Expression constructs

The human Myc-DDK-Kv4.2 construct was purchased from Origene (RC215266). All of the other expression constructs were made by PCR. Internal deletions and point mutations were generated using either the QuikChange Site–Directed Mutagenesis Kit (Stratagene) or the megaprimer method. PCR products were cloned into expression vectors pGEX 4T2 (Pharmacia) and pRK5 (Genentech), with Myc, flag or HA tags as we reported previously52. All constructs were verified by sequencing.

Chemicals

All chemicals were purchased: KA (Sigma, K0250), PTZ (Sigma, P6500), SB203580 (Tocris, 1202), PD 98059 (Tocris, 1213), SL327 (Tocris, 1969), S-AMPA (Tocris, 0254), Juglone (Millipore, 420120), PiB (Sigma, B7688), AmmTX3 (Alomone, 305). For injections, KA and PTZ were dissolved in saline; SB203580, SL327 and Juglone were dissolved in DMSO and 10% Tween 80.

Antibodies

Mouse anti-Kv4.2 (NeuroMab, 75-016) was used at 1:2000 for western blot, 1:200 for immunostaining, Rabbit anti-Kv4.2 (Sigma, P0233) was used at 1:2000 for western blot, rabbit anti-Kv4.2 (Sigma, HPA029068) was used at 1:200 for staining, pT602 (Santa Cruz, SC-16983-R) was used at 1:1000 for western blot, pT607 (Santa Cruz, SC-22254-R) was used at 1:500 for western blot, Pin1 (Santa Cruz, SC-46660) was used at 1:100 for staining, 1:1000 for western blot, Pin1 (Millipore, 07-091) was used at 1:3000 for western blot, p38 (Cell Signaling, 9212 s) was used at 1:1000 for western blot, p-p38 (Cell Signaling, 4511 s) at 1:1000 for western blot, DPP6 (Abcam, 41811) was used at 1:2000 for western blot, Myc (Millipore, 05-419) was used at 1:10000 for western blot, HA(Santa Cruz, SC-805) was used at 1:1000 for western blot, Actin (Sigma, A-1978) was used at 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse (Invitrogen, A-11029) was used at 1:500; Alexa Fluor 488 goat anti-rabbit (Invitrogen, A-11034) was used at 1:500; Alexa Fluor 555 goat anti-mouse (Invitrogen, A-21424) was used at 1:500; Alexa Fluor 555 goat anti-rabbit (Invitrogen, A-21429) was used at 1:500; Alexa Fluor 680 goat anti-mouse (Invitrogen, A-21057) was used at 1:10000; Alexa Fluor 680 goat anti-rabbit (Invitrogen, A-21076) was used at 1:10000; IRDye 800CW goat anti-mouse (Licor, 926-32210) was used at 1:10000, IRDye 800CW goat anti-rabbit (Licor, 926-32211) was used at 1:10000.

Mouse models

Kv4.2TA mice were generated using CRISPR-Cas9 techniques. Briefly, CRISPR sgRNA (CCTGTCGTCGCCTTCTGGGG) was made by in vitro transcription using ThermoFisher’s sgRNA synthesis service. B6D2F1 mice (JAX Stock No. 100006) were used as embryo donors for this study. For the injection step, sgRNA (20 µg / ml), Cas9 mRNA (100 µg/ml, Trilink Biotechnologies) and corresponding single strand oligos with mutations (100 µg/ml) were injected into the cytoplasm of fertilized eggs, which then were cultured overnight in M16 medium. Those embryos that reached the two-cell stage of development were implanted into the oviducts of pseudo-pregnant surrogate mothers (CD-1, Charles River). Mice born to these foster mothers were genotyped by PCR amplification followed by DNA sequencing to identify mice with correct mutations.

Mice were group housed in plastic mouse cages with free access to standard rodent chow and water. The colony room was maintained at 22 ± 2 °C with a 12 hr: 12 h light: dark cycle. Kv4.2TA mice were backcrossed at least three generations onto C57/Bl6J mice. All animal procedures were performed in accordance with guidelines approved by the National Institute of Child Health and Human Development Animal Care and Use Committee and in accordance with NIH guidelines.

Cell culture and transfection

HEK-293T cells used in biochemistry experiments were obtained from Dr. Paul Worley’s lab70. HEK-293T cells were cultured in DMEM medium containing 10% FBS. Transfections were performed with X-tremeGENE 9 according to the manufacturer’s specifications. Cells were harvested about 40 h after transfection.

Neuronal culture

Mouse hippocampal neuron cultures from embryonic day 18 (E18) pups were prepared as reported previously71. In all, 1 × 106 neurons were added to each well of a six–well plate (Corning) with cover slips coated with poly–L–lysine. Growth medium consisted of Neurobasal medium (Invitrogen) supplemented with 5% FBS (Hyclone), 2% B27, 1% Glutamine (Invitrogen), 100 U/mL penicillin, and 100 U / mL streptomycin (Invitrogen). Neurons were fed twice per week with glia-conditioned growth medium. DIV14-17 neurons were used for biochemistry experiments. Rat hippocampal neuron cultures from embryonic day 18 (E18) pups were prepared similarly as above. DIV14-17 neurons were used for the immunostaining experiment.

Tandem affinity purification-mass spectrometry (TAP-MS) assay

Kv4.2 was subcloned into the TAP tag vector that was obtained from Agilent (pCTAP, #240102). TAP-tagged Kv4.2 was then subcloned into the lentivirus vector (Dr. Paul Worley’s lab) to generate TAP-Kv4.2-IRES-GFP Lentivirus. TAP-Kv4.2-IRES-GFP Lentivirus and IRES-GFP control Lentivirus were generated using a standard protocol72. Rar hippocampal neuron cultures from embryonic day 18 (E18) pups were prepared as described above. Neurons were infected by TAP-Kv4.2-IRES-GFP Lentivirus or IRES-GFP control Lentivirus on the day of the culture and harvested at DIV14. TAP-Kv4.2 was purified using the TAP purification kit from Agilent (#240107) with some modifications. The samples were run on 10% SDS-PAGE gel (Novex/Invitrogen). The gels that contains the protein samples were excised, separated into high molecular and low molecular weight samples, and sent to the Taplin Mass Spectrometry Facility at Harvard University for in-gel digestion using trypsin and mass spectrometric analysis.

Peptide pulldown

The following peptides were synthesized: Non-Phospho for T602 and T607: KAIISIPTPPVTTPEGDDR; pT602: KAIISIP-pT-PPVTTPEGDDR; pT607: KAIISIPTPPVT-pT-PEGDDR; pT602, pT607: KAIISIP-pT-PPVT-pT-PEGDDR; Non-Phospho for S616: KEGDDRPESPEYSGG; pS616: KEGDDRPE-pS-PEYSGG. The peptides were conjugated to Affi-Gel 15 (Bio-Rad) according to the manufacturer’s instructions. Peptide-linked Affi-Gel was incubated for 3 h at 4 °C with Pin1 protein that was expressed in HEK-293T cells, and then washed clear for western blot analysis.

Co-immunoprecipitation and immunoprecipitation assays

Mouse brain tissues or HEK-293T cells were used in co–immunoprecipitation assays as previous reported in ref. 70. For Kv4.2-Pin1 co-IP experiment, co-IP buffer (1 X PBS, pH 7.4, with 0.8% Triton X–100, phosSTOP and Complete™ EDTA–Free protease inhibitors) were added (1:20 for brain tissues and 400 µl for a 6-well of HEK-293T cells), and the samples were sonicated. After centrifugation, the supernatant was mixed with 2–3 µg of Kv4.2 (NeuroMab, 75-016, or Sigma, P0233) or myc (Millipore, 05-419) antibodies for 3–4 h at 4 °C. Next, 40 µl of protein G magnetic beads (Bio-Rad, 161-4023) was added for an additional 2 h or overnight. The protein beads were washed three times with co-IP buffer. The protein samples were eluted with SDS loading buffer and analyzed by gel electrophoresis and western blotting. For Kv4.2-DPP6 co-IP experiment, co-IP buffer (1 X PBS, pH 7.4, with 1% Triton X–100, 0.2% Chaps, phosSTOP and Complete™ EDTA–Free protease inhibitors) were added (1:20 for brain tissues), and the samples were sonicated. After centrifugation, the supernatant was mixed with 3–4 µg of Kv4.2 antibody (NeuroMab, 75-016) for 3 h at 4 °C. Next, 50 µl of 1:1 protein G–Sepharose slurry (GE Healthcare, 17-0886-02) was added for an additional 3 h. The protein beads were washed three times with IP buffer. The protein samples were eluted with SDS loading buffer and analyzed by gel electrophoresis and western blotting. For Kv4.2 phosphorylation detection, brain tissues sonicated in co-IP buffer (1 X PBS, pH 7.4, with 1% Triton X–100, phosSTOP and Complete™ EDTA–Free protease inhibitors). After centrifugation, the supernatant was mixed with 3 µg of Kv4.2 (NeuroMab, 75-016) antibody for 3 h at 4 °C. Next, 50 µl of 1:1 protein G–Sepharose slurry (GE Healthcare, 17-0886-02) was added for an additional 3 h. The protein beads were washed three times with IP buffer. The protein samples were eluted with SDS loading buffer and analyzed by gel electrophoresis and western blotting.

Molecular modeling

Models of the bound phosphotyrosine-proline sequence motifs were developed from published structures. These models were primarily based on PDB: 2N10 (ref. 73) for the WW domain, and PDB: 2Q5A74 for the Catalytic domain. Modeling was done by an interplay of “manual” manipulation with UCSF Chimera75 and energy minimization (to prevent steric overlap and optimize salt-bridges and hydrogen bonds) with CHARMM76. Images were generated with UCSF Chimera.

Subtilisin proteolysis

Myc-Kv4.2 (co-transfected with p38) was expressed in HEK293T cells and purified with myc-magnetic beads (Thermo Scientific, #88842). Kv4.2 Phosphorylation at T602 and T607 after purification was measured by saturated phospho-specific and total Kv4.2 antibody pulldown combined with western blot. About 2/3 of purified Myc-Kv4.2 was phosphorylated at T607 and was used for proteolysis. Equal amount of Myc-Kv4.2 were then incubated with 100 ng of either GST, GST-Pin1, and GST-Pin1C113S in a buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl 2 , supplemented with phosphatase inhibitors. After 30 min incubation at room temperature, reaction mixtures were cooled on ice, and subtilisin (Sigma-Aldrich, P5380) was added for a further 1 min on ice. The reaction was stopped by the addition of boiling sample buffer, and the proteolytic fragments were resolved by 4–12% SDS-PAGE and visualized by western blot analysis.

Western blot and quantification

Protein samples were mixed with 4x LDS sample buffer (Invitrogen NP0007) and 10x sample reducing agent (Invitrogen NP0007) to a final concentration of 1×. Samples were loaded on 4–12% Bis-Tris gradient gel (Invitrogen 12-well, NP0322; 15-well, NP0323). The proteins were transferred to Immobilon-FL PVDF membrane (EMD Millipore, IPFL00010). The membrane was blocked with Odyssey blocking buffer (Li-COR, 927-40000) for 1 h at room temperature, followed by incubation with primary antibody in PBS overnight at 4 °C. The membrane was then washed with PBST (PBS, pH 7.4, and 0.1% Tween-20) three times and incubated with secondary antibody in PBS for another hour. After three washes with PBS, the membrane was scanned using an Odyssey imaging system (LI-COR) according to the manufacturer’s protocol. Quantification of western blots was carried out using the gel analysis function in ImageJ within the linear range of detection which is determined by using serial dilutions of a representative sample.

Immunostaining

Cultured hippocampal neurons (DIV10) were fixed with 4% PFA, and permeabilized with 0.2% Triton X-100 in PBS. Cells were then blocked with 10% horse serum at RT for 1 h and then incubated with mouse anti-Pin1 antibody (Santa Cruz, SC-46660, 1:100) and rabbit Kv4.2 antibody (Sigma, HPA029068, 1:200) at 4°C overnight. After washing, cells were incubated with anti-mouse-555 and anti-rabbit-488 secondary antibodies at RT for 1 h. After washing, cells were then mounted on slides with anti-fade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, P36962) and imaged using a Zeiss 710 laser scanning confocal microscope equipped with a ×63 objective.

Acute hippocampal slice preparation

For all electrophysiological recordings, adult male (5–7 weeks) mice were used. Mice were anesthetized in isoflurane and decapitated. Brains were removed and washed with ice-cold sucrose cutting solution. The sucrose solution was made up of the following (in mM): 60 NaCl, 3 KCl, 28 NaHCO 3 , 1.25 NaH 2 PO 4 , 5 Glucose, 0.5 CaCl 2 , 7 MgCl 2 . Brain hemispheres were dissected and mounted following a 45° cut of the dorsal cerebral hemisphere(s). Modified transverse slices (300 μm) were made by a Leica VT1200S vibrating microtome in ice-cold sucrose that was continuously bubbled with carbogen (95% O 2 /5% CO 2 ). Slices were recovered at 32 °C in sucrose solution for 30 min at which time the solution temperature was slowly lowered to room temperature where it remained for the remainder of the recording day.

Whole-cell current-clamp recordings

Following a 1-hour recovery in sucrose cutting solution, hippocampal slices were transferred to a recording chamber submerged in artificial cerebral spinal fluid (ACSF) with the temperature maintained at 33 °C (±1 °C). The ACSF contained the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 25 Glucose, 2 CaCl 2 , 1 MgCl 2 (pH 7.4). The recording chamber was continuously perfused with carbogen-bubbled ACSF at a rate of 2–3 mL/min. Somatic whole-cell patch-clamp recordings were performed on identified somata of hippocampal CA1 pyramidal neurons. Pyramidal neurons were identified using infrared Differential Interference Contrast (DIC) on an upright Leica Axioskop 2. Cells were patched with 3–6 MΩ borosilicate glass pipettes pulled from a Narishige vertical puller and filled with K+ Gluconate-based intracellular solution consisting of the following (in mM): 20 KCl, 125 K-Gluconate, 1 EGTA, 4 NaCl, 4 Na 2 ATP, 0.3 NaGTP, 10 HEPES, 10 Phosphocreatine with pH adjusted with KOH and HCl to a final value of 7.25–7.30 and an osmolarity of 290–300 mOsm.

Firing properties were measured from whole-cell recordings in the conditions described above unless otherwise noted (described in detail below). All data were recorded with a Multiclamp 700b amplifier (Molecular Devices) and a Digidata 1440 A digitizer. Signals were low-pass filtered at 5 kHz and digitized at 10 kHz using Clampex 10.7 software and were acquired in bridge balance mode to compensate series resistance. Liquid junction potential was not corrected for. Subthreshold membrane properties were measured after initial break-in in order to avoid alteration in equilibrium potentials and dialysis as a result of solution exchange. Whole-cell capacitance and series resistance were measured from Multiclamp 700B commander. A voltage step of −10 mV was initiated, and the decay tau of the whole-cell capacitive transient was used to calculate these parameters. Resting membrane potential was measured after switching to I = 0. Any recordings where series resistance exceeded 25 MΩ or resting membrane potential was greater (more depolarized) than −55 mV were discarded. Input resistance was calculated as the slope of the I–V curve in response to current steps from −50 to 50 pA in 50 pA steps (three steps in total). To evoke APs in patched CA1 pyramidal neurons, square 1 s current pulses were elicited in 50 pA steps with current injections ranging from −200 to +200 pA in 50 pA steps. In some cases, for instance in the measurement of rheobase, smaller step sizes of 20 pA were used to enhance the resolution of the average minimum current magnitude required to elicit APs. Three sweeps at each magnitude were elicited and the average response of the three sweeps was used for each cell. All measures of AP waveform were taken from the first spike in a train in response to a 200 pA injection, and inter-spike measurements, including inter-spike interval and after-hyperpolarization amplitude, were recorded between the first two spikes in a train elicited by a 200 pA square current injection (see associated figures).

Outside-out somatic patch voltage-clamp recordings of A-current

A-current was recorded in voltage-clamp mode from outside-out patches pulled from somata of identified pyramidal cells in hippocampal slices. Outside-out patch pipettes (3–6 MΩ) were filled with K+ Gluconate-based intracellular solution consisting of the following (in mM): 20 KCl, 125 K-Gluconate, 1 EGTA, 4 NaCl, 4 Na 2 ATP, 0.3 NaGTP, 10 HEPES, 10 Phosphocreatine. All recordings were performed at room temperature (24–25 °C). Tetrodotoxin citrate (TTX) (500 nM), Gabazine (2 µM), and CNQX (2 µM) were added to the extracellular bath (ACSF) in order to block voltage-gated Na+ channels and ligand-gated channels. A-current density was calculated using a standard subtraction protocol. In short, a pre-pulse step to −120 mV for 600 ms from a holding potential of -65 mV was initiated to relieve inactivation of A-type K+ channels. Voltage was stepped to +40 mV for 500 ms to measure total outward K+ current. A subsequent sweep consisted of a pre-pulse step to −30 mV to inactivate the transient current followed by a step to +40 mV to measure the non-inactivating current. Peak amplitude of the non-inactivating current was subtracted from total outward K+ current amplitude offline to isolate I A . Leakage and capacitive currents were digitally subtracted. Peak amplitude was normalized to current density by dividing by patch capacitance, which was measured following formation of the outside-out patch configuration and was analyzed with custom written code in MATLAB version R2018a (MathWorks). Inactivation kinetics were measured by fitting a single exponential to each isolated I A current trace and were analyzed with Graphpad Prism. Traces are averages of 10-30 sweeps. Recovery from inactivation was calculated using a double voltage-step (−120 to +30 mV) protocol with increasing interstep intervals between steps. Interstep intervals were (in ms): 5, 10, 15, 20, 25, 50, 100, 200, 500, and 1000. Recovery from inactivation was calculated as the ratio of the peak amplitude of the second current trace relative to the point where the initial A-current was fully inactivated and was normalized to the peak amplitude of the initial A-current. Single exponentials were fitted to the non-linear fitted curves of normalized recovery from inactivation and the taus from each individual recovery curve were averaged and compared. Voltage-dependent activation was recorded using a voltage step protocol consisting of a 600 ms prestep to −120 mV, followed by a series of 13 steps (10 mV each from −80 to 40 mV). Leakage and capacitive currents were digitally subtracted. The same protocol but with a 600 ms prestep from -65 mV to -30 mV was used to record non-inactivating currents, which were offline subtracted from the overall K+ currents to obtain inactivating A-type K+ currents. Currents were then converted into conductances, normalized to peak conductance at 40 mV, plotted against the holding voltage and fitted with a Boltzman-function to obtain V 1/2-activation and k activation . Voltage-dependent inactivation was recorded using a voltage-step protocol consisting of a series of 14 presteps (each 600 ms and 10 mV, from −120 to 10 mV) followed by an activating voltage step to 20 mV. Leakage and capacitive currents were digitally subtracted. Peak currents were measured for each step and the I/V relationship was fitted with a Boltzmann function to obtain V 1/2-inactivation and k inactivation . All data were recorded with a Multiclamp 700b amplifier (Molecular Devices) and a Digidata 1440A digitizer, were digitized at 10 kHz and low-pass filtered at 2 kHz using a Bessel filter.

Behavioral assays

Open field task

Novelty-induced locomotor activity was assessed in a novel open-field square arena (50 × 50 cm) constructed of white Plexiglas as previously described77. Mice were acclimated to the testing room for at least ~10 min and then placed in the arena and left to explore freely for 30 min. Sessions were performed once a day for two successive days. The distance traveled and time spent in different areas of the maze were measured. Results were analyzed with “ANY-maze” software (ANY-maze, Wood Dale, IL, USA). Data were compiled from two independent experimental cohorts. Male mice used in the first and second cohorts were ~8.5–10 and ~15–16 weeks old, respectively.

Morris water maze

The Morris water maze task was performed to evaluate hippocampus-dependent spatial navigation learning and memory78,79. The water maze consisted of a 120 cm circular pool (depth 50 cm), filled ~40 cm deep with 20–22 °C water containing a 10 cm wide square platform. External high contrast cues were placed on the interior of the pool above the water surface to aid with spatial navigation. Trials were video recorded and scored by ANY-maze software (ANY-maze, Wood Dale, IL, USA) for measures including latency to find the hidden platform, total distance traveled, and swim speed. The latency to the platform of the training trials was measured manually with a stopwatch.

General mouse handling was performed as follows: Mice were acclimated to the testing room for at least ~1 h before testing. Each mouse was placed into the water maze facing the wall in one of four possible quadrant positions, which was pseudo-randomly varied by training session. Mice were given 60 s to find the platform and a ~15 s platform rest interval. If a mouse was unable to find the platform in the allocated time, it was gently guided to the platform and allowed to rest for ~15 s. Mice were then patted dry with a cloth and put back into a warm cage for ~15 s after each trail. For data analyses, a latency time of 60 s was ascribed to mice that failed to reach the platform without guidance.

On Day 1 mice were trained in the visible platform version of the Morris water maze task to assess general swimming and visual ability. The platform was ~1 cm above the clear water surface with a red flag placed on the platform to increase its visibility. Each mouse underwent two visible platform training sessions and the location of the platform was varied between sessions. No significant difference in escape latency was apparent between genotypes. The water was made opaque with nontoxic white and red paint between Days 1 and 2. On Day 2 through Day 4 (sessions 1–6), mice were trained for the Hidden Platform protocol where the flag was removed from the platform and additional water was added to the pool to submerge the platform ~1 cm below the surface. Mice were given a total of 24 training trials (4 trials per session, two sessions per day for three successive days). On Day 5, the platform was removed, and mice underwent a 60 s probe trial to determine the amount of time spent exploring the target quadrant. On Day 6 and Day 7, mice were trained for reversal learning (2 sessions per day for 4 total sessions) where the Hidden Platform was moved to the opposite quadrant. On Day 8, the platform was removed, and the mice underwent a 60 s probe trial to determine the amount of time spent exploring the target quadrant. ~16–17-weeks-old male mice were used. Data were compiled from two independent experimental cohorts.

Lever press

Operant reversal learning was performed as previously described in ref. 49. Mice were food restricted to 85–90% of their free-feeding weight over several days prior to testing and throughout the experiment. Mice were trained to lever press on an FR1 schedule in daily sessions with an endpoint of 30 rewards or 30 min. When they collected all of the rewards in the allotted time for 3 consecutive days, animals progressed to RR2 sessions lasting 25 min, during which they could earn unlimited rewards. After 5 training days, levers were reversed such that the reinforced lever became non-reinforced and vice versa. Mice were given daily 15-minute reversal learning sessions for 5 days.

Statistical analysis

Biochemistry and behavior data were analyzed by Origin 2018b by two–tailed Student’s t test and two–way ANOVA, respectively. Electrophysiology data were analyzed by GraphPad Prism 7 (7.0d). For all measures of I A in outside-out somatic patches the experimenter was blinded to the genotype. For measures of firing properties, the experimenter was aware of these conditions. Sample sizes were not predetermined with any statistical methods but were chosen based on numbers reported in similar publications in the field. All statistical tests were two-tailed. Specifically, for electrophysiological analysis of I A and AP shape in WT and Kv4.2TA mice and pharmacological analysis (PiB treatment) in Kv4.2TA, unpaired t-test (with Welch’s correction) was used (passed normality testing). For electrophysiological analysis of the pharmacological impact on I A in WT slices a One-way ANOVA (ordinary), or One-Way ANOVA on Ranks (Kruskal–Wallis) was used and were corrected for multiple comparisons with Dunnett’s test (ordinary) or Dunn’s test (Ranks) respectively. The use of parametric or non-parametric analysis was determined after testing for normal distribution in the data using the D’Agostino & Pearson normality test for all electrophysiological data (alpha Level = 0.05). Non-parametric statistics were used if the data failed normality testing. For all analysis of pharmacological treatment effects in WT, significance was probed relative to control (vehicle). All analysis of firing frequency in response to sequential current steps, a two-way ANOVA with Sidak’s post hoc test was used. All the data are presented as mean ± SEM.

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.