Neuronal cell culture and reagents
Primary hippocampal and cortical neurons were dissociated from newborn or E18 rats, respectively, and maintained in culture for 1-3 weeks as described previously . Primary cerebellum cultures with enriched Purkinje neurons were made as previously described . Anti-KChIP1 monoclonal antibody was commercially generated . Anti-SV2 was a gift from Dr. K.M. Buckley. Anti-synaptophysin, anti-calbindin and anti-enhanced green fluorescence protein (EGFP) were purchased from Sigma (St. Louis, MO) and Clontech (Palo Alto, CA), respectively.
The cDNA encoding KChIP1 was cloned into pEGFPN (Clontech) and pcDNA3.1(-)MycHis (Invitrogen, San Diego, CA) by PCR to generate plasmids expressing C-terminally tagged KChIP1-EGFP and KChIP1-mycHis in mammalian cells. Primers for PCR were: 5'-gggaattcgccaccatgggggccgtcatgggcacc-3' (forward) and 5'-ggggatccacatgacattttgaaacagctggag-3' (reverse). The coding sequence of KChIP1 was cloned into pGEX4T2 (Amersham, Uppsala, Sweden) to express a GST-KChIP1 fusion protein. All plasmids were confirmed by sequencing. For expressing KChIP1 in neurons, EGFP and KChIP1-EGFP cDNAs were excised from pEGFPN2 and pEGFPN2-KChIP1 plasmids by Eco RI and Not I digestion, respectively. The fragments were then cloned into a Sma I digested pSFV1 plasmid (Invitrogen).
Fusion protein and antibody preparation
GST-KChIP1 fusion protein was produced and purified following the manufacturer's protocol. The GST-KChIP1 protein was used to immunize rabbits in order to raise polyclonal anti-KChIP1 antibodies. Antibody production was performed by Immungenex (San Diego, CA).
In situ hybridization
Non-radioactive in situ hybridization was performed essentially as described previously . Briefly, adult mice (2-4 months) were perfused and fixed with 4% paraformaldehyde. Brains were dissected and fixed overnight. Fifty-micron thick cryo-cut sections were obtained. Antisense and sense RNA probes were in vitro transcribed and labeled using KChIP1 cDNA fragments as templates and a mixture of nucleotides containing digoxygenin-UTP. Hybridization was carried out at 65°C overnight. Signals were detected with an alkaline phosphatase-conjugated goat anti-digoxygenin antibody and developed using NBT/BCIP. Immunostaining was performed after in situ hybridization. Sections were analyzed under either deconvolution or confocal microscopy.
We constructed a fusion protein between EGFP and KChIP1, and inserted the fused gene or EGFP alone under the CMV promoter into a Semliki Forest Virus vector (pSFV, Invitrogen). pSFV1/EGFP, pSFV1/KChIP1-EGFP, and virus Helper 2 DNA were linearized by Spe I. One μg DNA was transcribed in vitro using the mMessage mMachine kit (Ambion, Austin, TX). pSFV1 viral particles were generated according to instructions provided by Invitrogen. For infection of primary neurons, the original culture medium was changed to a serum-free medium. Viral particles pre-treated with chymotrypsin (Sigma) were added to the cultures at a 1 to 10 dilution. The infection was performed for 1 hour at 37°C, followed by replacement of the infection medium with conditioned culture medium. Infected neurons were monitored for expression of EGFP and used for patch-clamp recordings 18-30 h later.
Immunoprecipitation, immunoblotting, and immunofluorescence were performed essentially as described previously [30, 31]. Briefly, cells were rinsed once with PBS and lysed on ice in 0.7% NP-40 buffer (10 mM HEPES, pH7.5, 142.4 mM KCl, 5 mM MgCl2, 1 mM EGTA, and 0.7% NP-40). Insoluble cell debris was cleared by centrifugation at 14,000 rpm at 4°C for 30 min in an Eppendorf centrifuge, and the supernatants were collected for immunoprecipitation. Antibodies for immunoprecipitation (3 μg) and protein G beads (25 μl) were added to the lysates and mixed at 4°C overnight with a nutator. Following precipitation, protein-antibody-bead complexes were washed at least three times in 0.7% NP-40 buffer. The proteins were then separated on a 4-20% Tris-Glycine gel (Invitrogen), electro-transferred to PVDF membranes (Millipore, Billerica, MA), immunodectected with appropriate antibodies, and developed using an ECL kit (Amersham). Because the molecular weight of KChIP is similar to that of the IgG light chain, immunoprecipitation of KChIP1 was performed using beads to which the antibody was immobilized by a Seiz × Mammalian Immunoprecipitation kit (Pierce, Rockford, IL). For immunofluorescence staining, cells were grown on circular cover slips in 24-well culture dishes, fixed with 3.7% paraformaldehyde for 15 min, permeabilized with 0.1% Tween-20 for 10 min, and stained with primary antibody for 1 h. This was followed by incubation with a Cy2- or Cy3-labeled secondary antibody for 1 h. Cells were washed, cover slipped in anti-fade medium (Fisher, Pittsburgh, PA), and analyzed under confocal microscopy.
Brain sections were analyzed by immunohistochemistry as follows. Adult Sprague Dawley rats were anesthetized with nembutal and perfused with PBS followed by 4% formaldehyde in PBS (pH 7.4) using intracardiac catheterization. The brain was removed and fixed for an additional hour, after which it was rinsed in PBS, and 80 μm thick vibratome sections were cut. Brain sections were permeabilized in 0.1% Triton X-100, 1% normal donkey serum, and 1% cold water fish gelatin (Sigma) in PBS for 30 min. Sections were then incubated in primary antibody for 18 h at 4°C and washed in buffer, followed by further incubation with the appropriate secondary antibody (Jackson ImmunoResearch, West Grove, PA) in PBS for 1 hr at 4°C. Sections were rinsed in PBS, mounted in Gelvatol, and examined under confocal microscopy.
Expression and recording of Kv4.3 channels in Xenopus oocytes
cDNAs encoding Kv4.3, KChIP1, or KChIP1-EGFP were subcloned into the pCS2 plasmid. Capped RNAs were made using Message Machine RNA polymerase kits (Ambion) and verified by gel electrophoresis. Xenopus oocytes were co-injected with Kv4.3 (5 ng) and either EGFP (10 ng), KChIP1 (10 ng), or KChIP1-EGFP RNAs (20 ng), and maintained in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4). Kv4.3 potassium currents were recorded under two-electrode voltage clamp at a holding potential of -100 mV (Amplifier Model OC-725A, Warner Instrument, Hamden, CT). Recording electrodes were filled with 2 M KCl and had resistances between 0.3 and 1.0 mΩ. Currents were sampled at 5-10 kHz and filtered at 1-2 kHz. All recordings were performed at room temperature, and oocytes were perfused continuously with an external solution containing 96 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (pH 7.4). Data collection and analysis were performed using the pClamp 9.0 software program (Axon Instruments, Foster City, CA).
Whole-cell patch-clamp recording
Autaptic recordings were made from somas of isolated, single hippocampal neurons in low-density culture conditions with patch pipettes (4-6 MΩ resistance) that were filled with an internal solution consisting of (in mM): 140 potassium gluconate, 17.5 KCl, 9 NaCl, 1 MgCl2, 10 HEPES, and 0.2 EGTA, at pH 7.4 [28, 32]. The standard external solution contained 150 mM NaCl, 3 mM KCl, 10 mM HEPES, 5 mM glucose, 2 mM CaCl2, 50 μM D-2-amino-5-phosphonovalerate (APV), and 10 μM CNQX at pH 7.4. For the paired pulse facilitation experiments, 2 mM CaCl2 was replaced by 1 mM CaCl2 and 3 mM MgCl2. The currents were low-pass filtered at 2-5 kHz and digitally sampled at 10-20 kHz. Capacitative currents were subtracted and blanked. Illustrated traces represent an average of 4-8 responses. For experiments studying mIPSCs, TTX (1 μM) was added to block Na+ channels and resulting action currents. For recording K+ currents, we used an external solution comprised of 115 mM NaCl, 2.5 mM KCl, 1.5 mM MgCl2, 10 mM HEPES, and 0.1 mM BAPTA; 1 μM TTX and 3 mM CoCl2 were added to inhibit Na+ and Ca2+ currents, respectively. Potassium currents were evoked with a series of incremental voltage steps of 100 ms duration to 45 mV from a holding potential of -75 mV. Steady-state current amplitudes were measured 75 ms after the initiation of each voltage step and normalized to cell capacitance. For recordings of Ca2+-channel mediated Ba2+ currents, the external solution contained 160 mM TEA-Cl, 2 mM BaCl2, and 10 mM HEPES-CsOH. In addition, 1 μM TTX was added to block Na+ currents. A cesium gluconate-based solution was used as the internal solution . Voltage-activated Ba2+ currents were evoked by applying a 105 msec step to a test potential of 0 mV every 10 sec from a holding potential of -80 mV. The peak Ba2+ currents were calculated by subtraction of Cd2+- sensitive Ba2+ current and normalized to cell capacitance. Solution changes were made with fast-flow, gravity-fed flow tubes gated by valves. Data acquisition and analysis were made with pClamp 8 (Axon Instruments, Union City, CA) or a mini analysis program (Synaptosoft, Decatur, GA). Results are expressed as mean ± SE. All experiments were performed at room temperature.
Hot-plate and tail-flick test: KChIP1-/- mice were generated, genotyped and breed as described. In the hot-plate test, mice were placed on a standard thermal hotplate with a heated surface (55°C) (Columbus Instruments, Columbus, OH). The latency for nociceptive responses was recorded with a cutoff time of 30 seconds. The spinal nociceptive tail-flick reflex was evoked by radiant heat (Columbus Instruments, Columbus, OH) applied to the underside of the tail, and latencies were measured with a cutoff time of 10 seconds.
Rota-rod: Motor functions were tested using the Rota-Rod test (Med Associates, St Albans, VT,) as previously described . Briefly, animals were trained on a rotating drum and tested the following day with increasing velocity. Measures were taken of the duration each animal was able to maintain its balance walking on the rotating drum. The latency to fall was taken as a measure of motor function.
Open-field activity: The Activity Monitor system from Medical Associates (Med Associates, St Albans, VT,) was used to record locomotor activity as published previously . Briefly, each subject was placed in the center of the open-field and activity was measured for 30 minutes and was recorded via a camera and stored for offline analysis.
Elevated plus maze test: The elevated plus maze (Med Associates, St Albans, VT,) consists of two open arms and two closed arms situated opposite each other and separated by a 6 cm square center platform. Each runway is 6 cm wide and 35 cm long. For each test, the animal was placed in the center square and allowed to move freely for five minutes. Open arm entries were defined as the mouse having all four paws onto the open arm. The number of entries and time spent in each arm was recorded.
Light/Dark emergence task: The Light/Dark test was performed as previously . Briefly, the apparatus is a modified chamber (40 × 15.9 × 21.3 cm) separated into two compartments with a small opening (3.5 × 6 cm) between compartments. One compartment is completely dark, and the other is very bright. Mice were individually placed into the dark chamber of the box with the exit blocked for 5 seconds, after which the door lifted open and the mice were allowed to freely explore either compartment for 10 minutes. Time spent in the light chamber and light/dark compartment transitions were recorded.