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. 2001 Dec 15;537(Pt 3):801–809. doi: 10.1111/j.1469-7793.2001.00801.x

Evidence for the presence of a novel Kv4-mediated A-type K+ channel-modifying factor

Marcela S Nadal 1, Yimy Amarillo 1, Eleazar Vega-Saenz de Miera 1, Bernardo Rudy 1
PMCID: PMC2279007  PMID: 11744756

Abstract

  1. Subthreshold-operating transient (A-type) K+ currents (ISAs) are important in regulating neuronal firing frequency and in the modulation of incoming signals in dendrites. It is now known that Kv4 proteins are the principal, or pore-forming, subunits of the channels mediating ISAs. In addition, accessory subunits of Kv4 channels have also been identified. These either have no effect or slow down the inactivation kinetics of Kv4 channels. However, in many neuronal populations the ISA is faster, not slower, than the current generated by channels containing only Kv4 proteins.

  2. Evidence is presented for the presence in rat cerebellar mRNA of transcripts encoding a molecular factor, termed KAF, that accelerates the kinetics of Kv4 channels. Size-fractionation of cerebellar mRNA in sucrose gradients separated the high molecular weight mRNAs (4–7 kb) encoding KAF from the low molecular weight ones (1.5–3 kb) encoding factors that slow down the inactivation kinetics of Kv4 channels. The latter were identified as KChIPs using anti-KChIP antisense oligonucleotides.

  3. Both anti-KChIP and anti-Kv4 antisense oligonucleotides failed to eliminate KAF's activity from the high molecular weight mRNA fraction, thus suggesting that KAF might be a novel subunit(s) that can contribute to generating native ISA channel diversity.

  4. The time course of the currents expressed by KAF-modified Kv4 channels resembles more closely the time course of the native ISA in cerebellar granule cells.

The subthreshold-activating transient A-type K+ current (ISA) was first described as a fast K+ current that activates at membrane potentials that are below the threshold for Na+ spike generation (Connor & Stevens 1971a,b). The rapid, transient activation of these channels in the subthreshold range of membrane potentials causes delayed excitation, can prevent the action potential from reaching full amplitude, and determines the duration of the interspike interval, thus regulating the frequency of repetitive firing (Connors & Stevens 1971b; Rudy, 1988; Llinas, 1988; Baxter & Byrne, 1991). Recent studies have highlighted additional roles of this current in dendrites, including the temporal regulation of action potential back propagation, the integration of synaptic inputs, and the filtering of fast synaptic potentials (Schoppa & Westbrook, 1999; Johnston et al. 2000).

A number of experimental results, including antisense hybrid arrest and other gene elimination methods, have provided evidence that Shal-related (Kv4) K+ channel subunits are the main, pore-forming subunits of the channels underlying the classical somato-dendritic ISA in neurons, as well as the Ito in cardiac muscle (Baldwin et al. 1991; Dixon & McKinnon, 1994; Serodio et al. 1994; Tsunoda & Salkoff, 1995; Nakamura et al. 1997; Johns et al. 1997; Barry et al. 1998). However, although the currents produced by the expression of Kv4 pore-forming subunits in heterologous systems resemble ISA in native tissue, they display important functional differences with native currents. Moreover, subthreshold A-type K+ currents with a wide range of voltage-dependent activation and inactivation properties have been observed in cells (Rudy, 1988), and this diversity cannot be explained by the diversity of currents expressed by the products of the Kv4 genes known to date. This diversity of native A-type currents and the discrepancy between native and heterologously expressed A-channels could be explained by the existence of auxiliary subunits that modulate the activity of the pore-forming subunits.

Evidence for the existence of modulatory components of the channels underlying ISAs was first obtained from experiments showing that mRNA transcripts encoding an ISA expressed in Xenopus oocytes injected with rat brain mRNA could be separated into two distinct size fractions by sucrose gradient fractionation. One of the fractions, thought to contain mRNAs for the pore-forming subunits of the underlying channels, expressed a modified transient K+ current, while the second fraction, of smaller molecular size, encoded a modifying factor (Rudy et al. 1988). Following the identification of Kv4 proteins as the pore-forming subunits of the channels underlying ISAs, it was shown that the channels expressed by Kv4 cRNAs in Xenopus oocytes could be modified by factors encoded in rat brain mRNA that did not express currents by themselves under the same pulse protocols (Chabala et al. 1993; Serodio et al. 1994, 1996). Recently, a family of Ca2+-binding proteins called KChIPs was identified using yeast two-hybrid screens consisting of proteins that bind to the N-terminal region of Kv4 proteins, facilitate the expression, and modify the properties, of Kv4 channels in heterologous expression systems, and are likely to be components of native channels mediating ISAs (An et al. 2000). The effects of KChIPs on Kv4 channels resembled those reported for the small mRNA-size fraction described by Rudy et al. (1988).

However, there is at least one major difference between the currents expressed by Kv4 proteins in the presence of KChIP subunits and the native ISA recorded in the somata of several neuronal populations that prominently express Kv4 gene products. Two (Kv4.2 and Kv4.3) of the three known Kv4 genes are prominently expressed in brain (Serodio & Rudy, 1998). One of the effects of KChIPs on the currents mediated by Kv4.2 and Kv4.3 channels is to decrease the macroscopic rate of inactivation of the currents during depolarizing pulses (An et al. 2000; Nakamura et al. 2001). But the native ISA in several neurons inactivates, if anything, faster, and not slower, than the currents expressed by Kv4.2 and Kv4.3 subunits alone (see Table 1). This is nicely illustrated by the observations of Shibata et al. (2000) who reported that the currents expressed in cerebellar granule cells following transfection of Kv4.2 cDNA were slower than the native ISA. Other proteins have been shown to also interact with Kv4 channels in heterologous expression systems (KChAP: Kuryshev et al. 2000; Kvβ1 and Kvβ2: Yang et al. 2001; MirP1: Zhang et al. 2001). But these proteins either have no effect on channel kinetics (KChAP and the Kvβs) or also slow down channel inactivation (MirP1). Here we provide evidence for the existence of a factor(s), perhaps a novel subunit(s), encoded in cerebellar mRNA that accelerates the rate of macroscopic inactivation of Kv4 channels. The experiments suggest the presence of additional, unrelated components that can modify Kv4 channel kinetics differently from KChIPs. These components are likely to contribute to the generation of native ISA channel diversity.

Table 1.

Electrophysiological properties of ISA in neurons and heterologous expression systems

Voltage dependence
Activation Inactivation Kinetics (at room temperature)
V½ (mv) k (mv) V½ (mv) k (mV) tpeak (ms) t½ (ms) τrec (ms) Ref
Cerebellar NA NA −72.0 7.2 NA ∼13.1 (0 mV) 35.0 (−100 mV) a
granule cells −20.5* 13.5* −78.8 8.4 ∼1.3 (40 mV) 7.0–8.4 (40 mV) 40.0 (−90 mV) b
CA1 hippocampal −7.5 20.7 −72.1 8.8 NA ∼16.3 (40 mV) 13.0 (−110 mV) c
pyramidal cells −27.2 NA −85.5 NA NA ∼10.1 (50 mV) 32.0 (−110 mV) d
Kv4.2 −2.5 ± 2.6 19.4 ± 2.6 −65.7 ± 0.4 6.5 ± 0.4 7.3 ± 0.6 (40 mV) 22.2 ± 2.8 (40 mV) 131.7 ± 2.2 (−110 mV) e
Kv4.3 −10.1 ± 2.3 18.2 ± 2.6 −59.5 ± 0.5 5.9 ± 0.4 7.7 ± 0.8 (40 mV) 49.7 ± 4.2 (40 mV) 266.5 ± 10.8 (−110 mV) e
4–7 kb −33.9 ± 1.9 17.4 ± 1.4 −84.2 ± 0.4 5.2 ± 0.3 2.3 ± 0.2 (40 mV) 8.0 ± 1.6 (40 mV) 52.1 ± 1.9 (−110 mV) e
Poly(A)+-KChIP −35.4 ± 1.8 15.7 ± 1.3 −81.4 ± 0.2 4.9 ± 0.2 2.4 ± 0.1 (40 mV) 10.3 ± 0.4 (40 mV) 48.6 ± 2.3 (−110 mV) e
Kv4.2 + KChIP −19.5 ± 1.7 21.8 ± 2.2 −71.4 ± 0.2 6.7 ± 0.2 5.0 ± 0.4 (40 mV) 46.1 ± 4.4 (40 mV) 47.2 ± 2.4 (−110 mV) e
Kv4.2 + KAF −30.3 ± 2.6 20.2 ± 2.5 −79.5 ± 0.1 5.8 ± 0.1 2.2 ± 0.2 (40 mV) 9.7 ± 1.5 (40 mV) 67.7 ± 2.6 (−110 mV) e
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Activation V½ and k, midpoint and slope, respectively, of the conductance–voltage curve fitted to a first-order Boltzmann function. Inactivation V½ and k, midpoint and slope of the steady-state inactivation curve; tpeak, time for the current to reach its maximum value at the indicated voltage; t½, time at which half of the current has inactivated at the indicated voltage; τrec, time constant of recovery from inactivation at the indicated recovery voltage. Activation parameters are least reliable because g/gmax curves for ISA usually do not fit simple Boltzmann functions and the reported values depend on assumptions made given the lack of saturation of the g–V curve. NA, not available.

*

Calculated from the data in Fig. 8 of reference (a) using eqn (6) in that paper. References: a, Cull-Candy et al. (1989); b, Bardoni & Belluzzi (1993); c, Keros & McBain (1997); d, Klee et al. (1995); e, this paper

METHODS

Poly(A)+ RNA preparation and fractionation

Total RNA was isolated from freshly dissected cerebellum of 17- to 22-day-old Sprague-Dawley rats. The rats were decapitated under pentobarbital anaesthesia (80 mg (kg body weight)−1i.p.) and the brains quickly removed; all animal procedures were approved by the Medical School's Institutional Animal Care and Use Committee (IACUC). Poly(A)+ RNA was selected by oligo(dT) cellulose chromatography as previously described (Serodio et al. 1994, 1996), and was size fractionated in a linear 10-30 % sucrose gradient as described in Serodio et al. (1994) and Rudy et al. (1988).

Preparation of in vitro transcribed RNA and functional expression in Xenopus oocytes

Linearized recombinant plasmids containing full length Kv4.2 or Kv4.3 cDNAs were used as templates for in vitro transcription using the mCAP mRNA Capping Kit as described by the manufacturer (Stratagene). Oocytes were obtained from the ovarian lobes of female Xenopus laevis (Xenopus I, Ann Arbor, MI, USA) as previously described (Serodio et al. 1994). Briefly, the frogs were anaesthetized with 0.2 % MS-222 (Sigma, St Louis, MO, USA) and the ovarian lobes surgically removed and placed in calcium-free ND96 solution. The frog was then allowed to recover from surgery before being returned to the tank. After the third surgery, the frog was killed by decapitation under anaesthesia. Defolliculated stage V-VI oocytes were obtained by collagenase digestion and microinjected with the appropriate RNAs (total volume = 50 nl) and incubated for 1.5-3 days in ND96 solution at 18 °C as described by Serodio et al. (1994). Ionic currents were recorded under two-microelectrode voltage clamp with a Geneclamp 500 amplifier (Axon Instruments, 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, MgCl2 2.3 mm, Hepes 10 mm and CaCl2 0.5 mm; pH 7.4). Tetrodotoxin (TTX, Sigma) at a final concentration of 1 μm was added to the recording solution to block Na+ currents when recording oocytes injected with total poly(A)+ RNA.

Antisense hybrid arrest

Oligonucleotides (chemically synthesized by Sigma-Genosys) were treated with 0.01 μg μl−1 of proteinase K (Stratagene) for 1 h, phenol-extracted twice in RNAse-free conditions and ethanol-precipitated in the presence of MgCl2 and NaCl, as described in Serodio et al. (1994). The reaction mixture containing ∼0.3 μg μl−1 RNA and 0.2 μg μl−1 of antisense oligonucleotide in a 50 mm NaCl, 10 mm Tris-HCl (pH 7.5) buffer was incubated at 65 °C for 3 min, followed by a 30 min incubation at 37 °C, and then placed on ice until injection. When hybrid-arrested mRNAs and Kv4.2 or Kv4.3 cRNAs were injected in the same oocytes, the mRNA was injected first, and the cRNA injected 6 h later. The anti-Kv4s oligonucleotide (anti-all-Shal in Serodio et al. 1994) is a degenerate oligonucleotide with 100 % complementarity to all mammalian Kv4 RNA sequences known (Kv4.1, Kv4.2, and Kv4.3) and to Drosophila Shal. The sequence of the two degenerate oligonucleotides complementary to both mammalian Kv4 pore-forming subunits and the jellyfish γ-subunit JShalγ (U78641, Jegla & Salkoff, 1997) were: JShalγ1: GG(A/G)TCACG(A/G)TC(A/G)AAGAA(A/G)TA(C/T)TG and JShalγ2: GTCATGGTGACNA(C/T)NGT(A/G)TACCA (positions 78-85 and 358-365, respectively, of the coding sequence of JShalγ). The anti-KChIPs oligonucleotide (CTCCCCATCAT (A/G)TC (A/G)TA (A/G/T)AT) had 100 % complementarity to a conserved region of KChIP1, KChIP2 and KChIP3 (position 460-479 of the KChIP1 coding sequence; An et al. 2000). The sequence of the JShalγ forward primer used in conjunction with the JShalγ2 primer to amplify by RT-PCR Kv4 transcripts from rat cerebellar mRNA was: JShalγ3: CAGTACTTCTT(T/C)GA(T/C)(A/C)GNGA(T/C)CC. To confirm the specificity and determine the efficiency of the antisense treatments we used RT-PCR to amplify Kv4 or KChIP transcripts following antisense or control treatments. Antisense treatment is very efficient in Xenopus oocytes due to the presence in the oocytes of RNAse-H, which attacks RNA-DNA hybrids (Lotan, 1992). Consistent with this, following antisense treatment with the appropriate oligonucleotide the levels of Kv4 or KChIP mRNA became undetectable by RT-PCR.

RESULTS

Evidence for the presence of a Kv4 channel-accelerating factor (KAF) in cerebellar brain mRNA

For this study we used cerebellar mRNA because the cerebellum is dominated by one neuronal population, the granule cells, which are by far the most abundant neurons in this structure and are well characterized electrophysiologically (and known to have a very large ISA), and therefore their mRNA is likely to represent a large percentage of the mRNA obtained from cerebellar mRNA extracts. This facilitates comparisons between native currents and the currents expressed by cloned components in heterologous systems. Two of the three known Kv4 genes (Kv4.2 and Kv4.3) are prominently expressed in the cerebellar cortex, mostly in Purkinje and granule cells (Serodio et al. 1998). As previously shown for whole brain mRNA (Rudy et al. 1988) a fraction of the cerebellar RNA of ∼4-7 kb expresses a transient current (Fig. 1B) which differs from that observed with whole mRNA (Fig. 1A) in a number properties; most notably the current expressed by the 4-7 kb fraction reaches peak values sooner and declines faster (compare Fig. 1A and B). Treatment of the 4-7 kb mRNA fraction with antisense oligonucleotides complementary to a sequence common to all known mammalian Kv4 transcripts and Drosophila Shal (Fig. 1C) or the oligonucleotides (see Methods) complementary to mammalian Kv4 transcripts and the jellyfish γ subunit JShalγ (data not shown) inhibits the ability of this fraction to express transient K+ currents. These results demonstrate that the ISA expressed by the 4-7 kb mRNA fraction is produced by the Kv4 transcripts present in the fraction. Moreover, the antisense-treated fraction does not express any significant currents under the same pulse protocols (see Fig. 1C). These effects are specific, and are not observed when the mRNA fraction is processed in the absence of oligonucleotides (Fig. 1B) or in the presence of control oligonucleotides (data not shown).

Figure 1. Dissecting the molecular components of the channels mediating the ISA expressed from cerebellar mRNA.

Figure 1

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A, A-type K+ currents expressed in an oocyte injected with whole cerebellar mRNA. Unless otherwise noted all the currents shown were obtained by subtracting the currents obtained during depolarizing pulses from -70 to 40 mV (in 10 mV intervals) preceded by a 1 s prepulse to -40 mV from the currents obtained during the same pulses but preceded by a prepulse to -110 mV (VH= -90 mV). B, A-type currents in an oocyte injected with a 4-7 kb sucrose-gradient fraction of cerebellar mRNA. C, currents recorded in an oocyte injected with the 4-7 kb mRNA fraction following treatment with anti-Kv4 antisense oligonucleotides. D, A-type currents in an oocyte injected with Kv4.2 cRNA. E, A-type currents in an oocyte injected with a mixture of Kv4.2 cRNA and a 1.5-3 kb sucrose-gradient fraction of cerebellar mRNA. Inset: currents observed in an oocyte injected with the 1.5-3 kb mRNA fraction alone. F, A-type currents in an oocyte injected with a mixture of Kv4.2 cRNA and 1.5-3 kb mRNA pretreated with anti-KChIP antisense oligonucleotides.

The macroscopic inactivation of the ISA expressed by the 4-7 kb fraction was slowed down considerably when the 4-7 kb mRNA was co-injected with a mRNA fraction of smaller size mRNAs (∼1.5-3 kb) (data not shown). The 1.5-3 kb fraction also slowed down the ISA expressed by Kv4.2 (compare Fig. 1D and E) or Kv4.3 cRNAs (data not shown; Kv4.3 currents are slower than those expressed by Kv4.2, Serodio et al. 1996; see also Table 1). On its own the 1.5-3 kb fraction does not express any significant currents under the same voltage protocols (inset in Fig. 1E). Antisense treatment of the small size mRNA fraction with antisense oligonucleotides complementary to a sequence common to the three known KChIPs eliminated the ability of this fraction to slow down the kinetics of the ISA expressed by the 4-7 kb fraction (data not shown), by Kv4.2 (Fig. 1F) or by Kv4.3 (data not shown) cRNAs. This result identifies KChIPs as the factors encoded in the 1.5-3 kb fraction that modify the currents expressed by Kv4 transcripts.

If Kv4 pore-forming subunits and KChIP accessory subunits were the only molecular components present in cerebellar mRNA governing the kinetic properties of the ISA, one would predict that following antisense treatment of whole cerebellar mRNA with anti-KChIP antisense oligonucleotides, the RNA would express a current similar to that seen in oocytes expressing Kv4 cRNAs alone. However, the currents expressed by anti-KChIP antisense-treated cerebellar mRNA are faster than the currents expressed by Kv4.2 (Fig. 2A–C) or Kv4.3 (data not shown) cRNAs. These observations suggest that the cerebellar mRNA extract encodes for factors that accelerate Kv4 channel kinetics.

Figure 2. Evidence for the existence of mRNAs encoding a Kv4 accelerating factor(s) in cerebellar mRNA.

Figure 2

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A–C, the ISA expressed by anti-KChIP-treated cerebellar mRNA reaches peak values and declines faster than the ISA expressed by Kv4.2 cRNAs (the fastest Kv4 currents known), contrary to what would be expected if following KChIP removal the channels mediating the ISA were composed only of known Kv4 proteins. A, A-type currents in an oocyte injected with anti-KChIP antisense-treated cerebellar mRNA. B, A-type currents in an oocyte injected with Kv4.2 cRNA. C, the currents expressed by cerebellar mRNA (poly(A)+), Kv4.2 cRNA (Kv4.2) and anti-KChIP antisense-treated cerebellar mRNA (poly(A)+-KChIP) during a test pulse to +40 mV have been scaled to each other and superimposed. D and E, the Kv4 accelerating factor is present in 4-7 kb cerebellar mRNA. D, A-type currents in an oocyte injected with Kv4.2 cRNA and anti-Kv4 antisense-treated 4-7 kb cerebellar mRNA. The currents are faster than those expressed by Kv4.2 cRNA alone (compare with B). E, the currents expressed during a test pulse to +40 mV by Kv4.2 cRNA, 4-7 kb mRNA and a mixture of Kv4.2 plus anti-Kv4 antisense-treated 4-7 kb mRNA have been scaled to each other and superimposed.

The currents expressed by the 4-7 kb mRNA fraction also reach peak values and inactivate significantly faster than the currents expressed by Kv4.2 cRNAs (compare Fig. 1B and D; see also Fig. 2E). This suggests that mRNAs encoding the accelerating factor are present in 4-7 kb mRNA. To test whether the 4-7 kb fraction indeed contains transcripts encoding a component(s) that speeds up Kv4 currents, we explored the effects of co-injecting Kv4-antisense-treated 4-7 kb fraction with Kv4.2 cRNAs. Oocytes injected with such a combination expressed an ISA that was faster than that expressed by Kv4.2 alone, and resembled the currents expressed by the intact 4-7 kb fraction (Fig. 2D and E), or KChIP-antisense-treated whole cerebellar mRNA (Fig. 2A), confirming the presence of a component that accelerates the kinetics of Kv4 channels in this mRNA fraction. This putative component is unlikely to be an A-type K+ channel-forming protein, since, as described earlier, following Kv4-antisense treatment, the 4-7 kb fraction does not express any currents (Fig. 1C), while it retains the Kv4 accelerating activity.

We also explored the possibility that the accelerating factor is related to the jellyfish Shal‘γ subunit’, JShalγ (Jegla & Salkoff, 1997). This is an interesting Kv4 homologous protein that does not express currents on its own, but modifies the ISA expressed by functional Kv4 proteins, including increasing inactivation rates of very slowly inactivating Kv4 channels such as those expressed by jellyfish Shal and Kv4.1. The accelerating activity in 4-7 kb mRNA was unaffected by antisense treatment with the oligonucleotides complementary to mammalian Kv4 transcripts and the jellyfish γ subunit (JShalγ1 and JShalγ2; see Methods). These observations suggest that the accelerating factor is not related to JShalγ. Furthermore RT-PCR of rat cerebellar cDNA with primers designed to amplify all mammalian, Drosophila and jellyfish Shal transcripts (JShalγ3 and JShalγ2, see Methods) amplified only Kv4.2 and Kv4.3 transcripts. In addition, the effects of JShalγ differ considerably from those of the accelerating factor in that JShalγ produces a large decrease in the rate of recovery from inactivation of Kv4 currents (Jegla & Salkoff, 1997), while the accelerating factor increases the rate of recovery from inactivation (see below), as seen in native mammalian ISA (Table 1).

Size distribution and functional effects of the transcripts encoding the factor accelerating Kv4 channel kinetics

The photograph of a denaturing agarose electrophoresis of the RNA fractions from a representative sucrose gradient fractionation of cerebellar poly(A)+ mRNA is shown in Fig. 3A. Equivalent quantities from each fraction were injected into Xenopus oocytes and the expression of the three ISA channel-related activities (Kv4-mediated A-type currents, KChIP-like activity and the Kv4 accelerating factor) was quantified in each fraction. Each showed a specific distribution (Fig. 3B). Transcripts encoding Kv4 subunits and those encoding the Kv4-accelerating factor(s) (4-7 kb) overlapped, but the latter might be somewhat larger than those encoding Kv4 subunits.

Figure 3. Functional effects of KAF and KChIP on the currents mediated by Kv4.2 proteins.

Figure 3

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A, photograph of an agarose gel electrophoresis of the RNA fractions obtained in one the cerebellar poly(A)+ sucrose-gradient fractionations used in this study. B, distribution of A-type currents (ISA), KAF, the factor reducing the half-time of inactivation (t1/2) of Kv4.2 currents and KChIPs, the factor increasing the half-time of inactivation in the different sucrose-gradient mRNA fractions shown in A. Shown are averages (±s.e.m.) from 9 oocytes injected with each fraction. The average half-time of inactivation for the currents observed in oocytes injected with Kv4.2 cRNA alone is shown with the dotted line. C, peak A-type current values at +40 mV from 9 oocytes injected with Kv4.2 cRNA, Kv4.2 cRNA plus anti-Kv4 antisense-treated 4-7 kb mRNA and Kv4.2 cRNA plus 1.5-3 kb mRNA. D, recovery from inactivation of the A-type currents expressed by Kv4.2 cRNA, Kv4.2 cRNA plus anti-Kv4 antisense-treated 4-7 kb mRNA or Kv4.2 cRNA plus 1.5-3 kb mRNA. Shown are the currents recorded during test pulses to 50 mV following a test pulse to the same voltage separated by increasing time intervals at -110 mV. Vertical calibration: 0.5 μA for Kv4.2 alone and 1 μA for Kv4.2 + KAF or KChIP; Horizontal calibration: 250 ms. E, time course of the recovery from inactivation of the A-type currents expressed by Kv4.2 cRNA, Kv4.2 cRNA plus anti-Kv4 antisense-treated 4-7 kb mRNA or Kv4.2 cRNA plus 1.5-3 kb mRNA (n = 6). F, conductance-voltage relationship (g/gmax, filled symbols) and voltage dependence of steady-state inactivation (I/Imax, open symbols) of the A-type currents expressed by Kv4.2 cRNA, Kv4.2 cRNA plus anti-Kv4 antisense-treated 4-7 kb mRNA or Kv4.2 cRNA plus 1.5-3 kb mRNA (n = 7).

The novel factor, referred to in this paper as KAF (for Kv4 accelerating factor) had additional effects on Kv4.2 currents. It produced a ∼3- to 4-fold increase in the levels of the ISA expressed by a given amount of Kv4.2 cRNA (Fig. 3C), resembling the chaperone effect of KChIP proteins and other K+ channel-associated subunits (An et al. 2000). Similar to KChIPs it also accelerated the recovery from inactivation (Fig. 3D and E), although somewhat less than KChIPs, and produced negative shifts in the voltage dependence of steady state activation and inactivation that were larger than those produced by KChIPs (Fig. 3F, Table 1). Thus, the main difference between KChIPs and the new factor is in their effect on the kinetics of macroscopic inactivation of Kv4 currents: while KChIPs slow down the inactivation time course of Kv4.2 (Fig. 1D and E; Table 1; see also An et al. 2000; Nakamura et al. 2001) the novel factor enhances the rate of inactivation (Table 1). The KAF fraction had similar effects on the currents expressed by Kv4.3 cRNAs, but had no effect on the transient currents expressed by Kv1.4, a member of the Kv1 subfamily (data not shown).

The inactivation time course of the ISA mediated by Kv4 channels is complex and requires multi-exponential functions for adequate curve fitting as also seen in native ISA in neurons (Serodio et al. 1994, 1996; Nakamura et al. 1997, 2001). Exponential curve fitting to the inactivating portion of individual current traces following co-expression of Kv4.2 (or Kv4.3) with KChIP1 has shown that KChIP1 has relatively minor effects on the individual time constants, its major effect being to cause a switch in the relative contributions of the fast and slow components of inactivation (Nakamura et al. 2001). The substantial increase in the contribution of the slow component (from ∼15 % to ∼60 %) explains the slow-down of inactivation kinetics produced by KChIP proteins. In contrast, a similar analysis for KAF showed that the main effect of this factor is to decrease the value of the inactivation time constants with small effects on the relative contributions of the fast and slow components (Table 2).

Table 2.

Inactivation parameters of Kv4.2 currents

τ1 τ2 A B
Kv4.2 18.2 ± 1.4 107.4 ± 10.3 0.82 ± 0.008 0.18 ± 0.008
Kv4.2 + KChIP 22.1 ± 1.7 103.1 ± 4.4 0.41 ± 0.004 0.59 ± 0.004
Kv4.2 + KAF 8.1 ± 0.8 81.7 ± 4.9 0.91 ± 0.014 0.09 ± 0.014
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τ1 and τ2 are the time constants of inactivation and A and B are the corresponding fractional amplitudes determined from two-exponential curve fits to the inactivation portion of current traces obtained during depolarization pulses to 40 mV.

DISCUSSION

The functional importance of Kv4-mediated ISA in neurons and cardiac muscle emphasizes the significance of understanding the molecular composition of native ISA-generating channels. Evidence has been described for the presence in cerebellar mRNA of transcripts encoding a factor(s) (KAF) that accelerates the kinetics of Kv4.2 and Kv4.3 channels. KAF could be an accessory subunit, which, like KChIPs and other K+ channel accessory proteins, modifies the properties of the channels expressed by the pore-forming subunits (Coetzee et al. 1999; An et al. 2000). Alternatively, the accelerating factor could be an enzyme that regulates channel properties by post-translational modification. We favour the first hypothesis because some of the effects of KAF resemble those of other K+ channel accessory subunits, and in particular the putative chaperone-mediated increase in expression levels. The observations of Shibata et al. (2000; see Introduction), although indirect, are more consistent with the presence of a component which is required in stoichiometric amounts and is present in native cells in limited quantities, rather than an enzyme possessing catalytic activity. We should also point out that if KAF is an enzyme, it is extremely unlikely to be a protein kinase related to those (protein kinase A (PKA); protein kinase C (PKC); and the mitogen-activated protein kinase ERK) already known to phosphorylate and modulate Kv4 channels, since these enzymes produce effects on Kv4 channels (positive shifts in voltage dependence, channel inhibition) that are completely different from those produced by KAF (Nakamura et al. 1997; Hoffman & Johnston, 1998; Sweatt et al. 2001).

Further understanding of the function and mechanisms of action of KAF will require the cloning and characterization of the underlying molecules. Assays based on the results shown here could be used to clone cDNAs encoding KAF using functional cloning methods. The present data suggest that KAF is unlikely to be closely related molecularly to already known Kv4 channel-interacting subunits, and in particular KChIPs, because KAF activity survived anti-KChIP antisense treatment. Moreover, KChIPs, like other members of the recoverin/neural calcium sensor (NCS) family of Ca2+-binding proteins, are small proteins encoded by mRNAs of small molecular size (An et al. 2000). As discussed in Results, it is also unlikely that KAF is closely related to Kv4 proteins, including the jellyfish JShalγ subunit. However, KAF could be a silent, pore-forming subunit, equivalent to the Kv5-Kv9 proteins that interact with and modulate the channels formed by Kv2 subunits (reviewed in Coetzee et al. 1999).

Interestingly, the time course of the currents expressed by Kv4.2 plus KAF-containing Kv4-antisense-treated 4-7 kb mRNA resembles more closely the ISA recorded from cerebellar granule cells than the Kv4.2-KChIP combination (Table 1), suggesting that KAF is an important component of the native channels.

Acknowledgments

This research was supported by NIH Grants NS30989 and NS35215 to B.R.; NSF grant IBN0078297 to B.R.; and an American Heart Grant in Aid to E.V. We also wish to thank Dr William Coetzee and Dr Tomoe Nakamura for helpful discussions.

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