The procedures on animals conducted in this work were performed in strict accordance with Animal Welfare Act, the Public Health Services Animal Welfare Policy, and The National Institute of Health Guide for Care and Use of Laboratory Animals. The experimental protocol was approved by the Institutional Animal Care and Use Committees (IACUC) of Baylor College of Medicine (Protocol Number: AN-752). Following approved protocol, every effort was made to minimize suffering.
Tissue Procurement and Preparation of Brain Slices for In Situ Hybridization and Slice Recordings
Sprague-Dawley rats were anesthetized by isoflurane inhalation (IsoFlo, Abbott Laboratories), rapidly killed by cervical dislocation, and decapitated using a guillotine. Brain tissues from P21–28 rats were used for synthesizing cDNA used in amplification PCR or qRT-PCR. For in situ
hybridization, the whole brains of P12 rats were removed, frozen in isopentane, and sliced into 20 µm sections using a Jung CM3000 cryostat as previously described 
. The sections were mounted on poly-L-lysine-coated slides and processed for probing using rabio-labelled riboprobes.
For slice recordings, the whole brain from young P8–10 rats was divided using a blade into the two hemispheres and cerebellum. The blocks of tissue were immersed immediately in normal artificial cerebrospinal fluid (ACSF) (in mM: 124 NaCl, 44 KCl, 2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 2.6 NaHCO3, 1 glucose) saturated with 95% O2/5% CO2 at room temperature (RT, 21–23°C). After 30 min, a small block of cerebellar tissue is properly oriented and adhered to the cutting tray of the vibratome (VT1000S, Leica Microsystems Inc.) using cyanoacrylate glue (Vetbond, 3M), and the tray is submerged in ice-cold cutting solution (in mM: 182.6 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2, 1.2 NaH2PO4, 1 NaHCO3, 1 glucose). Cerebellar slices (300 µm) were cut in the coronal plane by the vibratome's automated function and stored at room temperature on a mesh submerged under normal ACSF saturated with 95% O2/5% CO2. After a 1 hr recovery period, a slice was transferred to the recording chamber perfused with normal ACSF saturated with 95% O2/5% CO2 at RT.
A 5′RACE-PCR was conducted on RNA isolated from the cerebellum of a p28 rat using the First Choice RLM RACE Kit (Ambion) as described previously 
. The DPP6a-specific probe used in in situ
hybridization was amplified from the 5′ RACE-PCR products, using the following PCR primers: (f) 5′-TGCTCTAGAGAGCTCACAGAAGCTTGGAGTACAG-3′
and (r) 5′-CCGGAATTCCTTATGGCCTTTCCCTGGAGGACAC-3′
, where the underlined nucleotides represent nested XbaI and EcoRI sites in forward and reverse primers, respectively. The probe was digested with XbaI and EcoRI and subcloned into pBluescript II KS(+) (Stratagene) to generate pBS/DPP6a-ribo. Radio-labeled antisense and sense riboprobes were synthesized from pBS/DPP6a-ribo linearized by EcoRI and XbaI, respectively, using the Riboprobe In Vitro Transcription Systems (Promega Corporation) in the presence of [35
S]-UTP (Perkin Elmer Life and Analytical Sciences).
To generate the normalization controls for qRT-PCR, cDNA fragments covering appropriate intron/exon boundaries (DPLPs: Exons 1–3; GAPDH: Exons 5–8) were cloned in series into pBluescriptII KS(+). Plasmids containing rat Kv4.2, human KChIP3a, and human KChIP4bL cDNAs were obtained as previously described 
. The complete rat DPP6a (rDPP6a) cDNA was cloned from a rat cerebellar cDNA library using overlap extension PCR. Rat DPP6S cDNA was generated by amplifying the DPP6S 5′ sequence from a rat cortical cDNA library and swapped into the rDPP6a construct with the help of an internal BglII site. Rat DPP6K 5′ sequence was amplified from a rat cerebellar cDNA library and swapped into the rDPP6a construct, similar to DPP6S. The DPP6K N-terminal deletion (ΔN16) mutant was generated by amplifying a PCR fragment encoding the truncation and swapping it into the DPP6K wild-type construct. All clones were constructed with strong 5′ Kozak sequences. Our original rat DPP6 clones differed at a single position from the Ensembl rat genomic sequence at E305V in the extracellular domain of DPP6a. We have mutated this site to the consensus and verified that this difference has no measurable effect on any functional properties (data not shown). Sequences for all DNA constructs were verified by automated sequencing (DNA sequencing facility, Baylor College of Medicine). RNA transcripts for oocyte expression were synthesized from linearized DNAs using mMessage mMachine high-yield capped RNA transcription kit (Ambion).
Brain mRNA Harvest, cDNA Synthesis, and Quantitative RT-PCR
Cerebellar samples (10 mg each) were collected from P21 rats, and total RNA was isolated using the RNAqueous-Micro kit (Ambion). Reverse transcription (RT) reactions were conducted with the Superscript III kit (Invitrogen) using random hexamers and following manufacturer's instructions.
Procedures for quantitative real-time PCR were adapted from Liss et al. (2001) 
. The following forward (f) and reverse (r) primers were used for nested PCR.
GAPDH (362 bp): (f) GTCTTCACCACCATGGAGA, (r) ATGACCTTGCCCACAGCCT
Kv4.2 (406 bp): (f) GACAGACAATGAGGATGTCA, (r) GTGGTAGATCCGACTGAAG
DPP10a (270 bp): (f) TGGTTTGTCTTGGAACTCTG, (r) CTCTAGTGACAGTCTTGTTTC
DPP10c (263 bp): (f) GAGGAAGTGTGAGCTCCGA, (r) CTCTAGTGACAGTCTTGTTTC
DPP10d (246 bp): (f) ACCCAGCAGGAACTTAGAG, (r) CTCTAGTGACAGTCTTGTTTC
DPP6a (239 bp): (f) AGTTTGCAAGGTAGAGGATC, (r) GAGACAGACTGGTATCTTCC
DPP6K (257 bp): (f) GTGCTCACCAGAACAATGGA, (r) GAGACAGACTGGTATCTTCC
DPP6L (353 bp): (f) GCTGTACCAAAGGTTCACC, (r) GAGACAGACTGGTATCTTCC
DPP6S (165 bp): (f) CAGGAAAATCTGTACAGCAG, (r) GAGACAGACTGGTATCTTCC
GAPDH (221 bp): (f) CCAAAAGGGTCATCATCTC, (r) ATCCACAGTCTTCTGAGTG
Kv4.2 (309 bp): (f) ACACTCCGAGTCTTTCGAG, (r) GCTCAGAGAGCAGATAGAC
DPP10a (140 bp): (f) CCATCACATCAAGTGTCAGC, (r) GGATGACAGACATTGTGATG
DPP10c (176 bp): (f) GATGACAGCCATGAAGCAG, (r) GGATGACAGACATTGTGATG
DPP10d (110 bp): (f) CAGAGAGGAACTGGGAAGT, (r) GGATGACAGACATTGTGATG
DPP6a (143 bp): (f) GTCCAATAACGTCAGGTGTC, (r) GGATGACTGAGGTGACAATC
DPP6K (178 bp): (f) GTGAGGTTCAAGACTCCAAG, (r) GGATGACTGAGGTGACAATC
DPP6L (125 bp): (f) GAGCGACTGTGACGAGGAG, (r) GGATGACTGAGGTGACAATC
DPP6S (123 bp): (f) TCTGTACAGCAGCAGGATCA, (r) GGATGACTGAGGTGACAATC
qRT-PCR was conducted using IQ SYBR Green Supermix (Bio-Rad) on a PTC-200 thermal cycler with a chromo-4 detection system (Bio-Rad) as described previously 
In Situ Hybridization
hybridizations with [35
S]-labeled riboprobes were conducted as previously described 
. In short, mounted sections were treated with proteinase K for 10 min, acetylated with acetic anhydride for 10 min, dehydrated with ascending concentrations of ethanol, and air dried. The sections were incubated with the riboprobes for 16–20 hrs at 60°C in the hybridization solution. The sections were then treated with RNase for 35 min to remove nonspecifically bound probes and washed 4 times under high stringency with solutions of decreased salinity at room temperature. Afterwards, they were washed once with SSC at high temperature for 35 min. Graded ethanol was then used to dehydrate the sections, and the sections were dried and laid apposed to X-ray film for 3 days at 4°C.
Heterologous Expression in Xenopus Oocytes and Two-electrode Voltage Clamp Recordings
Xenopus laevis frogs were anesthetized with 0.1% Tricane solution absorbed through the skin, and stage V–VI oocytes were surgically harvested and defolliculated by collagenase I treatment. Oocytes were injected with Kv4.2 cRNAs with or without auxiliary subunit cRNAs using a Nanoinjector (Drummond Scientific Company). Injected oocytes were incubated at 18°C for 1–2 days in standard ND96 solution (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4 adjusted with NaOH) supplemented with 5 mM Na-pyruvate and 5 µg/ml gentamycin.
The two-electrode voltage clamp technique was used to elicit whole-cell currents from injected oocytes. The microelectrodes had <1 MΩ tip resistance and were filled with 3 M KCl solutions. The voltage-clamp amplifier (Oocyte Clamp OC-725, Warner Instruments) was under the control of WinWCP software (John Dempster, University of Strathclyde, Glasgow, UK). The data were digitized and low-pass filtered (model 902, Frequency Devices) at various frequencies depending on the sampling rate. The capacitative transient and linear leak were subtracted off-line by scaling up transients at voltages without ionic currents (at −70 mV) and subtracting them from total currents. Recordings with offsets >3 mV were removed from data analysis, and the average leak current was <0.2 µA.
CG cells were visualized using a fixed-stage upright microscope (BX51WI, Olympus) equipped with dedicated water-immersion objectives designed with long working distances (LUMPlanFL, 60x/0.90 W). Imaging of neurons was conducted using infrared (IR) and differential interference contrast (DIC) techniques. Patch pipettes were fabricated from thin-walled borosilicate glass capillaries (TW150-4, World Precision Instruments, Inc.) using a Flaming/Brown micropipette puller (model P-97, Sutter Instrument Co.) and had a resistance of 1–2 MΩ. An Axopatch 200B integrating patch clamp amplifier (Axon Instruments) was used to direct voltage pulses, and the data were acquired using pClamp 7 (Axon Instruments) program on a desktop personal computer.
Pipettes were fire polished and had areas near their tips wrapped with parafilm to reduce electrode capacitance. Tight-seal whole-cell recordings were obtained using standard techniques. The patch electrodes were backfilled with solution containing (in mM): 120 K-gluconate, 20 KCl, 5 NaCl, 10 HEPES, 4 Mg2
-ATP, 0.3 Tris-guanosine 5′-triphosphate (GTP), 14 phosphocreatine, pH
7.25 after adjustment with KOH; 301 mOsm). Series resistance and capacitance were determined by optimal cancellation of the capacitative transient, and the series resistance was typically 3–5 MΩ. If the series resistance changed more than 10% of the initial value during recording, the recording was terminated or the data was discarded. The current signals were recorded using an Axopatch 200B and filtered at 5 kHz and sampled at 10 kHz. The liquid junction potential between the electrode and bath solutions was calculated to be ~−14.2 mV, and the data has been corrected for this voltage. All experiments were conducted at RT (22–23°C). Ohmic leak and capacitative currents were subtracted off-line using scaled-up versions of null traces at low voltages.
Isolation of ISA
Non-ISA currents were suppressed by modifications of the external bath solution and by pharmacological agents. The modified ACSF bath solution was low in Ca (in mM: 84 NaCl, 4 KCl, 0.1 CaCl2, 3.1 MgSO4, 1 NaH2PO4, 1 NaHCO3, 1 glucose; 300–320 mOsm) to reduce contamination by Ca currents and Ca-dependent K currents. Supplementation with 0.5 µM tetrodotoxin (TTX), 40 mM tetraethylammonium (TEA), and 100 µM 4-aminopyridine (4-AP) (Sigma-Aldrich) respectively block Na currents, non-inactivating K currents, and D-currents. TEA supplementation is accomplished by substituting TEA for equimolar amount of NaCl. In some experiments, 100 µM linopirdine (Sigma-Aldrich) and 100 µM ZD7288 (Tocris) were used to block M-current and H-current, respectively.
In addition, ISA was reliably isolated using a subtraction protocol that exploits the difference between the high- and low-threshold K currents. Currents were recorded in response to depolarizing steps to various voltages from a conditioning step at either −114 mV or −44 mV for 1 sec. The ISA is inactivated by prepulse to −44 mV, and the subtraction of the current after the −44 mV conditioning pulse from the current after the −114 mV conditioning pulse reveals the transient outward current during the test pulse. Tail reversal measurements gave ISA reversal potential at −80 mV. Calculated EK is −88.3 mV.
Data were analyzed with WinWCP (John Dempster, University of Strathclyde, Glasgow, UK), Clampfit 6 (Axon Instruments), and Origin softwares (OriginLab Corp.). Peak conductance (Gp
) was calculated as Gp
), where Ip
is the peak current, Vc
is the command voltage, and Vrev
is the reversal potential (−90 mV in ND96). The Gp
-V curves were described using the first-order Boltzmann function: Gp
)), where Gp
is the fraction of maximal conductance, Vm
is the membrane potential, Va
is the potential for half-maximal activation, and Sa
is the slope factor. Steady-state inactivation was also described assuming a simple Boltzmann distribution, with the corresponding parameters of I/Imax
, and Si
. Using Clampfit, the time courses of inactivation were fitted by a sum of two exponential terms, and the time courses of recovery from inactivation were described using a single exponential term initially and two exponential terms when the fitted curve deviated from experimental data significantly.
The number of DPP6 subunits per channel was examined by measuring the slowing of DPP6a-mediated fast inactivation via DPP6a
DPP6K mixing and comparing it to the predicted inactivation slowing when only one DPP6a subunit is present. DPP6a-mediated fast inactivation is markedly faster than DPP6K-mediated inactivation; therefore, significantly reducing the DPP6a contribution to the mix should discernibly slow fast inactivation. Calculations for the predicted maximum amount of slowing in fast inactivation assume the use of simple single-step inactivation models for both DPP6a- and DPP6K-mediated inactivation:
where O, Ia
, and IK
are open, inactivated (DPP6a), and inactivated (DPP6K) states, and kON
are rate constants for inactivation and recovery from inactivation proceeding to and from the indicated inactivated states. The kON
can be calculated from the following equations:
is the time constant for fast inactivation and fi
is the fraction inactivated by the fast mechanism. Kv4.2+KChIP3a channel with only DPP6a has a τi
of 6 ms with a fi
of 0.71 (). When only DPP6K is used, τi
has a value of 35 ms with a fi
of 0.74 ().
Biophysical Properties of Kv4 Channel Complexes with Various DPP6 Variants.
Since DPP6 proteins are known to form dimers 
, models were tested for both 2 DPP6 subunits per channel and 4 DPP6 subunits per channel. Two types of kinetic models were generated: a full proportionate ON-rate model (Model #1) and a DPP6a-only proportionate ON-rate model (Model #2):
represent the number DPP6a and DPP6K present, respectively. For Model #1, the ON-rate for Ia
is proportional to the respective number of DPP6a and DPP6K subunits present, and the OFF-rate is unchanged. For 4 DPP6's per channel, the predicted ka-ON
for DPP6a-mediated inactivation are respectively 30 s−1
per subunit and 48 s−1
per channel; for 2 DPP6's per channel, they are respectively 60 s−1
per subunit and 48 s−1
per channel. For 4 DPP6's per channel, the kK-ON
are respectively 5.8 s−1
per subunit and 7.5 s−1
per channel; for 2 DPP6's per channel, they are respectively 11.6 s−1
per subunit and 7.5 s−1
per channel. For Model #2, we assume that DPP6a specifically overlays an additional N-type inactivation process to a channel that would otherwise inactivate by a separate intrinsic inactivation process. In this case the intrinsic inactivation mechanism does not change much depending on DPP6 subunit composition (compare DPP6K and DPP6S kinetics and see 
), but the N-type inactivation process has an ON-rate that is proportional to the number of DPP6a subunits present. The intrinsic KON
is 21 s−1
and the intrinsic KOFF
is 7.5 s−1
. The DPP6a-specific kinetic rate constants are: for 4 DPP6's per channel, ka-ON
is 25 s−1
per subunit and ka-OFF
is 52 s−1
per channel; for 2 DPP6's per channel, ka-ON
is 50 s−1
per subunit and ka-OFF
is 52 s−1
per channel. Using the QuB software program (www.qub.buffalo.edu
), we constructed the different models assuming only one DPP6a subunit per channel. The predicted value and amplitude for the fast inactivation time constant were calculated using the Equilibrium P function.
For modeling the effects of DPP6K on steady-state inactivation, we used a linear model based on an equal energetic contribution for each incorporated DPP6K subunit. Based on the magnitude of the shift produced by four DPP6K subunits, the model assumes a −2.3 mV shift in the steady-state inactivation curve for each DPP6K subunit incorporated in the channel. To confirm that this model gives a linear shift in the Boltzmann midpoint based on the DPP6K mole fraction, we constructed a set of Boltzmann curves based on the DPP6a curve with a −2.3×NK mV shift in the DPP6a curve, where NK is the number of DPP6K subunits in the mixed tetramer. These curves were then summed using the calculated mole fractions for the different subunit compositions based on the expression mix used and then the summed data was fit with a first-order Boltzmann distribution. For experimental averages, data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined by comparing data sets using Student's two-tailed (independent) t-test and the significance level of p<0.05.