000 ms, −200 pA current step, respectively. “Sag” potential is the difference between peak and steady-state voltages.
All patch clamp recordings were analyzed using Clampfit 10.1 (Molecular Devices) and Microsoft Excel (Microsoft Corp., Redmond, WA). Statistical significance was evaluated using Student’s t-test (unpaired, two tails). p values are reported in the text or in the Figure Legends with values less than 0.05 considered significant.