Biochemical Journal

Research article

Position-dependent attenuation by Kv1.6 of N-type inactivation of Kv1.4-containing channels

Ahmed Al-Sabi , Seshu Kaza , Marie Le Berre , Liam O'Hara , MacDara Bodeker , Jiafu Wang , J. Oliver Dolly

Abstract

Assembly of distinct α subunits of Kv1 (voltage-gated K+ channels) into tetramers underlies the diversity of their outward currents in neurons. Kv1.4-containing channels normally exhibit N-type rapid inactivation, mediated through an NIB (N-terminal inactivation ball); this can be over-ridden if associated with a Kv1.6 α subunit, via its NIP (N-type inactivation prevention) domain. Herein, NIP function was shown to require positioning of Kv1.6 adjacent to the Kv1.4 subunit. Using a recently devised gene concatenation, heterotetrameric Kv1 channels were expressed as single-chain proteins on the plasmalemma of HEK (human embryonic kidney)-293 cells, so their constituents could be arranged in different positions. Placing the Kv1.4 and 1.6 genes together, followed by two copies of Kv1.2, yielded a K+ current devoid of fast inactivation. Mutation of critical glutamates within the NIP endowed rapid inactivation. Moreover, separating Kv1.4 and 1.6 with a copy of Kv1.2 gave a fast-inactivating K+ current with steady-state inactivation shifted to more negative potentials and exhibiting slower recovery, correlating with similar inactivation kinetics seen for Kv1.4-(1.2)3. Alternatively, separating Kv1.4 and 1.6 with two copies of Kv1.2 yielded slow-inactivating currents, because in this concatamer Kv1.4 and 1.6 should be together. These findings also confirm that the gene concatenation can generate K+ channels with α subunits in pre-determined positions.

  • Kv1 heterotetramer
  • Kv1.4-containing channel
  • N-terminal inactivation
  • N-type inactivation prevention (NIP)
  • inactivation
  • voltage-dependent gating

INTRODUCTION

Neuronal Kv1 (voltage-gated K+ channels) proteins largely arise from heterotetramerization of α subunits encoded by seven major genes (Kv1.1–1.6 and Kv1.8) with four auxiliary Kvβ subunits [13]. As each α subunit contributes distinct properties, the resultant channels reaching the plasmalemma vary considerably in their pharmacological and biophysical properties. After opening of these Kv1 channels by membrane depolarization, inactivation follows via two mechanisms. N-type inactivation mediated through the NIB (N-terminal inactivation ball) occurs by a ‘ball and chain’ process in which NIB occludes the inner mouth of the ion pore [4,5]. In addition, Kvβ1 subunits provide alternative N-terminal domains that confer rapid inactivation on non- or slow-inactivating Kv1 channels [68]. On the other hand, C-type inactivation arises from prolonged depolarization which leads to a local rearrangement and constriction of the channel at the outer mouth [911]. Kv1.4 is the only Kv1 member that shows both N- and C-type inactivation, yielding a distinctive A-type transient outward current [3].

Heteromerized Kvl.4 and l.2 subunits have been localized in axons and nerve terminals, which may form the molecular basis of a presynaptic A-type K+ channel involved in the regulation of neurotransmitter release [12]. Moreover, direct biochemical studies have revealed that Kv1.4 forms channel oligomers with Kv1.2 and 1.6 subunits in the brain [13]. The fast N-type inactivation endowed by Kv1.4 can be prevented by the presence of Kv1.6 through its NIP (N-type inactivation prevention) domain [14], giving rise to a much slower inactivating/sustained K+ current. As tetramerization of Kv channel subunits occurs in the endoplasmic reticulum [15], pinpointing their ordering in vivo has thus far eluded researchers because of an inability to predetermine the arrangement of their assembled constituents in the oligomers delivered to the cell surface. Therefore it is warranted to focus efforts on gaining a clearer understanding of the influences of subunit ordering, particularly as the resultant data could give insights into the modulation of neuronal transmission by Kv1 channels. Furthermore, such information is medically relevant given that delayed rectifier Kv1 subunits, but not Kv1.4, are down-regulated in the hippocampus of animal models prone to seizures [16], whereas Kv1.4 is up-regulated following chronic injury of the spinal cord [17].

In the present study, a recently designed expression platform [18] was utilized to express four Kv1 α constituents as a single ORF (open reading frame) in transfected HEK (human embryonic kidney)-293 cells, allowing predetermination of not just the combinations of α subunits, but also their actual arrangements in channels on the plasmalemma. This permitted a fundamental question to be answered, namely, whether subunit ordering influences the biophysical profiles of Kv1 channels. For this, advantage was taken of the NIP in Kv1.6 [14] being able to prevent fast inactivation of Kv1.4-containing channels. If NIP competes directly for the binding site of NIB, displacing it and giving rise to slow inactivation, no differences ought to be expected when these two α subunits are placed adjacently or distally in concatenated heteromers, with both of their currents inactivating slowly. On the other hand, if NIP function relies on it directly interacting with NIB, dissimilarities in inactivation rates may occur with a slow-inactivating current only occurring when both domains are optimally placed. Suboptimal placement might result in a fast-inactivating channel where NIP is positioned away from NIB and unable to modulate it. To address this important question, for the first time, inactivation, voltage-dependence of inactivation and recovery from inactivation were measured in three recombinantly-expressed tetramers with identical subunit composition, but distinct subunit ordering. A fourth tetramer, Kv1.4-(1.2)3 was constructed as a control, having one copy of Kv1.4 within the tetramer. The novel outcome of this approach questions the wisdom of predicting channel properties based on subunit content alone because their positioning greatly influences the channels' characteristics. Also, the data reaffirm convincingly that this concatenation of genes predetermines the positions of α subunits in the assembled functional channels on the cell surface.

MATERIALS AND METHODS

PCR amplification and assembly of Kv1.X constituents

Concatenation of four α subunits as a single ORF (Figure 1A) was accomplished using an inter-subunit linker derived from the UTRs (untranslated regions) of the Xenopus β-globin gene (GenBank® accession number J00978) [19]. The cDNAs for rat Kv1.2, 1.4 and 1.6 were kindly provided by Professor Olaf Pongs (Institute for Neural Signal Transduction, University of Hamburg, Germany). When sequenced, the Kv1.2 and Kv1.6 constructs corresponded to those previously published [3], whereas the Kv1.4 gene encodes a polypeptide sequence identical with that of rat Kv1.4 published on PubMed (GenBank® accession number NW_047657.2). Amplification of Kv1.2 and Kv1.6 was carried out using Kv-sequence specific primers which incorporated flanking XbaI–XhoI sites (Supplementary Table S1A, upper panel available at http://www.BiochemJ.org/bj/438/bj4380389add.htm), allowing their individual cloning into a previously modified UTR-containing intermediate plasmid pβUT, at XbaI–XhoI cloning sites [18]. A second round of PCR, using primers specific to the UTRs themselves, allowed amplification of the α subunit ORF contiguous with the flanking UTRs. Paired restriction sites were also added (Supplementary Table S1A, lower panel), facilitating position-specific direct cloning into pIRES2-EGFP (enhanced green fluorescent protein). The resultant Kv1.2 and 1.6 genes were individually subcloned into desired positions II to IV using paired sites for BglII/EcoRI, EcoRI/SalI, and SalI/BamHI respectively (Figure 1A), each now separated by a combined 78 bp linker including restriction enzyme sites. The Kv1.4 gene was amplified using specific primers (Supplementary Table S1B) and directly subcloned into position I between NheI and BglII sites. This gene was separated from the second subunit by a 42 bp linker including a BglII restriction site. Correct positioning of the genes in all pIRES2-EGFP plasmid constructs was confirmed by restriction analysis (Supplementary Figure S1 available at http://www.BiochemJ.org/bj/438/bj4380389add.htm) and complete DNA sequencing.

Figure 1 Concatenation of Kv1.X genes, expression and trafficking of complete heterotetrameric Kv1 channels to the plasmalemma of mammalian cells

(A) Schematic of concatenated constructs of Kv1 genes (symbolized), in a single ORF within the pIRES2-EGFP plasmid with requisite linkers and predicted subunit arrangements in the expressed proteins. NIB and NIP refer to the Kv1.4 N-terminal inactivation ball and its prevention domain in Kv1.6 respectively. (B) Transiently transfected HEK-293 cells expressing Kv1.4–1.2–1.2–1.2 (lanes 1, 5 and 9), Kv1.4–1.6–1.2–1.2 (lanes 2, 6 and 10), Kv1.4–1.2–1.6–1.2 (lanes 3, 7 and 11) or Kv1.4–1.2–1.2–1.6 (lanes 4, 8 and 12). Intact cells were biotinylated, detergent solubilized, precipitated with streptavidin–agarose beads and analysed by Western blotting using antibodies specific for Kv1.4 (lanes 1–4), Kv1.6 (lanes 5–8) or Kv1.2 (lanes 9–12); k, denotes a molecular weight marker (kDa). (C) Confocal fluorescence micrographs showing surface expression of heterotetrameric channels in transfected HEK-293 cells, after incubation with a monoclonal antibody reactive to either Kv1.2, Kv1.6 or Kv1.4. The labelling was visualized by using anti-species IgGs coupled to Alexa Fluor® 594. Control cells transfected with Kv1.4–1.2–1.6–1.2 (bottom right-hand panel) and non-transfected cells (bottom left-hand panel) were stained only with secondary antibody. Scale bar denotes 10 μm.

In vitro mutagenesis

NIP function of Kv1.6 was abolished by carrying out amino-acid substitutions identified by others [14]. Briefly, Kv1.6 was mutated using Pfx polymerase (Invitrogen) in the presence of primers to substitute NIP residues Glu27, Glu30 and Glu32 with alanines, thereby, yielding the mutant (Kv1.6 E27/30/32A) that was confirmed by full DNA sequencing. The resultant mutant Kv1.6 gene replaced wild-type counterparts in the mutant variants (Kv1.4–1.2–1.6mut–1.2 and Kv1.4–1.6mut–1.2–1.2).

Gene expression and verification of heterotetrameric constructs

The Kv1 channel constructs were expressed in HEK-293 cells (A.T.C.C.) for 24–48 h after transfection with Polyfect reagent (Qiagen). Cells were harvested, washed, resuspended at ~2–3×107 cells/ml in PBS (pH 7.4) and incubated with sulfo-NHS-LC-biotin [sulfo-succinimidyl-6-(biotinamido)hexanoate; 1 mg/ml; Pierce] at 4 °C for 30 min. The remaining reagent was quenched with 100 mM glycine for 30 min; samples were then solubilized in 2% (v/v) Triton X-100 with protease inhibitors for 1 h at 4 °C, and centrifuged at 10000 g for 1 h at 4 °C. The supernatant was incubated with streptavidin–agarose (70 μl slurry/ml; Pierce) overnight at 4 °C with rotation. After washing the pelleted streptavidin–agarose with ice-cold TBS (Tris-buffered saline; 50 mM Tris/HCl and 150 mM NaCl, pH 7.4) containing 0.1% Tween 20, bound proteins were dissolved in SDS/PAGE sample buffer and monitored by SDS/PAGE (4–12% gradient gel) followed by Western blotting with monoclonal Kv1 α subunit-specific antibodies (NeuroMab). The tetramers were visualized using a mouse secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch) and visulaized using the Immobilon Western Blot Substrate (Millipore). For fluorescence microscopy, HEK-293 cells were plated on poly-L-lysine-coated glass coverslips in 35 mm dishes and transfected using Polyfect (Qiagen) with the constructs encoding the representative heterotetramers (Kv1.4–1.2–1.6–1.2, Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.2–1.6). The samples were processed for staining 48 h post-transfection as previously described [20]. Following fixation with 4% PFA (paraformaldehyde) in PBS, the cells were washed, blocked in TBS (pH 7.4) and 4% (w/v) dried milk powder for 60 min and then permeabilized for 30 min with 0.1% Triton X-100 in the blocking buffer. Cells were incubated with mouse anti-Kv1.2, Kv1.6 or Kv1.4 IgGs for 1 h at room temperature. Washing was repeated prior to further incubation with goat anti-mouse IgG conjugated to Alexa Fluor® 594 (Invitrogen) for 1h. The samples were thoroughly washed in PBS prior to being fixed with Vectashield (Vector Laboratories). Control specimens were treated likewise except for omitting the first antibody. Confocal images were obtained with Observer Z1 Axion inverted microscope (LSM-710; Carl Zeiss). A 40 × 0.95 NA (numerical aperture) oil immersion objective was used for imaging with the pinhole diameter set for 1 airy unit. Fluor was excited with the 594 nm line of an Argon laser and emission bandpass was set at 583–734 nm.

Electrophysiological recordings and data analysis

Tetrameric Kv1 channel constructs were expressed in HEK-293 cells for 24–48 h after transfection as described above and split using a detaching agent, Accutase (Analab), on to glass coverslips before recording. All measurements were made using the whole-cell configuration of the voltage-clamp technique at a −90 mV holding potential, as outlined previously [18], except where specified. The recording pipettes were filled with an intracellular solution [95 mM KF, 20 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 11 mM EGTA, 10 mM Hepes, 2 mM Na2ATP (pH 7.2 with KOH), 10 mM glucose and 20 mM sucrose (300±10 mOsm)], with fire-polished tips having resistances between 1.5 and 3.0 MΩ. To ensure functionality of the NIB moiety, 2 mM glutathione (Sigma–Aldrich) was introduced as a reducing agent to the internal solution [14,21]. The external (bath) medium contained: 135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2 and 5 mM Hepes (pH 7.4 with NaOH). The liquid junction potential was not corrected. Only cells with series resistances <10 MΩ throughout the experiments were included in the present study. The series resistance was compensated (75–80%) to minimize voltage errors. Current signals were recorded with an EPC10 (HEKA Elektronik) patch-clamp amplifier. Leakage and capacitive currents were subtracted on-line using the P/4 subtraction protocol. Currents were filtered at 1 kHz, and sampled at 5 kHz (with the exception of 10 s pulses, which were sampled at 50 Hz). Time-dependent inactivation constants were determined during 10-s depolarization steps from −10 to 10 mV, in 10 mV increments, and the currents were fitted with double exponential functions [19]. For experiments on the recovery from inactivation, a variable interval-gapped pulse protocol was used: from a −90 mV holding potential, depolarization to +10 mV for 300 ms in an initial step was followed by a second identical pulse with gap intervals of 0–20 s. The ratios of the normalized peak currents at time intervals were plotted and fitted with a single exponential function to obtain time courses of recovery from inactivation. Measurement of steady-state inactivation involved a 10-s conditioning pulse applied in 10 mV increments from −100 to +10 mV, followed by a 500 ms test pulse at +10 mV (at −90 mV holding potential). Steady-state inactivation constants were calculated from the peak currents of the test pulses. Normalized inactivation curves were fitted to the Boltzmann function (I={1+exp[(VV1/2)/k]}−1), where V is the membrane (pre-pulse) potential, V1/2 is the potential at half-maximal inactivation and k is the slope factor. The results (means±S.E.M.; n is number of cells tested) were analysed using FitMaster (HEKA Electronik) and fitted by Igor Pro 6 (WaveMetrics). Statistical significance was evaluated by Mann–Whitney U test, as appropriate, for data from at least three independent experiments; P values of <0.05 were considered significant.

RESULTS

Channel concatemers of defined α subunit composition expressed following domain-specific assembly of gene cassettes into the pIRES2-EGFP plasmid

Through examining the properties of recombinantly expressed heterotetramers, a new strategy [18] has been validated for concatenating and expressing functional Kv1.X genes within a single ORF on the plasmalemma of mammalian cells. In the present study, two subunits, which play physiologically important, but opposing, roles in determining Kv1-channel inactivation, were selected to obtain proof of principle for retaining their ordering according to positions within the gene constructs (Figure 1A). Kv1.4, which can mediate N-type inactivation of mammalian Kv1 channels through its NIB [21,22] together with Kv1.6, which overrides this rapid inactivation via its NIP domain [14], were expressed in different positions with respect to each other. Two or three copies of Kv1.2 formed the other constituent of these heterotetramers. In one concatenated gene construct, Kv1.4 was separated from Kv1.6 with a single copy of Kv1.2 (Kv1.4–1.2–1.6–1.2), in another case Kv1.6 was placed immediately adjacent to Kv1.4 followed by two copies of Kv1.2 (Kv1.4–1.6–1.2–1.2), and the third tetramer had two copies of Kv1.2 between Kv1.4 and Kv1.6 (Kv1.4–1.2–1.2–1.6), giving three tetramers with identical subunit composition, but different ordering of their subunits. A fourth construct was assembled containing one copy of Kv1.4 along with three copies of Kv1.2 (Kv1.4–1.2–1.2–1.2); this acted as a rapid inactivating K+ channel which lacked the Kv1.6-containing NIP domain (Figure 1A).

All tetramer construction employed an inter-subunit linker derived from the Xenopus β-globin gene shown previously to be suitable [23]. Initial PCR of cDNA encoding Kv1.4, 1.6 or 1.2, yielded single bands on electrophoresis with the expected sizes of ~2.0, 1.6 and 1.5 kbp respectively. Flanking inter-subunit linkers and paired restriction sites, allowing cloning into the pIRES2-EGFP expression vector, were added to the amplified products of Kv1.2 and/or Kv1.6 as described [18]. The resultant heterotetramers, Kv1.4–1.2–1.6–1.2, Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.2–1.6, were assembled into pIRES2-EGFP with the Kv1.4 gene introduced at the start (position I) to conserve functionality of its NIB. Kv1.6, where present, was placed either adjacently (position II or position IV) or distally (position III) to the Kv1.4 sequence (Supplementary Figure S1). All of the concatenated constructs, upon transfection into HEK-293 cells and following surface biotinylation analysis, yielded an expressed protein band on SDS/PAGE at Mr ~280 kDa when probed with antibodies specific to Kv1.2, 1.4 or 1.6 (Figure 1B). This band represents full-length intact tetrameric protein with a smeared appearance due to the highly glycosylated nature of Kv1.4. A number of faint non-specific bands were observed with the Kv1.4 antibody at 140, 40 and 40 kDa, whereas a non-specific band was also visible with the Kv1.2 antibody at 200 kDa. The absence of these bands across all of the three blots indicate their non-specific nature.

Plasmalemmal targeting of intact concatenated K+ channels in active form

While biotinylation provided biochemical evidence of the presence of intact concatenated heterotetramer at the plasmalemma, immunostaining of transfected mammalian cells was performed to determine the extent of trafficking from the endoplasmic reticulum to the plasmalemma. Labelling of HEK-293 cells expressing Kv1.4–1.2–1.2–1.2, Kv1.4–1.2–1.6–1.2, Kv1.4–1.6–1.2–1.2 or Kv1.4–1.2–1.2–1.6 with mouse IgGs specific for Kv1.2, Kv1.6 or Kv1.4, followed by confocal fluorescence microscopy, detected clear immunostaining on the cell surface (Figure 1C). Untransfected cells (Figure 1C, bottom left-hand panel) and cells transfected with Kv1.4–1.2–1.6–1.2 and incubated with secondary antibody only (Figure 1C, bottom right-hand panel), showed no staining. Similar levels of labelling were observed for heterotetramers in which the wild-type Kv1.6 was substituted with a mutated Kv1.6 (results not shown).

Kv1.6 subunit prevents fast inactivation of K+ currents only when positioned adjacent to Kv1.4 in heteromeric channel proteins

When subjected to a 10-s depolarization step to +10 mV or −10 mV, the expressed Kv1.4–1.2–1.6–1.2 channel displayed a rapidly inactivating A-type K+ current (Figure 2A), which is surprising as this heteromer contains a NIP domain known to disallow N-type fast inactivation [14]. Such rapid decay suggests that distal positioning of the NIP-containing Kv1.6 relative to Kv1.4 in this heteromer attenuates NIP functionality. This fast-inactivation profile of Kv1.4–1.2–1.6–1.2 channel is similar to that of Kv1.4–1.2–1.2–1.2. In stark contrast, heteromers Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.2–1.6 yielded slow-inactivating currents (Figure 2A) as expected, due to the dominant-negative effect of the NIP domain in Kv1.6 [14]. Current traces from the three heteromeric channels were best fitted with a double exponential function, which revealed significant differences in inactivation rates (Figure 2B and Table 1). Accordingly, the K+ current resulting from heteromers where Kv1.4 and 1.6 are placed adjacently (Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.2–1.6), showed slower τ1inact and τ2inact values than those of Kv1.4–1.2–1.6–1.2 and Kv1.4–1.2–1.2–1.2, especially at more negative potentials (Figure 2B and Table 1). The fast-inactivating channels (Kv1.4–1.2–1.6–1.2 and Kv1.4–1.2–1.2–1.2) gave fairly constant τ1inact and τ2inact values at different potentials (Figure 2B and Table 1), while the slow-inactivating channel counterparts revealed variable τ1inact, but not τ2inact, values. Among the two slow-inactivating channels, differences in τ1inact values at more positive potentials can be correlated with shifting the Kv1.6 subunit from the second to the fourth position, which might affect the NIP functionality. A steady-state inactivation protocol demonstrated the influence of NIP positioning in the concatamers on the voltage dependence of inactivation. The ensuing results, fitted by a single Boltzmann function (Figure 2C), unveiled a difference in the midpoints for voltage-dependent inactivation. Heterotetramers where Kv1.6 and 1.4 subunits are in adjacent positions showed V1/2 values of −32 mV for Kv1.4–1.6–1.2–1.2, and −31 mV for Kv1.4–1.2–1.2–1.6 compared with −40 mV for Kv1.4–1.2–1.6–1.2 and −49 mV for Kv1.4–1.2–1.2–1.2; steady-state inactivation parameters are listed in Table 1. This shift in V1/2 of the Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.2–1.6 channels towards more depolarized potentials correlates with the slow inactivation due to adjacent positioning of the NIP, contrasting with that for the fast-inactivating Kv1.4–1.2–1.6–1.2 and Kv1.4–1.2–1.2–1.2 channels. Also, membrane potentials more negative than −70 mV were required to remove inactivation from all heteromers tested (Figure 2C). The recovery from inactivation was studied with a step from −90 to 10 mV, using a variable interval-gapped pulse protocol (see Figure 3A). Analysis of the time dependence of recovery from inactivation (Figure 3B) revealed that this is significantly faster for Kv1.4–1.6–1.2–1.2 followed by Kv1.4–1.2–1.2–1.6, than that observed for Kv1.4–1.2–1.6–1.2 (Figure 3B), as a result of the removal of Kv1.4 N-type inactivation by the adjacently placed Kv1.6 NIP. Likewise, the time dependence of recovery from inactivation for Kv1.4–1.6–1.2–1.2 was faster than that recorded for Kv1.4–1.2–1.2–1.2. On the other hand, the presence of a residual fast component in the K+ current of Kv1.4–1.2–1.2–1.6 might be affecting the τ value for recovery. The inactivation kinetic values are summarized in Table 1.

View this table:
Table 1 Summary of inactivation parameters for Kv1.4-containing heteromers expressed in HEK-293 cells

Results recorded are presented as means±S.E.M., n-values are in brackets. *, values are significant, P<0.05 (Mann–Whitney U test).

Figure 2 Inactivation kinetics of K+ currents are strongly dependent on the position of Kv1.6 relative to Kv1.4 in the concatamers

(A) Representative current traces, in response to a two-pulse steady-state inactivation protocol, for each depicted channel. (B) The histograms display the τ1 and τ2 rates of inactivation for the representative channels at different potentials. See Table 1 for the significant differences. *, P<0.05, Mann–Whitney U test. Error bars represent means±S.E.M. (C) The steady-state inactivation relationship, taken from normalized peak currents triggered by pre-pulse potentials and fitted with a single Boltzmann function (see the Materials and methods section). This plot shows a significant (P<0.05, Mann–Whitney U test) voltage shift for Kv1.4–1.6–1.2–1.2 (○, broken line) and Kv1.4–1.2–1.2–1.6 (▲, broken line) to more positive potentials compared with Kv1.4–1.2–1.6–1.2 channels (●, solid line) or Kv1.4–1.2–1.2–1.2 (Δ, solid line) channels, as a result of Kv1.6 NIP functionality. See Table 1 for values.

Figure 3 Recovery from inactivation is faster when Kv1.6 is adjacent to Kv1.4 in the concatamers

(A) Recovery from inactivation measured for Kv1.4–1.2–1.2–1.2–1.2, Kv1.4–1.2–1.6–1.2, Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.2–1.6, using variable interval-gapped pulse protocol (inset). (B) The resultant curves, fitted with a single exponential function show a significantly faster (P<0.05, Mann–Whitney U test) recovery from inactivation for Kv1.4–1.6–1.2–1.2 (○, broken line) and Kv1.4–1.2–1.2–1.6 (■, broken line) than observed for the Kv1.4–1.2–1.6–1.2 channel (●, solid line). This is due to the removal of Kv1.4 N-type inactivation by the Kv1.6 NIP domain. Notice the slow recovery from inactivation for Kv1.4–1.2–1.2–1.2 (△, solid line) channel compared with Kv1.4–1.6–1.2–1.2. Inactivation constants are shown in Table 1. Curves display the average values from at least 4 cells. Some error bars fall within the data symbols; the points in the broken lines are super-imposed; dotted lines indicate zero current. Error bars represent means±S.E.M.

Mutagenesis proved that attenuation of N-type inactivation by Kv1.6 is mediated by NIP

Having demonstrated modulation of the Kv1.4 NIB by Kv1.6 within concatenated heterotetramers of predetermined ordering, it was necessary to ascertain if these observed effects are entirely attributable to the NIP domain. Mutagenesis of residues in the Kv1.6 α subunit identified previously as crucial for NIP function [14], and replacing the wild-type with mutated subunit (E27/30/32A), yielded channels [Kv1.4–1.2–1.6(E27/30/32A)–1.2 and Kv1.4–1.6(E27/30/32A)–1.2–1.2)], which both decay rapidly (Figure 4A1). These inactivation time courses are similar to those observed with heteromers containing distally arranged wild-type Kv1.6 (Figure 4A1 and Table 1), confirming a complete loss of NIP function from Kv1.6 is possible either by mutation or distal positioning of the wild-type, each permitting fast inactivation of the K+ current. Both mutated channels showed similar values of inactivation for τ1inact and τ2inact at the different potentials tested (Figure 4A3 and Table 1). This is attributable to the fast inactivation mediated by Kv1.4 subunit in each heteromer where NIP function is attenuated. Such values compare well with those derived from the distally arranged heteromer (Kv1.4–1.2–1.6–1.2) which contains ‘wild-type’ Kv1.6, establishing that NIP in this position has no discernible effect on the NIB moiety. These results were supported by the observed restoration of voltage dependence for inactivation (V1/2 −37 mV for both channels; Table 1) and recovery from inactivation for either channel containing the mutated Kv1.6 subunit (Figures 4A2, 4B1 and 4B2) to values similar to that of Kv1.4–1.2–1.6–1.2 or Kv1.4–1.2–1.2–1.2 (Figure 4B2 and Table 1). This corroborative data highlight that only the NIP domain in Kv1.6 prevents fast inactivation, whose function is dependent on its position relative to the Kv1.4 subunit.

Figure 4 Position-dependent functioning of Kv1.6 is NIP-mediated: replacement of Kv1.6 with a mutant form abolished NIP activity, thereby restoring rapid inactivation

(A1) Representative current traces for channels with mutated Kv1.6 (E27/30/32A) incorporated (Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.6–1.2) and expressed in HEK-293 cells. (A2) Steady-state inactivation relationships obtained for these channels, fitted by a Boltzmann function [broken line for Kv1.4–1.6–1.2–1.2 (□) and solid line for Kv1.4–1.2–1.6–1.2 (■)], revealed near-identical profiles. Broken lines indicate zero current. (A3) The bar diagrams summarize the τ1 and τ2 rates of inactivation of both channels taken at different potentials. Error bars represent means±S.E.M. (B1) Typical currents showing recovery from inactivation of each heterotetramer fitted to a single exponential function. (B2) When plotted, the average data for each channel, from at least four cells gave superimposable curves; some error bars fall within the data points. Error bars represent means±S.E.M.

DISCUSSION

Arrangements of Kv1α genes in constructs determine subunit positions in the expressed channels

A new cloning platform was employed that afforded expression of four α subunits as a single protein, with predetermined subunit ordering relative to each other in the functional channels examined on the plasmalemma. Importantly, and in contrast with previous systems [24], the use of inter-subunit linkers appears to have ensured retention of the behaviour of pre-positioned subunits as illustrated by retention of NIP function of Kv1.6. Three such preassembled hetero-oligomers are presented, all with identical α subunit composition, but in different arrangements, containing a single Kv1.4 and 1.6 subunit plus two copies of Kv1.2. This offered the advantage over previous studies [14] in allowing the effects of their positioning to be examined. Heterotetramers (Kv1.4–1.2–1.6–1.2, Kv1.4–1.6–1.2–1.2 and Kv1.4–1.2–1.2–1.6) elicited K+ currents, with one displaying significantly different inactivation properties to the other two. Such dissimilarity, together with the uniform nature of the currents produced by each concatenated tetramer, established that the gene arrangements in the constructs dictate subunit ordering. A fourth heteromer (Kv1.4–1.2–1.2–1.2) possessing a single Kv1.4 subunit with three copies of Kv1.2 was used as a control of fast-inactivating heterotetramer; its lack of Kv1.6 subunit results in the fastest inactivation kinetics in comparison with the other heteromers tested.

Subunit ordering reveals position dependency of NIP function

This confirmed retention of subunit ordering and delivery of intact heterotetramers to the plasmalemma allowed the generation of channels exhibiting different types of inactivation by incorporating just one copy of Kv1.4 and 1.6, rather than two as examined previously [14]. Moreover, the concatenation of these subunits along with Kv1.2, combinations reported to co-exist in the brain [13], permitted elucidation of the importance of their ordering. The NIP in Kv1.6 proved functional only if placed immediately next to (position II or IV) its target Kv1.4 (position I) in the formed channel, yielding a slow-inactivating K+ current. Positioning Kv1.6 distal to Kv1.4 (position III) led to N-type fast inactivation, presumably because NIP is not optimally positioned to appropriately antagonize the activity of NIB. Furthermore, the same fast inactivation kinetics were observed with NIP-mutated forms of these channels, confirming that only the NIP domain is responsible for counteracting the function of NIB. Such position dependency of NIP activity could accord with a previous suggestion [14] that this domain does not act by occupying the acceptor site for NIB, instead, interacting directly with the latter.

NIP, NIB and inactivation outcomes

It was of interest to consider the observed inactivation profiles in relation to occupancy (or not) by NIB of its receptor site on the S6 segment of the channel, because this mechanism is the basis of N-type inactivation [4,5,22]. A reported inability of homotetrameric Kv1.6 [3,14] to produce a fast-inactivating K+ current implies that NIP is unable to bind to the inner pore, at least in a blocking fashion. The system validated in the present study for creating heteromers which exhibit fast (distal channels) or slow (adjacent channels) inactivation provides scope for evaluating their distinct biophysical properties. In this way, the adjacent concatamers were found to recover faster from inactivation than the rapidly inactivating distally arranged protein; this accords with the rate of recovery from inactivation being enhanced by a slow inactivation (C-type) mechanism [25]. It is noteworthy that the observed rates of fast inactivation for the mutated adjacent and distal concatemers are similar, indicative of the positioning of a non-functional NIP domain not impacting on their fast inactivation. Moreover, the fast inactivation behaviour of Kv1.4–1.6(E27/30/32A)–1.2–1.2 channels showed clearly that NIP and NIB domains are directly interacting upon depolarization, mainly by electrostatic interaction before the NIB reaches its receptor, by an undefined mechanism. One can speculate that conformational changes, initiated by the activation process, would be sensed by both charged motifs of the NIP and NIB domains facilitating their interaction. Positioning of both domains away from each other would prevent such interaction, as seen with both Kv1.4–1.2–1.6(E27/30/32A)–1.2 and Kv1.4–1.2–1.6–1.2 channels.

In situ hybridization and immunohistochemical localization studies suggested that Kv1.2, Kv1.4 and Kv1.6 proteins may coexist on the membranes of several types of central neurons in mammals [13,16,2628]. In rat brain membranes, heteromultimeric Kv1 channels, with direct association of Kv1.4 and Kv1.6 subunits, were observed using subunit-specific antibodies in immunoaffinity experiments [13,14]. Collectively, these findings suggest that the gating contributions of Kv1.4 subunits can be modified by differential positioning of the NIP domain of Kv1.6 subunit in neuronal Kv1 channels.

In co-expression studies, recombinant Kvβ1 has been shown to confer rapid inactivation, via its distinct NIB domain, on all tested members of Kv1 α subunits except Kv1.6, suggesting that Kvβ1 subunit could also be an important modulator of K+ channel complexes [6,7]. In fact, the Kv1.2, 1.4, and 1.6 α subunits in rat brain membranes can be co-immunoprecipitated with Kvβ1 [29]. These findings indicate that, in neurons, the gating of Kv1 heteromers can be modified by NIB/NIP domain(s) and/or auxiliary β1 subunits. In either case, the Kv1.6 subunit position in the heteromeric Kv1 channel could play a key role in tuning the inactivation process, and thus shaping the firing pattern of the neuron.

It is tempting to speculate on the functional relevance of NIP action in diseased states. Seizure activity in an animal model has been linked to the spatio-temporal changes in the expression of the Kv1 subfamily within the hippocampus [16], where the levels of delayed rectifier Kv1 channels (including Kv1.2 and Kv1.6) are reduced with minor changes in Kv1.4. This curious alteration might be a physiological response to seizure events in which Kv1.4 inactivation by NIP gets diminished, thereby dampening unwanted depolarizations during attacks. Extensive investigations would have to be performed to assess the in vivo functional implications of NIP and its placement within native heterotetramers.

It is clear from the results presented in the present paper that: (a) concatenation of Kv1 subunits results in the predicted assembly of functional channels at the plasmalemma; (b) subunit ordering crucially influences channel properties; (c) composition alone is not sufficient for predicting the characteristics of native channels; and (d) NIP–NIB interaction(s) occur at a site distinct from that of the NIB-binding site in the inner portion of the ion pore.

AUTHOR CONTRIBUTION

Ahmed Al-Sabi co-designed the experiments, performed the electrophysiological recordings, data analysis and interpretation of the results. Seshu Kaza and Jiafu Wang built and evaluated the heterotetrameric constructs. Marie Le Berre and Liam O'Hara expressed and characterized the heterotetrameric proteins. MacDara Bodeker proposed the work, contributed to designing the experiments and built precursors of the channel constructs. J. Oliver Dolly conceived the research, obtained funding and participated in the experimental design and interpretation of the results. All authors participated in writing the paper.

FUNDING

This work was funded by a Principle Investigator grant (09/1N.1/B2634) and a Research Professorship award (04/RP1/B345) by Science Foundation Ireland to J.O.D. and a PRTLI 4 grant from the Irish Higher Education Authority for the Neuroscience sections of ‘Target-driven therapeutics and theranostics’.

Acknowledgments

The authors thank Professor Olaf Pongs (Institute for Neural Signal Transduction, University of Hamburg, Germany) for generously providing Kv1 constructs, Dr Jon Sack (International Centre for Neurotherapeutics, Dublin City University, Ireland) for valuable comments on the paper and Dr Saak Ovsepian (Neuro Imaging Unit, Dublin City University, Ireland) for help with the confocal microscopy and comments on the paper.

Abbreviations: EGFP, enhanced green fluorescent protein; HEK, human embryonic kidney; Kv1, voltage-gated K+ channel; NIB, N-terminal inactivation ball; NIP, N-type inactivation prevention; ORF, open reading frame; TBS, Tris-buffered saline; UTR, untranslated region

References

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