Differential modulation of Kv4 kinetics by KCHIP1 splice variants

Molecular and Cellular Neuroscience 24 (2003) 357–366

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Differential modulation of Kv4 kinetics by KCHIP1 splice variants Diane Van Hoorick, Adam Raes, Wim Keysers, Evy Mayeur, and Dirk J. Snyders* Laboratory for Molecular Biophysics, Physiology, and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium Received 2 January 2003; revised 5 May 2003; accepted 7 May 2003

Abstract The ␤-subunits of the KChIP family modulate properties and expression level of Kv4 channels. We report the cloning of the first splice variant of KChIP1 (KChIP1b) which contains an extra exon, rich in aromatic residues, in the amino terminus. Both splice variants interacted equally well with Kv4.2 subunits based on confocal imaging and upregulation of current density (more than five-fold). No effects on the voltage dependence of activation or inactivation were noted. However, the effects on the kinetics of recovery from inactivation were opposite: KChIP1b induced a slow component in the recovery (␶ ⬃ 1.2 s), in contrast to the increased recovery rate (␶ ⫽ 125 ms) with KChIP1a. Accordingly, frequency-dependent accumulation of inactivation was enhanced by KChIP1b but reduced by KChIP1a. Since Kv4.2 channels are involved in protection against back propagating action potentials in dendritic spines, a differential expression of either splice variant could shape the dendritic function. © 2003 Elsevier Science (USA). All rights reserved.

Introduction Ion channels are integral membrane proteins that regulate ionic membrane permeability and control various physiological functions such as neuronal signaling, muscle contraction, hormone secretion, cell volume, and ionic homeostasis. The native K⫹ channels consist of a tetramer of pore-forming ␣-subunits, frequently associated with auxiliary subunits that can modulate the intrinsic function of the ␣-subunits. In pyramidal cells of the CA1 region of the hippocampus, for example, the electrical properties are determined by the simultaneous expression of Kv2, Kv3, and Kv4 family members (Martina et al., 1998). Channels containing Kv4 subunits activate at subtreshold membrane potentials and mediate rapidly inactivating transient outward currents. In the nervous system these A-type channels decrease the amplitude of back propagating action potentials, reduce the ability to initiate action potentials in dendrites, and reduce the magnitude of EPSP (Hoffman et al., 1997; Yuste, 1997; Schoppa and Westbrook, 1999). In cardiac myocytes the Kv4 subunits underlie the transient outward

* Corresponding author. Fax: ⫹00-32-03-820.25.41. E-mail address: [email protected] (D.J. Snyders).

current, ITO, which shapes the early phase of the cardiac action potential. As such ITO may influence the balance of currents during the plateau phase and thus modulate the duration of the action potential and refractoriness of myocardial tissue (Campbell et al., 1995; Dixon et al., 1996). The properties of Kv4 channels in heterologous expression systems are similar to, but not identical to those of neuronal A-type currents or cardiac ITO. The differences could be due to an association with auxiliary subunits or other posttranslational modifications in vivo. Indeed, association of KChIP proteins with Kv4 subunits has been reported (An et al., 2000; Bahring et al., 2001a; Rosati et al., 2001). This family of ␤-subunits is related to Ca2⫹ binding proteins and is characterized by 4 EF-hand motifs. These KChIP subunits display a modulatory action on Kv4 channels. It has been suggested that KChIP’s act as chaperones, thus elevating the expression level of channels at the plasma membrane and the Kv4 current density. In rodents (rat, mouse) the transmural gradient of ITO across the left ventricular wall is due to a gradient of Kv4.2 mRNA levels. However, in larger mammals (dog, human) the gradient in ITO density is due to differential expression of KChIP2 (Rosati et al., 2001). In addition to effects on the level of expression at the plasma membrane, KChIP’s have been reported to modulate the

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kinetics of inactivation and/or recovery from inactivation (An et al., 2000; Holmqvist et al., 2001). In general, induction of inactivation is slower but recovery is faster compared to Kv4.2 alone. Thus, these ␤-subunits provide a more subtle modulation of the cellular excitability. In hippocampal CA1 pyramidal neurons a gradient in transient A-type current has also been reported; there is a high level of A-type current in the dendrites that decreases toward the soma (Hoffman et al., 1997). The exact nature of the selective targeting of channels towards one end of a neuron has not been elucidated but association with auxiliary subunits could be responsible for such a process. A more comprehensive view on the functions and interactions of auxiliary subunits and their splice variants will enhance our insight in the molecular basis of in vivo currents. Here we report the cloning of a KChIP1 splice variant with distinct kinetic effects on Kv4.2 channels. Opposing effects of KChIP1a and KChIP1b on Kv4.2 inactivation kinetics were observed, resulting in an opposite frequency dependence of accumulation of inactivation.

Results KChIP1b, a novel KChIP1 splice variant The full-length mouse clone of KChIP1 was PCR amplified from mouse brain cDNA with primers P1 and P2 (see Experimental methods). Electrophoretic analysis of the HindIII\BamHI digestion indicated the presence of two fragments (Fig. 1A), one of which contained 33 bp more than expected (Fig. 1C). Analysis of the mouse and human genome sequence revealed that the novel 33-bp sequence represented a previously unrecognized exon in the large intron between exons 1 and 2. Indeed, this sequence was flanked by the correct splice acceptor and donor sites. The smaller formerly identified clone was renamed KChIP1a while the longer, newly identified, splice variant was named KChIP1b. The additional exon was designated exon 1b which leads to the genomic structure as shown in Fig. 1B. In the human genome exon 1 and exon 1b are separated by a large intron of at least 133 kb while the following seven exons (2– 8) are distributed over approximately 23 kb. KChIP1 is located on chromosome 5 in humans and on chromosome 11 in mice. The extra exon 1b is rich in amino acids with aromatic side chains (5 of 11). According to secondary structure prediction programs the majority of the inserted amino acids in KChIP1b are arranged in a ␣-helical conformation (Kneller et al., 1990; Cuff et al., 1998; Jones, 1999). In the proposed secondary structure of KChIP1b this leads to an additional ␣-helix, compared to KChIP1a. The tissue distribution of mRNA expression for each splice variant was assessed using cDNA panels with PCR primers that were specific for each splice variant (see Experimental methods). This analysis unexpectedly showed a differential expression pattern for both splice variants. After

separation on a 1.5% agarose gel an intense band of KChIP1b was detected in brain, kidney, liver, placenta, skeletal muscle, small intestine, and testis. KChIP1a showed high expression in brain, kidney, lung, pancreas, leukocytes, and prostate (Fig. 1D). This difference in expression profile could be due to a tissue-specific expression of exonic or intronic splicing enhancers and silencers (Maniatis and Tasic, 2002). The differential presence of either of these proteins could regulate the inclusion or exclusion of exon 1b. KChIP splice variants are recruited to the plasma membrane by Kv4.2 channels The subcellular localization of the identified splice variants and their interaction with Kv4.2 and Kv4.3 subunits was investigated with confocal imaging of GFP-tagged KChIP proteins. Both splice variants were fused with GFP at the carboxy terminus and expressed in HEK293 cells. When expressed alone, each splice variant displayed a cytoplasmic localization, characterized by a uniform staining of the cytoplasm while no membrane localization could be detected (Fig. 2A and C). The KChIP proteins were also excluded from the nucleus and no apparent staining of the ER was noted, since there was minimal overlap with the expression of DsRed-ER. However, upon cotransfection with Kv4.2, a known interaction partner of KChIP1 proteins (An et al., 2000; Nakamura et al., 2001; Beck et al., 2002), we observed relocalization of both KChIP1a and KChIP1b. Fig. 2B and D shows that the green fluorescence of the KChIP1x proteins was now localized at the level of the plasma membrane, apparently recruited there through an interaction with the membrane targeted channels formed by Kv4.2 subunits. Comparison of the panels in Fig. 2 indicates that KChIP1b interacts with Kv4.2 at least as well as KChIP1a. Similar recruiting to the plasma membrane was observed when KChIP1b was coexpressed with Kv4.3 (data not shown). KChIP1b increases Kv4.2 current density The effects of KChIP1b on current density were tested using a stable cell line expressing Kv4.2 in Ltk⫺ cells (Yeola and Snyders, 1997) in order to reduce the inevitable cell-to-cell variability of transient transfections. Typical Atype or ITO-type currents were recorded under standard voltage clamp protocols. The amplitude of the peak current at ⫹50 mV was used as an index of the current density. After 24 h of induction this cell line displayed a modest current density (Fig. 3A, Table 1 ) while upon transfection of either KChIP1a or KChIP1b a significant increase in current density was observed (Fig. 3C and D). The upregulation by KChIP1b is not statistically different from the upregulation by KChIP1a (P ⬎ 0.05).

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Fig. 1. Genomic structure and sequence alignment of KChIP1a and KChIP1b. (A) Two fragments with different electrophoretic mobility were detected upon restriction enzyme digestion of PCR-amplified KChIP1 sequences from mouse brain. The lower band corresponds to the original KChIP1 (now KChIP1a) while the slower migrating band represents the novel splice variant KChIP1b. (B) The eight exons of KChIP1a (black boxes) are distributed over at least 156 kb in the human genomic DNA. The additional exon (1b) of KChIP1b is shaded in gray. The solid line represents the splicing order for KChIP1a while the dashed line depicts the insertion of the additional exon for KChIP1b. (C) DNA sequence of the additional exon of KChIP1b flanked by splice acceptor and donor consensus sequences in the human and mouse sequence. (D) cDNA panels from different human tissues were tested for the presence of KChIP1a or KChIP1b using PCR primers specific for each splice variant. After separation on a 1.5% agarose gel intense bands were detected in brain and kidney tissue for both splice variants. KChIP1a showed additional strong signals in lung, pancreas, leukocytes, prostate, and thymus tissues. KChIP1b on the other hand was present in liver, pancreas, skeletal muscle, small intestine, and testis. Primers for the housekeeping gene G3PDH were used as positive control while water was added instead of template for the negative control. The last lane contains a 100-bp DNA ladder with the more intense band being 600 bp. (E) Amino acid alignment of mouse KChIP1a, KChIP1b. and frequenin. Identical residues are shaded black and conservative substitutions are shaded gray. The inserted sequence in KChIP1b has a relatively high number of amino acids with aromatic side chains but no specific additional domains or signal sequences could be detected. Note the considerable amount of homology between KChIP1 proteins and frequenin, with the EF-hand motifs indicated as detected in the crystal structure of frequenin.

Modulation of Kv4.2 activation and inactivation kinetics The voltage dependence of activation was obtained using 5-s step pulses between ⫺80 and ⫹30 mV. From the I-Vrelation of the peak current (Fig. 3B) it follows that coexpression of KChIP1a or KChIP1b had no effect on the apparent threshold for activation. The voltage dependence of activation was determined by normalizing the peak current amplitude by the Goldmann–Hodgkin–Katz equation to correct for the outward rectification behavior of the Kv4.2 channels (Clay, 2000). The resulting data were fitted with a Boltzmann function. The midpoint of activation (Fig. 3E) and the slope did not show significant differences upon cotransfection of either KChIP1a or KChIP1b (Table 1).

The voltage dependence of inactivation was obtained from the normalized peak currents during the test pulse (at ⫹30 mV) as a function of the 5-s prepulse voltage (Fig. 3A, C and D) and was fitted with a Boltzmann function. Fig. 3F shows that there was no significant effect on the midpoint or slope of inactivation in the presence of KChIP1a (Table 1). Upon transfection of KChIP1b there was a small hyperpolarizing shift noted compared to Kv4.2 alone. Both splice variants slowed the time course of inactivation; however, they did so in a different manner as illustrated qualitatively in Fig. 4A. The time course of Kv4.2 inactivation, examined in the absence or presence of KChIP1x, was characterized by a double exponential process. In the case of Kv4.2 the fast time constant (34 ⫾

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Fig. 2. Subcellular localization of KChIP1 splice variants. GFP-tagged versions of either splice variant were transfected without or with Kv4.2 and the subcellular distribution was investigated using confocal imaging. An ER-marker, DsRed-ER, was added to all transfections. The confocal images of GFP-tagged KChIP1x subunits showed a cytoplasmic localization (A and C). There was virtually no overlap with the red fluorescence of the ER-marker DsRed-ER, while the KChIP1 proteins were also excluded from the nucleus. Contransfection of either splice variant with Kv4.2 resulted in prominent staining at the level of the plasma membrane (B and D). Virtually all of the green fluorescence was now localized at the level of the plasma membrane indicating that Kv4.2 recruited both KChIP1a and KChIP1b equally well.

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Fig. 3. Effects of KChIP1b on voltage dependence of activation and inactivation. Typical recordings of Kv4.2 alone (A) or in the presence of KChIP1a (C) or KChIP1b (D) using the pulse protocol depicted in A (top). A, C, and D were scaled to the same scale bar. The peak amplitude during the first pulse of 5 s (⫺80 to ⫹30 mV) was used to calculate the peak IV relation shown in B. The current during the second pulse to ⫹30 mV was used to obtain the voltage dependence of inactivation. E shows the voltage dependence of activation for the respective conditions. The midpoint of inactivation was shifted for KChIP1b by approximately 8 mV in the hyperpolarizing direction (F). Cotransfection of KChIP1a did not result in a difference.

3 ms, n ⫽ 8) was responsible for the larger part of the inactivation process while the slow time constant (185 ⫾ 17 ms, n ⫽ 6) accounted for the remaining 25% of the total amplitude. We observed a slowing of the inactivation consistent with previous reports on the effects of KChIP1a on Kv4.2 inactivation. This slowing was not accompanied with a significant difference in the time constants but was due to a significant increase in the contribution of the slow component (Table 1). However, in the presence of KChIP1b the slow time constant did increase significantly, in contrast with KChIP1a, while the fast time constant remained invariant. Additionally, the contribution of the slow component increased and the combination of both effects resulted in a more extensive slowing of inactivation in the presence of KChIP1b compared to KChIP1a.

KChIP1b slows recovery from inactivation The recovery from inactivation was examined by a twin pulse protocol. Inactivation was induced during a 1000-ms step to ⫹50 mV and quantified by a test pulse to ⫹50 mV after a variable interval at ⫺90 mV. Typical recordings are shown in Fig. 4B, C, and D. The resulting recovery process behaved monoexponentially in the case of Kv4.2 alone (Fig. 4B) or coexpressed with KChIP1a (Fig. 4C), but resulted in an acceleration of the recovery with KChIP1a present (Table 1, Fig. 4E). In contrast, transfection of KChIP1b induced a second exponential component in the time course of recovery from inactivation (Fig. 4D). The time constant of this slow component was larger than 1 s and this resulted in a recovery process at least four times slower compared to Kv4.2 alone or with KChIP1a. The overall effect of

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Table 1 Functional effects of the two KChIP1 splice variants on Kv4.2

Density pA/pF n Activation V1/2 (mV) k n Inactivation V1/2 (mV) k n Inactivation ⫹50 mV ␶1 (ms) ␶2 (ms) %2 n Recovery ⫺90 mV ␶1 (ms) ␶2 (ms) %2 n a b

Kv4.2

⫹ KChIP1.a

26.5 ⫾ 3.0 16

21

⫹ KChIP1.b

212 ⫾ 47a

115 ⫾ 16a 18

2.9 ⫾ 1.9 13.3 ⫾ 0.8 7

⫺5.0 ⫾ 2.2 13.8 ⫾ 1.1 5

2.4 ⫾ 3.6 13.4 ⫾ 1.0 6

⫺46.8 ⫾ 1.2 6.1 ⫾ 0.2 8

⫺47.5 ⫾ 0.9 4.5 ⫾ 0.2 12

⫺55.4 ⫾ 1.7b 6.0 ⫾ 0.3 6

34 ⫾ 3 185 ⫾ 17 25 ⫾ 1

42 ⫾ 3 160 ⫾ 7.5 36 ⫾ 3a

35 ⫾ 2 258 ⫾ 19a,b 35 ⫾ 2a

8

6 242 ⫾ 22

na na 10

6 119 ⫾ 8a

na na 11

⫺60 mV an additional phenomenon became apparent. At ⫺60 mV there was already a considerable amount of closed-state inactivation as apparent from the steady-state inactivation curve (Fig. 3F) (Bahring et al., 2001b; Beck and Covarrubias, 2001). This results in a balance between two processes, inactivation and recovery from inactivation at ⫺60 mV. These two features resulted in a constant reduction of the current to 50% in the presence of KChIP1b (Fig. 5E) between 1- and 0.1-Hz stimulation frequencies. For KChIP1a the influence of inactivation was less pronounced with a constant current reduction of 30%. At frequencies of 1 and 3 Hz the opposing effects of both splice variants were maintained. To investigate the closed-state inactivation a protocol as shown in Fig. 6A was used. No time-dependent current was detected by using steps from ⫺90 to ⫺50 mV, consistent

106 ⫾ 10a 1199 ⫾ 94b 34 ⫾ 4 6

Significant difference compared to Kv4.2 (P ⬍ 0.05). Significant difference compared to KChIP1a (P ⬍ 0.05).

KChIP1b was thus opposite to the effect exerted by KChIP1a on Kv4.2, i.e., net slowing instead of an acceleration of the recovery from inactivation, as illustrated in Fig. 4E. In the case of Kv4.2 alone 95% recovery was obtained at ⬃660 ms which was substantially reduced (⬃330 ms) in the presence of KChIP1a. In sharp contrast, the same recovery was observed only after ⬃1800 ms in the presence of KChIP1b. The expression of the different splice variants thus has a profound influence on the time course of recovery from inactivation. Frequency-dependent accumulation of inactivation To relate the differential effect of both splice variants on the recovery from inactivation to more physiological circumstances we applied pulse trains of short depolarizations (10 ms) at various frequencies and examined the amount of use-dependent accumulation of inactivation. Upon the application of these pulse trains, the current amplitude reached a steady state within 30 pulses (Fig. 5A–C). At high frequencies all Kv4.2 channels were inactivated at the end of the pulse train (30 or 50 Hz), independent of the presence of either KChIP1 splice variants (Fig. 5D). With a holding potential of ⫺70 mV and at intermediate frequencies (3 or 1 Hz), the effect of the splice variants was clearly distinct. Kv4.2 channels showed approximately 30% accumulation of inactivation when stimulated at 3 Hz. In the presence of KChIP1a we observed only a 5% reduction under these conditions. In contrast, coexpression of KChIP1b reduced the current amplitude by 60%, consistent with the slower biexponential recovery from inactivation. When the same experiments were conducted with a holding potential of

Fig. 4. Kinetics of induction and of recovery from inactivation. A shows the typical time course of the inactivating current traces under the different experimental conditions as indicated. For comparison the traces were normalized to the peak current amplitude. Inactivation proceeded more slowly in the presence of KChIP1a or KChIP1b. Recovery from inactivation for Kv4.2 alone (B) or in the presence of KChIP1a (C) or KChIP1b (D) was fitted with appropriate exponential functions. (E) The recovery from inactivation was obtained by plotting the ratio of the amplitude during the second test pulse to the initial maximal amplitude against the interval between the two consecutive pulses. The solid line is the exponential fit with the time constants shown in Table 1. Note the slowing of the recovery with KChIP1b.

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Fig. 5. Frequency dependence of inactivation. Cells were stimulated with pulses to ⫹50 mV at varying frequencies while the effect of the holding potential was tested. A (Kv4.2), B (KChIP1a), and C (KChIP1b) are typical recordings at 3 Hz with a holding potential of ⫺70 mV. D and E show the relative amount of residual current after 30 pulses at the different frequencies with potentials of ⫺70 and ⫺60 mV, respectively. KChIP1b enhanced accumulation of inactivation compared to Kv4.2 alone while KChIP1a reduced it, especially at 3 Hz (D).

with the activation curve (Fig. 3E). However, the peak current amplitude recorded during the subsequent test pulse to ⫹50 mV displayed an exponential decay as a function of

Fig. 6. Closed-state inactivation in the presence or absence of KChIP1 proteins. Pulses to ⫹50 mV were applied after different time intervals (0 to 1400 ms) at ⫺50 mV. Between the consecutive pulses cells were clamped at ⫺90 mV in order to recover all the channels from inactivation. A shows the pulse protocol, with only the 1st and the 11th pulses depicted for clarity. The arrow in C points to the corresponding 11th trace. In all cases a decrease of the peak current amplitude was noted, indicating the presence of closed-state inactivation. The solid line (envelope of peaks) is an exponential fit of the peak amplitude, after different time intervals at ⫺50 mV, with time constants of 616 and 611 ms for Kv4.2 with KChIP1a and Kv4.2 with KChIP1b, respectively.

the prepulse duration. This indicates that inactivation did develop at ⫺50 mV in the absence of detectable current, i.e., closed-state inactivation (Bahring et al., 2001b; Beck and Covarrubias, 2001) (Fig. 6). After 1.4 s at ⫺50 mV Kv4.2 channels showed only a 20.4 ⫾ 3.8% (n ⫽ 5) reduction in peak current amplitude. Upon coexpression with KChIP1b on the other hand, the peak current amplitude was reduced with 45 ⫾ 3% (n ⫽ 6) and with 46 ⫾ 5% (n ⫽ 8) in the presence of KChIP1a. This reduction in the presence of either splice variant was significantly different from Kv4.2, but not between KChIP1a and KChIP1b. To estimate the rate of closed state inactivation, the peak current amplitudes were plotted as a function of the interval at ⫺50 mV and were fitted with a monoexponential function. In the case of the stable cell line of Kv4.2 the combination of a small current amplitude and a small reduction by closed state inactivation (⬃20%) did not allow an accurate fit of the exponential decay. However, upon coexpression of KChIP1a or KChIP1b with Kv4.2 we obtained time constants of 615 ⫾ 52 ms (n ⫽ 8) and 610 ⫾ 39 ms (n ⫽ 6), respectively.

Discussion The KChIP proteins belong to the family of Ca2⫹ binding neuronal Ca2⫹ sensor (NCS) proteins which are characterized by the presence of four EF-hands (Burgoyne and Weiss, 2001). Since there is a considerable amount of homology in the EF-hand region of the NCS proteins, it is

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quite likely that the crystal structure of the KChIP proteins will be similar to that of members of the NCS family, such as frequenin (Fig. 1). Unlike the rest of the NCS proteins, the KChIP-family members have longer N-termini with most of the diversity concentrated herein. The differences between KChIP1a and KChIP1b result from an extra exon of 11 amino acids inserted in the N-terminus. Noteworthy is the fact that the inserted sequence is rich in aromatic amino acids and that this exon may introduce an additional ␣-helix in the N-terminus. This suggests that a specific structure of the less conserved N-terminal section of KChIP1x is required for the differential modulation of Kv4 inactivation kinetics. Likewise, the N-terminal end of KChIP4 was found to be important although in this case it partially abolished the inactivation of Kv4.3 (Holmqvist et al., 2002). The splice variants of KChIP2 also differ in the N-terminal end but act similarly (upregulation, slowing of inactivation, and faster recovery) (An et al., 2000; Bahring et al., 2001a; Ohya et al., 2001; Patel et al., 2002; Wang et al., 2002). Only the KChIP2T splice variant lacked the enhanced recovery from inactivation (Deschenes et al., 2002). In contrast, our results for the KChIP1 splice variants point to more profound differences in the recovery kinetics: the slowing of the recovery kinetics with KChIP1b has not been observed for any of the other KChIP splice variants. As such, our results are rather unique as there is a complete reversal of the effect on recovery of inactivation due to the insertion of 11 amino acids. Unfortunately, a full comparison of the KChIP effects of different researchers is hampered by the fact that the studies were performed with different ␣-subunits (Kv4.1, Kv4.2, Kv4.3), different KChIP subunits, and/or different expression systems (oocytes versus mammalian cells). Nevertheless, a striking functional effect of KChIP proteins is the upregulation of Kv4 channel density at the plasma membrane. This effect is not restricted to heterologous expression studies. For example, in the hearts of larger mammals (canine, ferret, and human) a transmural gradient for KChIP2 has been demonstrated that matched the transmural density in ITO (Rosati et al., 2001; Patel et al., 2002). Also, the expression of ITO was lost in transgenic mice expressing a defective KChIP2 subunit (Kuo et al., 2001). Both observations indicate that KChIP2 is a major regulator of the ITO density in the heart. As a consequence the members of the KChIP family can be regarded as important ␤-subunits involved in the expression of A-type and/or ITO currents and might be responsible for the spatial gradients observed throughout several cell types and tissues. The original KChIP1 (now KChIP1a) was shown to interact with Kv4.2 and Kv4.3 (An et al., 2000), as confirmed by our results. This interaction was suggested to be important for the expression and/or functional aspects of the A-type current (Nakamura et al., 2001; Beck et al., 2002) as (1) Kv4 channels are regarded as the basis of most neuronal A-type currents and (2) KChIP proteins are strongly expressed throughout the human brain (Ohya et al., 2001). The new KChIP1b splice variant might be at least as important

as KChIP1a for the A-type current as it is also strongly expressed in brain, it interacts stably with Kv4.2 channels (demonstrated by confocal imaging), and it strongly increases the current density. However, the two splice variants displayed marked differences in inactivation such as time course of induction, accumulation, and recovery. Because rapid inactivation of A-type currents is an important factor in neuronal excitation (Pongs, 1999), the presence of the KChIP1a versus the KChIP1b splice variant could differentiate excitation effects in vivo. The characteristic rapid phase of inactivation of Kv4 channels is caused by both the N- and the C-terminal domains as demonstrated by deletion mutants (Jerng and Covarrubias, 1997) which was interpreted as a stabilization of the open state (Bahring et al., 2001b). Both splice variants also slowed the inactivation of Kv4.2, similar to N- and C-terminal deletions and consistent with a stabilization of the open state. Since C-terminal truncations of Kv4.1 significantly decreased the rate of recovery (Jerng and Covarrubias, 1997), this suggests that the modulatory but differential effects of KChIP1b on the recovery of inactivation may also involve an interaction with the C-terminal part of the Kv4.x channel. Presynaptic A-type channels are characterized by a slow recovery of inactivation (Roeper et al., 1997) and are probably involved in dampening action potentials at low frequencies (Pongs, 1999). Although presynaptic A-type currents are not necessarily all encoded by Kv4 subunits (Sheng et al., 1992, 1993; Rhodes et al., 1997), the association of Kv4.2 with KChIP1b caused slowing of the recovery of inactivation, comparable to presynaptic levels (␶sec ⬎ 1 s). As a consequence the resulting accumulation of inactivation (as shown by pulse trains in Fig. 5) was increased by KChIP1b, thus decreasing the amount of A-type current. Additionally, the A-type current amplitude increases with the distance from the soma, thereby preventing the back -propagation of action potentials (Hoffman et al., 1997). In both cases the presence of a KChIP subunit could be responsible for the fine-tuning of the intrinsic properties of the A-type current in these cells. In conclusion we have isolated and characterized a splice variant of KChIP1 that interacts with members of the Kv4 family of K⫹ -channel subunits, increases channel density, and alters the biophysical properties of the current. Both have distinct and opposite effects on the recovery of inactivation which could be an important factor in splice-variant-specific fine tuning of excitability in the brain and heart.

Experimental methods Isolation of KChIP1b Mouse KChIP1a and KChIP1b cDNAs were obtained from a mouse brain cDNA library isolated in our laboratory. The following primers with flanking restriction sites were used for amplification: 5⬘ -cttaagcttAAGACGCACACAAGTCTTC-3⬘ and 5⬘-ataggatccaTTACATGACATTTTG-

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GAACAGC-3⬘. These primers were based on the EST sequences with Accession Nos. BG293918 and BE652426. PCR products were cut with HindIII and BamHI before directional ligation into the pEGFP-vector (Clontech). GFPtagged KChIP proteins were constructed by removing the stop codon using standard site-directed mutagenesis (Quickchange, Stratagene) with primers 5⬘-TTCCAAAATGTCATGaAATTGGATCCACCGGTCG-3⬘ and 5⬘-CGACCGGTGGATCCAATTtCATGACATTTTGGAA-3⬘.

inactivation and recovery from inactivation were fitted with a single or double exponential function as appropriate, using a nonlinear least-squares (Gauss-Newton) algorithm. Results are expressed as means ⫾ SEM. Analysis of variance with appropriate post hoc comparisons was used to compare the differences in mean values; P ⱕ 0.05 was considered significant.

Expression analysis

The sequences of mouse KChIP1a and KChIP1b were submitted in GenBank with Accession Nos. AY050526 and AY050525, respectively.

A cDNA panel covering different tissues was obtained from Clontech (human cDNA panels I and II). PCR analysis was performed with primer sets specific for each splice variant, which was achieved by using part of the exon 1b sequence as forward primer for amplication of KChIP1b and a primer with sequence bridging exon 1 and 2 (thus excluding exon 1b) as forward primer for KChIP1a amplification. The same 3⬘ reverse primer was used for the amplification of both KChIP1a and 1b. For the expression analysis 35 PCR cycles were applied for both splice variants. PCR products were analyzed on a 1.5% agarose gel. Transfection and confocal imaging Culturing conditions of the HEK293 cells, transfection with cDNA, and the generation of the stable cell line have been described previously (Snyders and Chaudhary, 1996; Yeola and Snyders, 1997). For the confocal images HEK293 cells were cultivated on coverslips. Cotransfections were performed with a 1:10 ratio of GFP-tagged KChIP1x DNA versus Kv4.2 or Kv4.3. The endoplasmic reticulum was visualized with the DsRed-ER localization vector as described previously (Ottschytsch et al., 2002). Confocal images were obtained on a Zeiss CLSM 510, equipped with an argon laser (excitation 488 nm) for the single track visualization of GFP and DsRed. Whole cell current recording, pulse protocols, and data analysis Current recordings were made with an Axopatch-200B amplifier (Axon Instruments, Foster City, CA) in the whole cell configuration of the patch– clamp technique as described previously (Snyders and Chaudhary, 1996). Details of the pulse protocols are specified in the figure legends. The voltage dependence of channel opening was calculated from the peak current amplitude at different potentials, normalized using the Goldmann–Hodgkin–Katz equation (Clay, 2000). For the voltage dependence of inactivation the peak current amplitude at ⫹50 mV was plotted as a function of the prepulse voltage. The voltage dependence of activation and inactivation were fitted with a Boltzmann equation according to y ⫽ 1/(1 ⫹ exp(⫺(E ⫺ Eh)/k)), where Eh represents the voltage at which 50% of the channels are open or inactivated and k the slope factor. Kinetics of

Accession numbers

Acknowledgments We thank Jean-Pierre Timmermans for the use of the confocal microscope. D.V.H. is a fellow with the Vlaams Instituut voor de Bevordering van het WetenschappelijkTechnologisch Onderzoek in de Industrie (IWT). This work was supported by NIH-NHLBI Grant HL59689 and by grants from the Flanders Institute for Biotechnology (VIB) and the Belgian Science Foundation (FWO) GO421102N. This research was performed within the framework of the Interuniversity Attraction Poles (IUAP) program P5/19 of the Federal Office for Scientific, Technical and Cultural Affairs (OSTC), Belgium.

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