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Research Report

Differential characterization of three alternative splicedisoforms of DPPX☆

Marcela S. Nadal a, Yimy Amarillo a, Eleazar Vega-Saenz de Mieraa, Bernardo Rudya,b,⁎aDepartment of Physiology and Neuroscience, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USAbDepartment of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA

A R T I C L E I N F O

☆ GenBank Accession numbers: DPPX-S: M7DPPX-D: CB585942, CB579034, CB586631; Rat⁎ Corresponding author. Department of Phys

Medicine, 550 First Avenue, New York, NY 10E-mail address: rudyb01@med.nyu.edu (B

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.03.106

A B S T R A C T

Article history:Accepted 22 March 2006Available online 9 June 2006

Transient subthreshold-activating somato-dendritic A-type K+ currents (ISAs) havefundamental roles in neuronal function. They cause delayed excitation, influence spikerepolarization, modulate the frequency of repetitive firing, and have important roles insignal processing in dendrites. We previously reported that DPPX proteins are keycomponents of the channels mediating these currents (Kv4 channels) (Nadal, M.S., Ozaita,A., Amarillo, Y., Vega-Saenz, E., Ma, Y., Mo, W., Goldberg, E.M., Misumi, Y., Ikehara, Y.,Neubert, T.A., Rudy, B., 2003. The CD26-related dipeptidyl aminopeptidase-like proteinDPPX is a critical component of neuronal A-type K+ channels. Neuron 37, 449-461). TheDPPX gene encodes alternatively spliced transcripts that generate single-spanningtransmembrane proteins with a short, divergent intracellular domain and a largeextracellular domain. We characterized the modulatory effects on Kv4.2-mediatedcurrents and the rat brain distribution of three splice variants of the DPPX subfamily ofproteins. These three splice isoforms – DPPX-S, DPPX-L, and DPPX-K – are expressed inadult rat brain and modify the voltage dependence and kinetic properties of Kv4.2channels expressed in Xenopus oocytes. Analysis of a deletion mutant that lacks thevariable N-terminus showed that the N-terminus is not necessary for the modulation ofKv4 channels. Using in situ hybridization analysis, we found that the three splice variantsare prominently expressed in brain regions where Kv4 subunits are also expressed. DPPX-K and DPPX-S mRNAs have a widespread distribution, whereas DPPX-L transcripts areconcentrated in few specific areas of the rat brain. The emerging diversity of DPPX splicevariants, differing only in the N-terminus of the protein, opens up intriguing possibilitiesfor the modulation of Kv4 channels.

© 2006 Elsevier B.V. All rights reserved.

Keywords:Potassium channelKv4 channelSubthreshold activating A-typepotassium current

Abbreviations:DPP, dipeptidyl peptidaseISA, subthreshold activated A-typeK+ currentsKChIPs, potassium channelinteracting proteinKv channel, voltage-gatedpotassium channelPCR, polymerase chain reactionUTR, untranslated regionEST, expressed sequence tag

1. Introduction

We have shown that the aminopeptidase-like protein DPPXaccelerates the kinetics of inactivation and recovery of

6427; DPPX-L: M76426; DPPgenomic contig: NW_0476iology and Neuroscience016, USA.. Rudy).

er B.V. All rights reserved

inactivation of transient, subthreshold activating A-type K+

currents (ISA) (Nadal et al., 2003). These somatodendriticcurrents play important roles in regulating the excitability ofneurons, including latency to the first spike and duration of

X-K: CF743412, CB518788, BI603302, BX647966; DPPX-E: NP_034205;85.1.and Department of Biochemistry, New York University School of

.

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the interspike interval (Connor and Stevens, 1971; Hille,2001), action potential waveform (Bardoni and Belluzzi, 1993),signal integration in dendrites (Hoffman et al., 1997; Johnstonet al., 2000), regulation of firing frequency and dopaminerelease (Liss et al., 2001), and the timing of synaptic inputs(Schoppa and Westbrook, 1999). The ability of ISA to performthese various physiological roles relies upon its uniquekinetic properties such as rapid inactivation, subthresholdactivation and inactivation, and rapid recovery from inacti-vation at hyperpolarized membrane potentials. These prop-erties, in turn, are determined by the nature of the moleculesthat form the A-type channels. A number of studies haveshown that the pore forming subunits of the voltage-gatedK+ channels underlying ISA are Kv4 subunits; of which Kv4.2and Kv4.3 are predominantly expressed in rodent brain(reviewed in Jerng et al., 2004a). Kv4.3 subunits also formthe channels responsible for the transient outward K+current in human cardiomyocytes (Ito), which is essentialfor the early repolarization phase of the cardiac actionpotential (Dixon and McKinnon, 1996; Nakamura et al.,1997; Xu et al., 1999).

Many ion channels are protein complexes that includepore-forming subunits and associated or auxiliary proteins,which can have a major impact on the function of thechannel. These associated partners can change dramatical-ly the electrophysiological properties of the channels, aswell as control their cell surface expression and subcellularlocalization. The specific localization of Kv4 channels tothe somatodendritic compartment is crucial for the impor-tant roles of these channels in integration of synapticinputs and signal processing in dendrites, includingattenuation of backpropagating action potentials (Hoffmanet al., 1997). Two auxiliary subunits are believed to be keycomponents of Kv4 channel complexes in neurons: KChIPs(An et al., 2000; Rhodes et al., 2004), and the dipeptidylaminopeptidase-like proteins DPPX (Nadal et al., 2001, 2003)and DPP10 (Jerng et al., 2004b; Ren et al., 2005; Zagha et al.,2005).

DPPX and DPP10 proteins are single-spanning transmem-brane proteins with a short, divergent intracellular amino-terminal domain and a large extracellular sequence, whichcontains two highly conserved domains: a β-propeller and anon-catalytic αβ-hydrolase fold (Kin et al., 2001; Strop et al.,2004; Wada et al., 1992). We reported previously that DPPXincreases the current magnitude and modulates the voltage-dependence and kinetic properties of Kv4 channels such thatthey more closely resemble the fast kinetics of the nativechannels in neurons (Nadal et al., 2003).

Five alternatively spliced variants of DPPX that differ onlyin the intracellular N-terminus can be identified in the rodentgenome database; however, the functional consequences ofthe alternative splicing are unknown. It was shown that thepresence of an HA tag in the N-terminus of DPPX-S haddramatic effects on the modulation of the properties of Kv4channels (Jerng et al., 2004b), emphasizing the importance ofstudying whether the variable N-terminus of DPPX splicevariants may have differential effects on the modulation ofthese channels.

In this study, we compared the effects of three DPPX splicevariants prominently expressed in brain, DPPX-S, DPPX-L, and

DPPX-K, on the kinetic and voltage-dependent properties ofKv4.2 channels expressed in Xenopus oocytes. To further studythe effect of the isoform-specific domain on themodulation ofKv4 channels, we characterized the effects of a DPPX deletionmutant lacking the variable N-terminus.

We also used in situ hybridization to compare thedistribution of the three isoforms in adult rat brain. Thisshowed specific but overlapping mRNA expression with somedistinct patterns of distribution. The combined expression ofthe three isoforms, which we confirmed using a probe to thecommonDPPX sequence, coincidedwith the expression of Kv4subunits in the rat brain regions analyzed (Serodio and Rudy,1998).

2. Results

2.1. DPPX alternatively spliced variants

A search of the GenBank EST database using the last 784residues of the rat coding sequence of DPPX-S (M76427)revealed the sequence of five alternatively spliced productsof the DPPX gene: DPPX-S, DPPX-L, DPPX-E (the embryonicform; Hough et al., 1998), DPPX-D, and DPPX-K. All five splicevariants differ only in the intracellular N-terminal sequence:the predicted proteins have short divergent N-termini with 17(DPPX-K, DPPX-D), 19 (DPPX-S), 20 (DPPX-E), and 75 (DPPX-L)amino acids preceding a common juxtamembrane region of 14amino acids (Fig. 1A). DPPX-S (“short”) and DPPX-L (“long”)were the first isoforms of DPPX identified (de Lecea et al., 1994;Kin et al., 2001; Wada et al., 1992). The embryonic alternativelyspliced version, DPPX-E, was isolated from mouse E8.5 cDNA(Hough et al., 1998). DPPX-D was so named because the onlyESTs found were amplified from dorsal root ganglia poly(A)+

RNA: CB585942, CB579034, CB586631. DPPX-K (named after thehigh content of lysines in the N-terminus) is apparentlyexpressed in rodent adult brain as indicated by the ESTs data(CF743412, CB518788) and therefore could be a potentialmodulator of Kv4 channels in neuronal populations wherethese channel subunits are expressed. This study is focusedon comparing the functional properties and mRNA distribu-tion in rat brain of DPPX-S, DPPX-L, and DPPX-K. DPPX-E andDPPX-D were not studied further because present dataindicate that they are not expressed in adult rodent brain.

2.2. Structure of the DPPX gene

The rat DPPX gene has five divergent 5′ exons, each encodingfor the N-terminus of one alternatively spliced variant,followed by 25 exons shared by all isoforms (Fig. 1B), asdetermined by two sequence comparison of the rat cDNAsequences with the rat genomic sequence contained inRn4_WGA2208_3 (GenBank Accession Number NW_047685.1)using BLAST. The 29 exons are distributed along more thanone million bp (Fig. 1B). Intron–exon junctions adhere closelyto known donor and acceptor splice consensus sequences.This analysis predicts five transcripts of 2403 bp (DPPX-K,DPPX-D), 2409 bp (DPPX-S), 2577 bp (DPPX-L), and 2412 bp(DPPX-E) that encode for polypeptides of 801 (DPPX-K, DPPX-D), 803 (DPPX-S), 859 (DPPX-L), and 804 (DPPX-E) amino acids.

Fig. 1 – Primary sequence alignment of the divergent N-termini of DPPX isoforms and structure of the DPPX gene. (A)Comparison of the divergent N-termini of DPPX isoforms. Exon junction is indicated by asterisk and the initial segment of thetransmembrane domain (TMD) by an arrow. Shading shows conserved amino acids. The numbers in the right indicate thenumber of amino acids shown in the alignment followed by the total number of amino acids of the open reading frame for eachsplice variant. Alignment was produced with Clustal W with the following rodent sequences: M76427 (DPPX-S); M76426(DPPX-L); CF743412 (DPPX-K); CB585942 (DPPX-D); NP_034205 (DPPX-E). ΔN indicates the sequence of the variable N-terminusdeletion mutant ΔN-DPPX. (B) Genomic organization of DPPX gene. Exons are represented by boxes, ATG signals the startingcodons and the stop codon is indicated by TAA. The scale indicates distance between exons. The N-termini of DPPX splicevariants have the following predicted proteinmotifs in their specific N-termini: DPPX-K has a Protein kinase C phosphorylationsite (Woodgett et al., 1986): TaK (positions 9–11). DPPX-S has two Protein kinase C phosphorylation sites (Woodgett et al., 1986):TaK (positions 3–5) and SgK (positions 10–12), and a Casein kinase II phosphorylation site (Pinna, 1990): TakE (positions 3–6).DPPX-L also has two Protein kinase C phosphorylation sites (Woodgett et al., 1986): TgK (positions 9–11) and TsR (positions14–16), and a Casein kinase II phosphorylation site (Pinna, 1990): SdcD (positions 69–72).

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2.3. Rat tissue distribution of DPPX transcripts

It has been determined by Northern blot analysis that DPPX-Sand DPPX-L are expressed in rat brain (Kin et al., 2001).We alsoused Northern blot analysis to investigate the expression ofDPPX-K transcripts in rat tissues using as a probe DPPX-K-specific sequences obtained by PCR amplification as describedin Experimental procedures (blast search in GenBank non-redundant databases found no relevant similarity to othersequences). The DPPX-K gene product is expressed in ratbrain, resulting in a major band of 4.8 kb (Fig. 2A). Two otherfaint bands of lower molecular weight, 4.2 kb and 2.8 kb, werealso observed. No signal was detected in heart, spleen, lung,liver, skeletal muscle, kidney, or testis. The Northern blotanalysis with a probe for all DPPX isoforms (nucleotides 423to1519 of M76426) showed two bands only in brain (3.6 kb and4.8 kb, Fig. 2B), in agreement with previous reports (3.8 kb and4.5 kb inWada et al., 1992) and 3.6 and 4.4 kb in (de Lecea et al.,1994; Hough et al., 1998). The upper band was stronger andapproximately corresponds in size to the major band of DPPX-K and those reported for DPPX-S and DPPX-L (4.5 kb and 4.7 kb,respectively; Kin et al., 2001).

2.4. Functional comparison of the effect of alternativelyspliced DPPX isoforms on Kv4.2-mediated A-type currents inXenopus oocytes

As previously reported for DPPX-S (Nadal et al., 2003), DPPX-K(Fig. 3, Table 1) and DPPX-L (Table 1) also had large effects oncurrent magnitude and channel kinetics when co-expressedwith Kv4.2 subunits in Xenopus oocytes.

A-type currents were up to 20 times larger and reached amaximum approximately 2.5 times faster when depolarizedto +40 mV in oocytes injected with both Kv4.2 subunits andDPPX-K at a ∼1:1 molar cRNA ratio than in those injected withKv4.2 alone (Fig. 3B, Table 1). DPPX-K also accelerated the rateof macroscopic inactivation of the Kv4-mediated transient A-type current during increasingly depolarizing steps (repre-sentative examples of traces obtained at different voltagesfrom oocytes injected with Kv4.2 cRNA alone or Kv4.2+DPPX-K cRNAs are shown in Figs. 3A and C, respectively). The timeto reach half-inactivation (t0.5) upon depolarization to +40 mVwas significantly faster for Kv4.2 plus DPPX-K (11.9 ± 1.4 ms,n = 21) than for Kv4.2 alone (23.2 ± 1.9 ms, n = 13) (Table 1). Co-expression of Kv4.2 with DPPX-K produced negative shifts of24 mV in the conductance–voltage relation and of 15 mV inthe voltage dependence of steady-state inactivation (Fig. 3D,Table 1). DPPX-K also increased the rate of recovery frominactivation (Figs. 3E and F). The τ of recovery at −110 mV was79.2 ± 0.6 ms (n = 11) for the currents produced by Kv4.2 plusDPPX-K versus 147.6 ± 6.7 ms (n = 8) for the currents producedby Kv4.2 alone (Fig. 3F; see a representative example of theeffect of DPPX-K in Fig. 3E).

Although not required for forming the channel complex,the N-termini of the different splice variants may neverthe-less interact with the pore-forming subunits and havedifferential effects on the kinetic properties of the channel.To test this, we compared systematically the effect of DPPX-K, DPPX-S, and DPPX-L on the modulation of Kv4.2-mediatedA-type currents expressed in Xenopus oocytes. We found thatall splice variants have similar effects on the kinetics andvoltage dependence of Kv4.2 channels on most parameters

Fig. 2 – Tissue distribution of DPPX-K in rat. Northern blotanalysis of adult rat poly(A+) RNA (2 μg/lane) from differenttissues hybridizedwith probes specific for DPPX-K (A) and forsequences common to all DPPX isoforms (B). RNA loadingwas assessed by hybridization with β actin. Left: size ofRNAs. The blotwas exposed to X-ray film for 8 days (DPPX-K),3 days (DPPX), and 12 h (actin).

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analyzed and summarized in Table 1. These results suggestthat the variable N-termini of DPPX isoforms do not play amajor role in the modulation of Kv4 channels by DPPX. Toconfirm this, we made a deletion mutant that lacks theisoforms-specific N-terminus of DPPX splice variants: ΔN-DPPX (see Fig. 1 for the N-terminus sequence).

Co-expression of Kv4.2 with the different splice variants orthe deletionmutant increased the rate of activation comparedto expression of Kv4.2 alone. There was an approximate 2.5-fold reduction in time to reach peak current at 40 mV in thepresence of all the isoforms and the deletionmutant ΔN-DPPX(Table 1).

Furthermore, all isoforms and the deletion mutant hadsimilar and pronounced effects on the kinetics of inactivation(Tables 1 and 2).

The DPPX-K, -S, and -L isoforms produced similar hyper-polarizing shifts in the voltage dependence of the activation

(Fig. 4A, Table 1). The values were in the range of what wasreported previously (Nadal et al., 2003). We observed 27.4, 21.0,and 20.8mV hyperpolarizing shifts in the V1/2 (voltage yieldinghalf-maximal conductance) of Kv4.2 channels with DPPX-K,DPPX-S, and DPPX-L, respectively (Fig. 4A, Table 1). The N-terminus deletion mutant produced a similar shift (25.8 mV,Fig. 4A, Table 1). All the different splice variants and the N-terminus deletion also shifted the voltage dependence ofKv4.2 channel inactivation in the hyperpolarizing direction(Fig. 4B, Table 1).

Differences among the alternatively spliced isoformswere observed in two properties. There was a significantdifference on the rate of recovery from inactivation, wherethe rates of Kv4.2 in combination with DPPX-K or the N-terminus deletion mutant – although faster than Kv4.2alone – were significantly slower than the rates observed inthe presence of DPPX-S or DPPX-L (P < 0.001, Table 1). Therewere also differences in the magnitude of the shift in thevoltage dependence of steady-state inactivation (Fig. 4B,Table 1). Not only were the differences between DPPX-L andeither DPPX-S or DPPX-K statistically significant (figurelegend of Fig. 4), but also the trend is in the same directionas the differences in the rate of recovery from inactivation:the faster the recovery, the smaller the shift in steady-stateinactivation.

2.5. Rat brain distribution of DPPX-K, DPPX-L, andDPPX-S mRNAs

We use radioactive in situ hybridization to compare thepattern of expression of DPPX-K mRNA in rat brain with theexpression patterns of DPPX-S and DPPX-L (Fig. 5). Using aprobe complementary to all splice variants (DPPX), we foundthat the levels and pattern of expression of the three splicevariants combined matched those of the common probe(DPPX) in most nuclei (Fig. 5). More importantly, expressionof DPPX transcripts (or the sum of the expression of the threedifferent splice variants, Fig. 5) seemed to fully overlap theexpression of Kv4 subunits in all brain regions analyzed(Serodio and Rudy, 1998).

DPPX-S had the most widespread pattern of expression,followed by DPPX-K (Fig. 5). DPPX-L, in contrast, was stronglyexpressed only in olfactory bulb, cerebellum, and the habe-nula, was weak in thalamus and hippocampus, and hadnegligible expression in the rest of the brain (Fig. 5). DPPX-Sand DPPX-K comprised most of the overall DPPX signal, yetDPPX-L also contributed significantly in olfactory bulb andcerebellum.

In the olfactory bulb, there was an overlapping pattern ofexpression of the three splice variants in the glomerular,mitral, and granule cell layers (Figs. 5A–D), with a strongersignal from DPPX-L, specially in the granule cell layer (Fig. 5C).

In the caudate putamen, where Kv4.2 is strongly expressed,the signal observed with the DPPX probe (Fig. 5H) seemed tocome mainly from DPPX-S (Fig. 5F). In the globus pallidus, thesignal was weaker than in the caudate (note the differencebetween CPu and GP, Fig. 5H) and seemed to be contributedmostly by DPPX-K (Fig. 5E).

In the neocortex, the signal observed in layers 2–3 and layer5 with the DPPX probe (Figs. 5D, H, and L) came mainly from

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the expression of DPPX-S (Figs. 5F, J, and N), whereasexpression of DPPX-K was diffuse and weakly distributedamong all layers (Figs. 5A and E).

Fig. 3 – Functional expression of DPPX-K in Xenopus oocytes. Kvoocytes injected with Kv4.2 cRNA alone (A) or Kv4.2 plus DPPX-K cunder two-electrode voltage clamp. Oocytes were voltage-clampeto −110 mV to remove channel inactivation, followed by depolarizPeak current at +40mV (Imax) is plotted against time to peak (timeKv4.2 cRNA alone (open circles) or in combination with DPPX-K (relation and steady-state inactivation (I/Imax) for the A-type currecircles) or in combination with DPPX-K (solid circles). Data are sh(see Table 1). (E) Recovery from inactivation of the A-type currentcRNAs at a ~1:1 molar ratio. The currents were recorded during tseparated by increasing time intervals at −110 mV. (F) Time courKv4.2-mediated A-type currents in oocytes injected with Kv4.2 palone (open circles) (mean ± SE, n = 11 for Kv4.2 plus DPPX-K and

In the hippocampus, DPPX was uniformly expressedthroughout CA1–CA3 (Fig. 5L), closelymatching the expressionpattern of Kv4s (Serodio et al., 1996). DPPX-S contributed most

4.2-mediated A-type K+ currents in representative XenopusRNA at a ~1:1molar ratio (C) obtained by whole cell recordingd at a holding potential of −90 mV and hyperpolarized for 1 sing test pulses from −80 to 40 mV in increments of 10 mV. (B)peak) during depolarization to +40mV in oocytes injected withsolid circles). (D) Normalized conductance–voltage (G/Gmax)nts recorded in oocytes injected with Kv4.2 cRNA alone (openown as mean ± SE and were fitted with Boltzmann functionss in a representative oocyte injected with Kv4.2 plus DPPX-Kest pulses to + 40 mV following a pulse to the same voltagese of the recovery from inactivation at −110 mV oflus DPPX-K cRNAs at a ~1:1 molar ratio (solid circles) or Kv4.2n = 8 for Kv4.2 alone).

Table 1 – Voltage dependence and kinetic properties of Kv4.2-mediated A-type currents in Xenopus oocytes

Voltage dependence Kinetics(at room temperature)

n

Activation Inactivation

Von, mV V0.5, mV k, mV V0.5, mV k, mV tpeak, ms t0.5, ms τrec, ms

Kv4.2 −50 to −40 −4.1 ± 2.2 21.5 ± 2.5 −64.1 ± 0.6 7.5 ± 0.5 6.5 ± 1.1 (40 mV) 23.2 ± 1.9 (40 mV) 147.6 ± 6.7 (−110 mV) 8Kv4.2 + DPPX-S −60 to −50 −25.1 ± 1.5 18.6 ± 1.5 −76.9 ± 0.8 4.7 ± 0.5 2.5 ± 0.3 (40 mV) 10.5 ± 1.0 (40 mV) 49.0 ± 0.7 (−110 mV) 8Kv4.2 + DPPX-L −60 to −50 −24.9 ± 1.1 16.1 ± 1.0 −74.6 ± 0.6 4.9 ± 0.3 2.3 ± 0.4 (40 mV) 9.2 ± 1.3 (40 mV) 42.6 ± 1.7 (−110 mV) 6Kv4.2 + DPPX-K −60 to −50 −31.5 ± 1.6 17.2 ± 1.4 −78.8 ± 0.9 4.6 ± 0.4 2.5 ± 0.2 (40 mV) 11.9 ± 1.4 (40 mV) 79.2 ± 0.6 (−110 mV) 11Kv4.2 + ΔN DPPX −60 to −50 −29.9 ± 1.6 19.7 ± 1.4 −80.8 ± 1.0 4.6 ± 0.5 2.3 ± 0.2 (40 mV) 11.7 ± 2.1 (40 mV) 71.5 ± 2.4 (−110 mV) 10

Current properties (averages ± SEM for the indicated number of cells, n) obtained in Xenopus oocytes injected with the indicated cRNAs (molarratio ∼1:1). Activation: Von, membrane potential at which currents first become apparent; V1/2 and k, midpoint and slope, respectively, of theconductance–voltage curve fitted to a first-order Boltzmann function. Inactivation: V1/2 and k, midpoint and slope of the steady-stateinactivation curve. All kinetic parameters are reported at room temperature. Activation parameters are less reliable because the conductance–voltage curve of ISAs often does not saturate in the range of potentials used to fit the curve to a Boltzmann function.

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of the signal (Fig. 5J), followed by DPPX-K (Fig. 5I), and to alesser extent, DPPX-L (Fig. 1K).

In the thalamus, expression of DPPX-K and DPPX-Sshowed a complementary pattern of expression in somenuclei (Figs. 5I and J, and Fig. 6). For example, DPPX-K wasmost strongly expressed in the dorsal lateral geniculate (DLG,Fig. 5I); whereas DPPX-S was predominantly expressed in theventral lateral geniculate (VLG, Fig. 5J). DPPX-K was stronglyexpressed in the ventroposterolateral, mediodorsal, andlaterodorsal thalamic nuclei (VPL, MD and LD, Fig. 6A),while DPPX-S was strongest in the ventroposteromedialthalamic nucleus (VPM, Fig. 6B). DPPX-S (Fig. 5J) and DPPX-L(Fig. 5K) were expressed in some of the intralaminar andmidline thalamic nuclei, such as the parafascicular andcentrolateral nuclei (PF-CL). The only splice variantexpressed in reticular thalamus was DPPX-S (Fig. 6B), whichwas also very strong in habenula (Figs. 5J and 6B). DPPX-Swas also the dominant signal in the medial geniculate andthe anterior pretectal nucleus (MG and APT, Fig. 5N).

Both DPPX-K and DPPX-S were expressed in the amygdala(Ag, Figs. 5I and J), yetDPPX-S transcripts gavea stronger signal.

DPPX-K was expressed in hypothalamic and mammillarynuclei (M, Fig. 5I), where there was also some complementarysignal from DPPX-S transcripts (Fig. 5J). The three splicevariants labeled the zona incerta (ZI, Figs. 5 I–K, see also Fig. 6).

The substantia nigra pars compacta expressed exclusivelyDPPX-S transcripts at high levels (SNC, Fig. 5N).

Both the superior and inferior colliculus showed a verystrong signal with the DPPXprobe thatmay be accounted by the

Table 2 – Time constants of inactivation and their relativecontributions

τ1 τ2 A1 / (A1 + A2) n

Kv4.2 14.28 ± 0.5 102.3 ± 7.9 0.88 ± 0.02 8Kv4.2 + DPPX-S 9.08 ± 1.6 53.9 ± 31.6 0.88 ± 0.09 8Kv4.2 + DPPX-L 8.1 ± 0.4 26.3 ± 2.2 0.73 ± 0.17 6Kv4.2 + DPPX-K 10.4 ± 1.7 63.3 ± 19.9 0.88 ± 0.07 11Kv4.2 + ΔN-DPPX 8.6 ± 1.3 47.2 ± 24.9 0.84 ± 0.11 10

Averages ± SEM for the indicated number of cells (n) of the fast (τ1)and slow (τ2) time constants of the development of inactivation at+40 mV and their corresponding amplitude ratios A1 / (A1 + A2), asdetermined by biexponential fits.

combined expression ofDPPX-K andDPPX-SmRNAs (Figs. 5A, B,and D). DPPX-S was expressed in the olfactory tuberculum (Fig.5B). DPPX-K, DPPX-S, and, to a lesser degree, DPPX-L wereexpressed in the pontine nucleus (Figs. 5A–C). All three isoformswere expressed in the piriform cortex (Figs. 5E–G).

The mRNAs of the three splice variants were stronglyexpressed in cerebellum, in particular in the granule cell layer(Figs. 5A–C and Q–S). The pattern of expression of DPPX-K (Fig.5A), and to some extent that of DPPX-L also (Fig. 5C), appearedto follow the rostrocaudal gradient of Kv4.2 transcripts in thegranule cell layer (Serodio and Rudy, 1998). It is interestingthat the gradient matches the patterned distribution of Kv4.2and not that of Kv4.3 since it has been suggested that, inneurons expressing predominantly Kv4.2 or Kv4.3 subunits,DPPX may form complexes preferentially with Kv4.2, whereasDPP10 with Kv4.3 (Zagha et al., 2005).

3. Discussion

We report the functional characterization and comparativemRNA distribution in rat brain of three isoforms of the DPPXgene that are strongly expressed in brain: DPPX-K, DPPX-S, andDPPX-L. DPPX proteins shift the voltage dependence andmodify the kinetic properties of Kv4-mediated channels suchthat they more closely resemble subthreshold-activated A-type channels in neurons, thereby explaining the discrepancybetween A-type currents expressed by native channels andKv4-mediated currents expressed in heterologous expressionsystems. In addition to the differences described here (Fig. 4,Table 1), the existence of different alternative splice variantsof DPPX with divergent N-termini (Fig. 1A) suggests that theremight be a yet undiscovered layer of modulation of Kv4channels by this emerging subfamily of proteins.

3.1. DPPX splice variants have divergent N-termini

We searched the ESTs database for alternatively splicedvariants encoded by the DPPX gene. Alignment of the proteinsequences and analysis of the rat genomic versus the cDNAsequences indicated that there are five isoforms that utilizethe same splice site and differ only in the intracellular N-terminus (Fig. 1A).

Fig. 4 – Comparison of the effect of DPPX splice variants on the voltage dependence of activation and inactivation of Kv4.2channels in Xenopus oocytes. Normalized conductance–voltage (G/Gmax) relation (A) and steady-state inactivation (I/Imax) (B) forthe A-type currents recorded in oocytes injected with Kv4.2 cRNA alone (○) or in combination with DPPX-S (♦), DPPX-L (▲),DPPX-K (●), or the deletion mutant ΔN-DPPX (■). Data are shown as mean ± SE and were fitted with Boltzmann functions (seeTable 1). For the shift in steady-state inactivation, independent t tests were performed pair wise between each splice variantandDPPX-L,which displayed the smallest shift; the differenceswere statistically significant for DPPX-S andDPPX-K (P < 0.0001),but not for ΔN-DPPX (P = 0.02).

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The potential phosphorylation sites for PKC and for caseinkinase II present in the intracellular domains of DPPX-K, -S,and -L (figure legend of Fig. 1) could add levels of modulationof A-type channels to the existing regulation by PKA- or PKC-coupled pathways on the ERK phosphorylation of Kv4.2subunits (reviewed in Birnbaum et al., 2004). The fact thatthe N-terminus of the different DPPX splice variants isbestowed with different combinations of phosphorylationsites (Fig. 1) may point to differential roles of these isoformson the modulation of Kv4 channels by second messengercascades.

3.2. Structure of the rat DPPX gene

The rat DPPX gene has 29 exons distributed along more thanone million bp (Fig. 1B) and is localized to chromosome 4,whereas the human DPPX gene is in chromosome 7 (7q36.2(Yokotani et al., 1993) and the mouse DPPX gene is linked tomarkers in the proximal end of chromosome 5 in a region ofconserved synteny (Wada et al., 1993). There are 5 knownvariants of exon 1, each corresponding to one alternativelyspliced isoform (Fig. 1B).

3.3. DPPX-S, DPPX-L, and DPPX-K splice variants areexpressed in rat brain

Northern blot analysis of the tissue distribution of DPPX-K inrat shows a main band of 4.7 kb in brain (Fig. 2A). DPPX-S andDPPX-L also label a 4.4–4.7 kb band in Northern blots from ratbrain (de Lecea et al., 1994; Hough et al., 1998; Kin et al., 2001;Wada et al., 1992), yet a more sensitive study using Southernblot analysis of PCR products suggested that DPPX-S may alsobe expressed in kidney, testis, ovary, and prostate (Wada et al.,1992). The banding pattern of these three splice variantsoverlapped with the upper band (4.7 kb) observed with theprobe for all DPPX splice variants (Fig. 2B). The lower band

obtained with this probe (3.6 kb, Fig. 2B) matched previousNorthern blot studies and has not been assigned to any DPPXsplice variant yet (de Lecea et al., 1994; Kin et al., 2001;Wada etal., 1992). The predominant expression of DPPX in rat brain hasbeen confirmed byWestern blot with antibodies against the C-terminus of the protein (Kin et al., 2001; Nadal et al., 2003;Zagha et al., 2005).

In human, significant amounts of DPPX-S were found inbrain (Yokotani et al., 1993) and heart, as well as testes,pancreas, lung, kidney, and ovary, as determined by quanti-tative RT-PCR (the result from heart was also confirmed byWestern blot) (Radicke et al., 2005). The reported levels ofexpression of DPPX in heart are lower than in brain andlimited to ventricles (Radicke et al., 2005).

3.4. The role of the variable N-termini on the functionaleffects of DPPX on Kv4 channels

Qualitatively, all three spliced variants examined in thisstudy had similar effects on the kinetic and voltage-dependent properties of Kv4 channels expressed in Xenopusoocytes. Co-expression of Kv4.2 with DPPX-K, DPPX-S, orDPPX-L resulted in larger currents with faster activationand inactivation, faster recovery from inactivation, andproduced a shift in the voltage dependence of activationand inactivation to hyperpolarized potentials. Furthermore,a DPPX mutant lacking the variable N-terminus affectedchannel function in a similar fashion (Table 1). In allcases, the resultant current more closely resembles sub-threshold-activated A-type K+ currents in neurons (Nadalet al., 2003).

However, there were consistent quantitative differencesbetween the effects of the spliced variants on two of thefunctional parameters explored here: the rate of recovery frominactivation and the voltage dependence of steady-stateinactivation. DPPX-S and DPPX-L produced significantly faster

Fig. 5 – Distribution of DPPX splice variants mRNAs in rat brain. X-ray autoradiograms of sagittal (A–D) and coronal sections ofan adult rat brain at level of forebrain (E–L), midbrain (M–P), and cerebellum and medulla (Q–T) hybridized with 35S-labeledprobes specific for the DPPX-K, DPPX-S, and DPPX-L transcripts and with a probe that recognizes all isoforms (DPPX). Ag,amygdala; APT, anterior pretectal nucleus; arc, arcuate nucleus; CA1, CA2, CA3, CA1–2 and 3 hippocampal subfields,respectively; CPu, caudate putamen; Cx-2/3, cortical layers 2 and 3; Cx-5, cortical layer 5; DG, dental gyrus; DLG, dorsolateralgeniculate nucleus; Gl, glomerular layer of the olfactory bulb; GP, globus palidus; Gr, granule cell layer of the olfactory bulb; Hb,habenula; IC, inferior colliculus; ICj, Islands of Calleja; M, mammillary nuclei; MG, medial geniculate nucleus; Mi, mitral celllayer of the olfactory bulb; PF-CL, parafascicular and centrolateral thalamic nuclei; Pir, piriform cortex; SNC, substantia nigrapars compacta; VLG, ventrolateral geniculate nucleus; VPM, ventral posteromedial nucleus. The scale bar corresponds to 10mmin the sagittal sections, 5 mm in the coronal sections E to P, and 4 mm in the coronal sections Q to T.

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rates of recovery from inactivation than DPPX-K. The rate ofrecovery from inactivation in the presence of DPPX-K wassimilar to that obtained when Kv4.2 was co-expressed withthe mutant lacking the N-terminus (Table 1).

Interestingly, differences among the splice variants on theshift of the voltage dependence of steady-state inactivation(Fig. 4B, Table 1) followed the same tendency as the recoveryfrom inactivation, i.e., the smaller shift corresponds to thefastest recovery rates. The differences on voltage-dependenceare likely the result of the differences in inactivation recovery,since the acceleration of the rates of recovery is expected tooppose the negative shift produced by faster inactivationrates.

The results with the alternatively spliced N terminiand with the mutant lacking the variable N-terminussuggest that this region of DPPX has minimal effects onthe structural changes induced by this accessory proteinon the Kv4 subunits, except for those changes thatinfluence inactivation recovery. Furthermore, this suggeststhat the structural changes on Kv4 channels mediatingthe effects on inactivation recovery are somewhat inde-pendent from those producing other changes in Kv4channel function.

It has been reported that addition of an HA-tag to thecytoplasmic N-terminus of DPPX-S markedly slowed thekinetics of inactivation of Kv4 channels (Jerng et al., 2004b).

Fig. 6 – Distribution of DPPX-K and DPPX-S mRNAs in rat thalamus. Sections at the level of the thalamus from adult rat brainhybridized with 35S-labeled probes specific for the DPPX-K (A) and DPPX-S (B), illustrating the complementary pattern ofexpression of these two transcripts. Hb, habenula; ic, internal capsule; LD, laterodorsal nuclei; MD, mediodorsal nuclei; RT,reticular thalamus; VPL, ventral posterolateral nucleus; VPM, ventral posteromedial nucleus; ZI, zona incerta. The scale barcorresponds to 1 mm.

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Our results suggest that the N-terminal region does notinteract directly with the Kv4 pore-forming subunits. Thereported effects of tagged DPPX might be due to effects of theHA tag on the DPPXmolecule and its interactions with the Kv4subunits or might reflect effects of the HA sequence itself onthe channel protein.

Our results indicate that variations in the N-terminalsequence of DPPX splice variants do not significantly influenceKv4.2 channel function, yet it remains to be determinedwhether theymay still do so by affecting the interaction of theKv4 channels with other auxiliary subunits, such as KChIPs.Although no interaction between KChIPs and DPPX has beendemonstrated, the various N-terminal sequences of DPPXmaydifferentially affect the interaction of KChIPs with the channelcomplex.

3.5. Distribution of DPPX splice variants mRNA in ratbrain

We compared the expression patterns of DPPX-K, DPPX-S, andDPPX-L splice variants by radioactive in situ hybridization inrat brain. DPPX-S and DPPX-K had the most widespreadexpression, whereas DPPX-L was concentrated to limitedareas, such as olfactory bulb, cerebellum, and the habenula(Figs. 5C, K, and S). DPPX-S and DPPX-K were also veryprominent in olfactory bulb and cerebellum, in addition tohippocampus, thalamus, and cortex (Figs. 5A and B). Inthalamus, DPPX-K and DPPX-S seem to show a complemen-tary pattern of expression (Figs. 5I and J, and Fig. 6). Thecombined expression of these three splice variants overlappedin all areas labeled with a probe of a common DPPX sequence.The distribution of DPPXmRNAs obtained with this probe is inagreement with previous in situ hybridization studies using aprobe common to all splice variants (de Lecea et al., 1994;Wada et al., 1992).

The pattern of expression also seemed to match theexpression of Kv4 subunits, especially Kv4.2 (Serodio andRudy, 1998). This is in agreement with our previous resultssuggesting that, in areas expressing predominantly Kv4.2 orKv4.3, Kv4.2 is present in the same regions as DPPX whereasKv4.3 co-localizes with DPP10 (Zagha et al., 2005). However,there is one regionwhere DPPX is strong, the SNC (DPPX-S, Fig.5N), which only expresses Kv4.3 (Serodio and Rudy, 1998;Serodio et al., 1996), and more specifically, Kv4.3L (Liss et al.,2001).

The physiological roles of subthreshold-activated A-typeK+ channels in delayed excitation, regulation of firingfrequency, and dendritic function (Baxter and Byrne, 1991;Connor and Stevens, 1971; Hoffman et al., 1997; Johnstonet al., 2000; Liss et al., 2001; Llinas, 1988), rely on theirelectrophysiological properties as well as on their patternof expression and cellular localization, all of which arenotably influenced by the presence of DPPX proteins in thechannel complex. The localization of these channels tosomatodendritic compartments is crucial for their roles inintegration of synaptic inputs and dendritic signal proces-sing (Hoffman et al., 1997; Johnston et al., 2000). A strikingexample of this is the role that the gradient of subthresh-old-activated A-type current along dendrites of hippocam-pal neurons plays in regulating the backpropagation ofaction potentials (Hoffman et al., 1997). Also, Kv4 channelsconcentrate in clusters at synaptic contacts in hypotha-lamic neurons (Alonso and Widmer, 1997). Thus, the cell-specific expression of the DPPX splice isoforms couldconfer Kv4 channels with differential modulation atvarious levels. The variable DPPX N-termini could bestowthe channel with different mechanisms of subcellularlocalization, bind to different intracellular signaling mole-cules, and/or acutely regulate the number of channels atthe cell surface.

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4. Experimental procedures

4.1. Isolation of DPPX-K and design of probes for Northernblot and in situ hybridization

To isolate the specific N-terminus sequence of DPPX-K, thefollowing primers were designed: 5′-TGCTCTAGAGGTTCG-GACTTGGG-3′ (forward) and 5′-GTCTTCACTGAAGA-GATCTTCTACAGTGACC-3′ (reverse). These primers wereutilized in polymerase chain reactions using a humanexpressed sequence tag (EST) (BI603302, IMAGE cDNA clones5287406, Invitrogen) as template (there is a 100% homologybetween human, rat, and mouse sequences at the amino acidlevel). The PCR conditions were: 2 cycles of 48 °C annealing,68 °C extension, and 94 °C denaturation, each step for 30 s,followed by 30 cycles of 64 °C annealing, 68 °C extension and94 °C denaturation, each step for 30 s. The 256 bp band wassequenced, digested with XbaI and BglII, and subcloned intothe XbaI and BglII sites of pSGEM-DPPX to reconstitute the full-length sequence for in vitro transcription and expression ofcRNAs in Xenopus oocytes. The ΔN-DPPX mutant was con-structed by PCR using the following primers to create astarting methionine right before nucleotide 264 of M76426: F:5′-GGAGGAGATGGAGCTGGTGGGGAGT-3′ and R: 5′-CTAAA-ACTTCCGGCCATCCTTG-3′. The resulting 1119 bp fragmentwas then digested with EcoRI/BglII and subcloned into EcoRI(partial)/BglII sites of the pSGEM-DPPX vector.

The splice-specific probes were amplified by PCR fromcDNA (DPPX-S, accession number M76427; DPPX-L, accessionnumber M76426) or from rat genomic DNA (for DPPX-K) usingprimers complementary to sequences present in the first exoncoding for the 5′UTR and the N-terminus specific for eachsplice variant (see below). The hybridization probe to all splicevariants (DPPX) was amplified using primers complementaryto sequences downstream the alternative splice junction(nucleotides 423 to1519 of M76426). The PCR conditions were:2 cycles of 55 °C annealing, 68 °C extension and 94 °Cdenaturation, each step for 1 min, followed by 30 cycles of59 °C annealing, 68 °C extension and 94 °C denaturation, eachstep for 1 min. Both strands of all PCR products weresequenced.

Probe

Primers Size of PCRfragment (bp)

S

F: 5′-TCTTCTTCTGCCGAGCCCGT-3′ 278 R: 5′-GCTCCTGATCCTGCTGCTGTA-3′

L

F: 5′-GCTCAGCACCCAGGCGACTC-3′ 428 R: 5′-CAGCTCGTCCTCCTCGTCACA-3′

K

F: 5′-ACCAGCACTGTCAGCCAAGC-3′ 406 R: 5′-AGCTCCATCACGTTGCCCTG-3′

DPPX

F: 5′-GGAGGAGATGGAGCTGGTGGGGAGT-3′ 1096 R: 5′-CTAAAACTTCCGGCCATCCTT-3′

4.2. Northern blot analysis

Rat Multiple Tissue Northern Blot (BD Biosciences Clontech)with 2 μg of polyA+ RNA per lane. Probes specific for DPPX-K(K) and a region of the coding sequence common to all splicevariants (DPPX) were labeled with [32P]dCTP using Rediprime II

RandomPrime Labelling System (AmershamBiosciences). Theblots were hybridized in QuickHyb (Stratagene) with 1 × 106

cpm/ml (5 ng of DNA/ml) for 1 h at 68 °C. They weresubsequently washed twice with 2× SSC, 0.1% SDS at roomtemperature for 15 min each, followed by a final wash with0.1× SSC, 0.1% SDS at 60 °C for 30 min, and then exposed to X-ray film (Kodak) at −70 °C for 10 days (K) or 3 days (DPPX). Blotswere hybridized first with the K-specific probe, then strippedby heating the membrane at 95 °C in 0.1 SSC, 0.1% SDS for 10min and hybridized with the DPPX specific probe, thenstripped a second time and hybridize with an β actin probefor lane content.

4.3. In situ hybridization studies

In situ hybridization was performed by using the methodsdescribed in Weiser et al. (1994). All solutions were preparedwith RNase-free water and reagents. Briefly, 60-day-old malerats were perfused intracardially with 100 ml of cold salinesolution (0.9% NaCl with 0.5% NaNO2 and 1000 u Heparin),followed by 300 ml of cold 4% paraformaldehyde solution in0.1 M phosphate buffer, pH 7.4. The brains were carefullyremoved, cut in blocks and postfixed for 2 h. After postfixing,the brains were placed in 30% sucrose overnight. A freezing-microtome was used to obtain 40 μm thickness slices. Floatingsections were prehybridized at 50 °C for 2 h (50% formamide,2× SSC, 10% Dextran Sulfate, 4× Denhardt's, 50 mM dithio-threitol, and 0.5 mg/ml denatured salmon sperm DNA), andthen hybridize overnight in the presence of 107 counts of 35S-radiolabeled, heat-denatured probe. Sections were thenwashed in decreasing concentrations of SSC buffer at 48 °C(2×, 1×, 0.5×, 0.25× and 0.1×) and, following a final wash in0.05 M phosphate buffer at room temperature, were handmounted on slides, air-dried and exposed to Kodak XAR-5 X-ray film. For anatomical description, we followed the nomen-clature of the Paxinos Atlas (Paxinos, 1995).

4.4. Preparation of in vitro transcribed RNA andfunctional expression in Xenopus oocytes

Experiments were carried out as described previously (Nadalet al., 2001, 2003). Briefly, linearized recombinant plasmidscontaining full-length Kv4.2 or DPPX cDNAs were used astemplates for in vitro transcription using the mCAP mRNACapping Kit as described by the manufacturer (Stratagene).Oocytes were obtained from the ovarian lobes of femaleXenopus laevis (Xenopus I, Ann Arbor, MI, USA). The frogs wereanaesthetized with 0.2% MS-222 (Sigma, St. Louis, MO, USA)and the ovarian lobes surgically removed and placed incalcium-free ND96 solution. Defolliculated stage V–VI oocyteswere obtained by collagenase digestion, microinjected withthe appropriate cRNAs (the Kv4.2:DPPX-K molar cRNA ratiowas ∼1:1), and incubated for 1.5–3 days in ND96 solution at18 °C. Ionic currents were recorded under two-microelectrodevoltage clamp with a Geneclamp 500 amplifier (Axon Instru-ments, Foster City, CA, USA) at room temperature (20–23 °C).The recording chamber was continually perfused with low-Cl−

ND96 (sodium methanesulfonate 96 mM, KCl 2 mM, MgCl22.3 mM, HEPES 10 mM and CaCl2 0.5 mM; pH 7.4). Handling ofthe animals followed the corresponding protocol guidelines

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and steps were taken to minimize pain and discomfort of theanimals.

Acknowledgments

We thank Eddie Zagha and Pilar Molina Portella for theircomments. This work was supported by NIH grants NS30989and NS045217, and NSF grant IBN 0314645.

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