Modes of Regulation of Shab K+ Channel Activity by the Kv8.1 Subunit*

(Received for publication, October 1, 1996, and in revised form, December 30, 1996)

Miguel Salinas , Jan de Weille , Eric Guillemare , Michel Lazdunski Dagger and Jean-Philippe Hugnot

From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The Kv8.1 subunit is unable to generate K+ channel activity in Xenopus oocytes or in COSm6 cells. The Kv8.1 subunit expressed at high levels acts as a specific suppressor of the activity of Kv2 and Kv3 channels in Xenopus oocytes (Hugnot, J. P., Salinas, M., Lesage, F., Guillemare, E., Weille, J., Heurteaux, C., Mattéi, M. G., and Lazdunski, M. (1996) EMBO J. 15, 3322-3331). At lower levels, Kv8.1 associates with Kv2.1 and Kv2.2 to form hybrid Kv8.1/Kv2 channels, which have new biophysical properties and more particularly modified properties of the inactivation process as compared with homopolymers of Kv2.1 or Kv2.2 channels. The same effects have been seen by coexpressing the Kv8.1 subunit and the Kv2.2 subunit in COSm6 cells. In these cells, Kv8.1 expressed alone remains in intracellular compartments, but it can reach the plasma membrane when it associates with Kv2.2, and it then also forms new types of Kv8.1/Kv2.2 channels. Present results indicate that Kv8.1 when expressed at low concentrations acts as a modifier of Kv2.1 and Kv2.2 activity, while when expressed at high concentrations in oocytes it completely abolishes Kv2.1, Kv2.2, or Kv3.4 K+ channel activity. The S6 segment of Kv8.1 is atypical and contains the structural elements that modify inactivation of Kv2 channels.


INTRODUCTION

Voltage-gated K+ channels (Kv) serve a wide range of functions including the regulation of cardiac pacemaking, action potentials, and neurotransmitter release in excitable tissues, as well as hormone secretion, cell proliferation, cell volume regulation, and lymphocyte differentiation in non-excitable tissues (2). The diversity of K+ channel functions is reflected by the diversity of K+ channel structures. Delayed rectifier K+ channels are constituted by 2 types of subunits called alpha  and beta  (3). A large number of genes encode the different alpha  pore-forming subunits (18 genes cloned in mammals (4)), and an increasing number of beta -auxiliary subunits are being discovered (5-8). Different gene expressions in different cells or at different times of development associated with the possible formation of heteromultimeric channels containing different types of alpha - and beta -subunits probably allow individual cells to acquire their own characteristics of K+ current properties.

Sequence similarities among members of the Kv family were initially used to define 4 subfamilies (Kv1, Kv2, Kv3, Kv4) of alpha -subunits (9-12). The members within a given family share a large percentage of sequence identity (>70%) while this percentage falls to ~40% among members of different subfamilies. Four additional types of alpha -subunits (Kv5.1 (IK8 (13)), Kv6.1 (K13 (13)), Kv7.1 (14), Kv8.1 (1)) have been recently cloned.

All subunits belonging to the Kv1 to Kv4 subfamilies have been functionally expressed in Xenopus oocytes. Elicited K+ currents display a large variety of electrophysiological characteristics reminiscent of the variety of K+ currents recorded in vivo. Particularly, large variations in inactivation characteristics have been observed ranging from tau inact ~30 ms for a very fast inactivating channel, such as (Kv1.4) (15), to tau inact ~15 s for a slow inactivating one, such as Kv2.1 (16). K+ channel inactivation occurs by at least two distinct mechanisms. The N-type inactivation is usually quite rapid with a time constant in the milliseconds range. It occurs by a "ball and chain" mechanism involving the NH2-terminal cytoplasmic domain, which acts as a tethered blocker that occludes the pore channel in its open state and causes inactivation (17-19). C-type inactivation is generally slower and appears to involve the COOH-terminal sequence of the alpha -subunits (20-22).

Although the Kv5.1 and Kv6.1 proteins apparently have the structural hallmarks of functional K+ channel alpha -subunits, their expression in oocytes fails to induce K+ currents (13). The Kv8.1 subunit also displays the structural characteristics of a K+ channel subunit and is highly expressed in the brain. As Kv5.1 and Kv6.1, it does not lead to expression of K+ currents when produced in Xenopus oocytes. However, upon coexpression it is able to specifically abolish K+ currents generated by channels formed by Kv2.1, Kv2.2, and Kv3.4 subunits (1), and this inhibition is associated with the formation of multimers with these other subunits.

This paper extends previous work which was limited to Kv8.1 expression in Xenopus oocytes. It reports an analysis of Kv8.1 expression in the COS mammalian cell line. In this system, the Kv8.1 subunit is normally retained in cytoplasmic compartments. It requires coexpression with Kv2.2 to bring the subunit to the plasma membrane. Kv8.1 then induces no inhibition of the Kv2.2 current in this system but instead produces a drastic modification of the kinetic properties of Kv2.2 and particularly of its inactivation. The same effect can in fact be seen on Kv2.1 and Kv2.2 currents in Xenopus oocytes expressing moderate levels of Kv8.1, while a total inhibition is seen with higher levels of Kv8.1. Therefore, depending on its level of expression, Kv8.1 can either modify the kinetics of the Kv2 channels or completely abolish their activity. Site-directed mutagenesis has been used to show that Kv8.1 effects on the Kv2 current are mediated by the presence of singular amino acids located in the S6 domain of the Kv8.1 subunit.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

COSm6 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and supplemented with 10% fetal calf serum and antibiotics (60 µg/ml penicillin, 50 µg/ml streptomycin). One day before transfection, 105 cells were plated onto 35-mm Petri dishes for an electrophysiology experiment and 15 × 103 cells onto 15-mm plates for indirect immunofluorescence microscopy. The cells were transfected by a modification of the DEAE-dextran/chloroquine method (23) using 0.6 µg of supercoiled DNA per cm2 of cell culture. For electrophysiology studies, we co-transfected a plasmid encoding the CD8 receptor, which allows direct visualization of transfected cells by antibody-coated beads (24).

Electrophysiology on COS Cells

Voltage-clamp experiments were carried out using the whole-cell suction-pipette technique. The intracellular (pipette) solution contained 150 mM KCl, 1 mM MgCl2, 2 mM EGTA, 10 mM HEPES-KOH, pH 7.2. The extracellular solution was 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES-NaOH, pH 7.3. Pipettes were coated with Sylgard resin to reduce their capacity. Electrical signals were digitized and stored on a hard disk by a personal computer for further analysis. Experiments were carried out at room temperature (24 ± 2 °C).

Indirect Immunofluorescence Microscopy

The cells that were grown on glass coverslips were fixed for 15 min with 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS).1 After rinsing twice with PBS, cells were permeabilized by incubation for 10 min with 0.1% Triton X-100. The sites of nonspecific binding were blocked by 2 h of incubation with 5% goat serum, 2% bovine serum albumin in PBS at room temperature. The cells were then incubated for 2 h with a mixture of M2 monoclonal antibody (1:50 dilution, Eastman Kodak Co.) or polyclonal antibodies against the Kv1.5 subunit (1:100)2 with a BS solution (2% bovine serum albumin in PBS), followed by washing with PBS and incubation for 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse Ig (1:100, Sigma) or fluorescein (5-[4,6-dichlorotriazin-2-yl)amino]fluorescein)-conjugated F(ab')2 fragment of a goat anti-rabbit Ig (1:100, Immunotech) in BS. After washing in PBS then in 1 mM Tris-HCl, pH 7.5, cells were mounted in Vectashield Medium (Vector Laboratories, Inc.) and observed with a Leitz Aristoplan microscope (Wild Leitz) using an interference blue (fluorescein isothiocyanate) filter and a 40× or oil-immersion 100× lens.

Plasmid Constructions and Mutagenesis

The Kv8.1 hamster cDNA was cloned into the pRc/CMV vector (Invitrogen) for expression in COS cells. To introduce mutations in this plasmid, a two-step polymerase chain reaction method using sense and antisense mutant primers was used (25). The dominant-negative mutant Kv8.1Delta , obtained by this technique, bears a deletion from residue 426 to the COOH-terminal end of the protein. After each mutation the Kv8.1 open reading frame was entirely resequenced (dye terminator kit, Applied Biosystems). The construction of NH2-terminal tagged Kv8.1 subunit was described previously (1). To create a COOH-terminal tagged Kv8.1 protein the same scheme was used, but the polymerase chain reaction fragment was cleaved with ClaI restriction enzyme and inserted in a HindIII- (filled with Klenow DNA polymerase I large fragment) and ClaI-digested Flag-pRc/CMV vector.

Expression of the Kv2.2 subunit in COS cells was realized with a pCDNAI vector. To create NH2-terminal tagged Kv2.2 protein, a polymerase chain reaction fragment (PWO Taq polymerase, Boehringer Mannheim) containing Kv2.2 open reading frame and a 3'-ApaI restriction site was cloned in a HpaI- and ApaI-digested Flag-pRc/CMV vector.

Kv2.1 subunit mutations were generated by oligonucleotide-directed mutagenesis on a single-stranded template (26) derived from Bluescript II vector containing Kv2.1 cDNA (27). All mutational changes were checked by sequence determination.

cRNA Synthesis, Injection, and Electrophysiological Measurement in Xenopus Oocytes

Preparation of oocytes, cRNA synthesis, injection (50 nl), and electrophysiological measurements have been previously described (1, 28). We have previously reported that in oocytes as well as in cell lines high magnitude currents could show pronounced modifications when compared with currents of lower intensity (28, 29). Therefore, in all this work, attention was paid to always compare currents of similar and relatively low intensity.


RESULTS

Modification of Kv8.1 Subcellular Localization upon Kv2.2 Coexpression in COS Cells

Despite numerous attempts to express the Kv8.1 subunit in various expression systems including Xenopus oocytes and Chinese hamster ovary and COS cell lines, K+ currents could never be recorded (1). One possible reason for this lack of success is an inadequate cellular localization of the expressed Kv8.1 protein. To check that point, an 8-amino acid epitope, which can be recognized by a monoclonal antibody, was added to the Kv8.1 protein, and protein localization was examined by indirect immunofluorescence on Triton-permeabilized COS cells (Fig. 1). Transfected cells expressing the Kv8.1 protein tagged on the NH2-terminal or on the COOH-terminal end show a very strong fluorescent staining localized at the perinuclear region as well as in a fine reticular network extending through the cytoplasm (Fig. 1, A, B, and C). In the many experiments that have been done, a surface labeling of Kv8.1 was never observed. Confocal microscopy experiments also failed to detect the Kv8.1 protein at the plasma membrane (data not shown).


Fig. 1. Immunofluorescence localization of Kv subunits in COS cells. A and B, cells expressing NH2-tagged Kv8.1 subunit; C, COOH-tagged Kv8.1 subunit; D, Kv1.5 subunit; E, NH2-tagged Kv2.2 subunit; F and G, NH2-tagged Kv8.1 and Kv2.2 subunits. Detection is performed with M2 monoclonal antibody except in D where an anti-Kv1.5 rabbit polyclonal antibody was used. Arrowheads indicate perimembranous fluorescence.
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To verify that the COS cells were able to correctly express well known functional K+ channel alpha -subunits and integrate them at the plasma membrane, cells were also transfected with a Kv1.5-expressing vector (30). Protein detection in this case was made with polyclonal antibodies specific for Kv1.5. As shown in Fig. 1D, Kv1.5-expressing cells, which produce K+ currents with the expected biophysical properties (30), show a typical surface labeling unlike Kv8.1-expressing cells.

An NH2-terminal tagged Kv2.2 alpha -subunit was also prepared. Electrophysiological recording of cells transfected with this subunit show typical delayed outward rectifier K+ currents (not shown), and as expected, immunodetection indicates a clear surface membrane localization (Fig. 1E) in addition to a perinuclear accumulation.

As the Kv8.1 subunit is able to interact physically with the Kv2.2 protein (1), the subcellular localization of Kv8.1 was examined in cells transfected with a mixture (3:1 molecular ratio) of Kv2.2 and tagged Kv8.1-expressing plasmids. Immunofluorescent detection then revealed a different Kv8.1 localization pattern. A minority of cells showed a fluorescence distribution resembling that obtained with Kv8.1 alone, i.e. an apparent lack of surface membrane localization. In contrast, the majority of cells displayed a fluorescent staining resembling that observed for the Kv2.2 localization, i.e. a perimembranous staining combined with a cytoplasmic retention (Fig. 1, F and G). The conclusion is that the Kv8.1 subunit is transported to the plasma membrane when it is coexpressed with the functional Kv2.2 subunit.

The Kv8.1 Subunit Modifies Kv2.2 Current Characteristics

The effect of Kv8.1/Kv2.2 coexpression was then explored electrophysiologically. Expression of the Kv2.2 subunit alone resulted in a typical delayed outward rectifier K+ current with a slow inactivation process (Fig. 2B). Expression of Kv8.1 alone did not give rise to a detectable K+ current (not shown). Cells were then transfected with a mixture of Kv2.2 and Kv8.1-expressing vectors using 1:5 to 1:50 molecular ratios. Increasing amounts of Kv8.1 decreased the intensity of the Kv2.2 current (Fig. 2A). However, this decrease was not specific because co-transfection of Kv2.2-expressing cells with increasing amounts of beta -galactosidase produced the same effect (Fig. 2A). This lack of specificity was confirmed by a coexpression of Kv2.2 with a non-functional deleted form of the Kv1.5 subunit (Kv1.5Delta ) (30) which, in addition to being non-functional, cannot associate with Kv2.2 since Kv1 and Kv2 subunits cannot form heteropolymers (9, 31, 32). Again the same decrease of Kv2.2 current was observed with a Kv2.2/Kv1.5Delta mixture in a 1:50 ratio (Fig. 2A). To verify that Kv2.2 and Kv8.1 subunits interact in COS cells we created a truncated form of the Kv8.1 subunit, Kv8.1Delta , in which half of the S6 domain was deleted. This modified subunit, which conserves the NH2-terminal part that is required for interaction with the Kv2.2 subunit (1) but lacks a part of the S6 domain that is known to be essential for K+ channel expression (30), acts as a potent dominant-negative subunit since cells cotransfected with a mixture of the Kv2.2 and Kv8.1Delta plasmids (1:5 ratio) showed no detectable K+ current (Fig. 2A).


Fig. 2. Coexpression with Kv8.1 modifies activation and inactivation of Kv2.2 currents in COSm6 cells. Voltage-dependent currents, evoked by a step from -60 mV to +30 mV, were recorded. A, intensities of the Kv2.2 current in the presence of increasing amounts of Kv8.1-containing plasmids, while maintaining the Kv2.1 plasmid amount constant, reduces the average peak current. Co-transfection of Kv2.2 with other plasmids containing beta -gal or Kv1.5Delta also results in a decrease in peak current. Kv8.1Delta abolishes the Kv2.2 current. B, examples of K+ currents recorded at +30 mV. Note the slowly activating component and reduced current inactivation when Kv2.2 is coexpressed with Kv8.1 (5:1 molecular ratio of Kv8.1/Kv2.2 ). In this representation currents were scaled so that their peak currents are at the same level. C, voltage dependence of the steady-state activation. Steady-state inactivation was measured by recording the peak outward current (ordinate) in response to voltage steps from a holding potential of -60 mV to varying potentials (abscissa). D, the activation phases of K+ currents were fitted with Ae * exp(t/tau e) + Ah * (1 - exp(t/tau h)4. Co-transfection of Kv2.2 with Kv8.1 introduces a slow exponential component Ae. The ratio of Ae over Ah is shown as a function of membrane potential. E, co-transfection of Kv2.2 with Kv8.1 shifts the steady-state inactivation curve by 11 mV to the hyperpolarizing direction. Steady-state inactivation was measured by recording the peak current (ordinate) in response to a step from a 30-s conditioning prepulse and varying potential (abscissa) to +30 mV. F, an estimation of inactivation kinetics was made by taking the ratio of the current inactivated during a 10-s pulse to +30 mV over the peak current. Co-transfection of Kv2.2 with Kv8.1 reduces the inactivation ratio from 42 ± 5% to 16 ± 2% (p < 0.01%).
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When compared with control cells expressing Kv2.2 alone, COS cells in which Kv2.2 and Kv8.1 are coexpressed display new electrophysiological properties. A typical Kv2.2/Kv8.1 current trace is shown in Fig. 2B. The steady-state activation curve of the Kv8.1/Kv2.2 current has a midpoint potential of 13.1 mV and a slope parameter, k, of 17.1 mV. These values are similar to those of the Kv2.2 current (midpoint, 7.1 mV; slope, 17.9 mV) (Fig. 2C). However, activation kinetics are clearly modified. The time course of the delayed outward Kv2.2 current can be fitted with the Hodgkin-Huxley model (33) (Fig. 2D). The Kv2.2 currents modified by the coexpression with Kv8.1 cannot be adequately fitted with the same equation, and an additional exponential term is required. The ratio of the amplitude of this exponential component compared with the Hodgkin-Huxley component is shown as a function of the depolarization potential in Fig. 2D. Coexpression of Kv1.5Delta instead of Kv8.1 with Kv2.2 had no effect on the kinetics of the Kv2.2 current (not shown).

The inactivation of the K+ current expressed by Kv2.2 is drastically changed by the coexpression of Kv8.1 (Fig. 2, E and F). The midpoint potential for Kv2.2 inactivation (E0.5(inact)) is -37.1 mV, the slope parameter, k, is 13.0 mV, and 43% of the current is inactivated during a 10-s pulse to +30 mV. The inactivation of the Kv8.1/Kv2.2 current is less complete (16%) after the same pulse and displays a different voltage sensitivity (E0.5(inact) at -48.1 mV) together with a slightly less steep voltage dependence (k = 9.3 mV). Fig. 2F shows that these changes of inactivation are specific to the expression of Kv8.1 and are not observed upon coexpression with beta -galactosidase or Kv1.5Delta .

Kv8.1 Expression in Xenopus Oocytes Has a Dual Effect: Modification of Kv2 Currents at Low Levels of Expression and Total Inhibition of Kv2 and Kv3 Currents at Higher Concentrations

It has been shown previously that in Xenopus oocytes the coexpression in a 3:1 molecular ratio of Kv8.1 cRNA with Kv2.1, Kv2.2, or Kv3.4 transcripts leads to a total inhibition of K+ currents (1). To estimate the minimal amount of Kv8.1 required to produce an inhibitory effect, varying amounts of Kv8.1 cRNA were injected while maintaining levels of Kv2.1, Kv2.2, and Kv3.4 cRNA constant. Results are shown in Fig. 3A.


Fig. 3. Effect of Kv8.1 coexpression with Kv2.1, Kv2.2, and Kv3.4 subunits in Xenopus oocyte. A, influence of the molecular ratio of Kv8.1 cRNA/Kvx cRNA on K+ current inhibition (n = 10). Current amplitudes were measured at +30 mV, and 100% of currents corresponds to Kvx current amplitudes without Kv8.1. Kvx corresponds to Kv2.2 (bullet ), Kv2.1 (square ), and Kv3.4 (open circle ). B, C, and D, normalized mean current traces (n = 10) measured at +30 mV from oocytes co-injected with Kv8.1 cRNA and Kv3.4 cRNA (B, Kv3.4/Kv8.1, 1:1; C, Kv2.1 cRNA (Kv2.1/Kv8.1, 1:0.1); or D, Kv2.2 cRNA (Kv2.2/Kv8.1, 1:0.03)). E and F, voltage-dependent inactivation curves of Kv2.1, Kv2.2, Kv2.1 + Kv8.1 (1:0.03), and Kv2.2 + Kv8.1 (1:0.03) current. The duration of conditioning prepulses was 55 s, and test pulses were +30 mV.
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Residual currents remaining after a 50-70% reduction of Kv2.1, K2.2, and Kv3.4 currents by Kv8.1 are shown in Fig. 3, B, C, and D. Their biophysical characteristics are given in Table I. The kinetics of the residual Kv3.4/Kv8.1 current are not different from those of the control Kv3.4 current (same E0.5(act), E0.5(inact), % inactivation). However, Kv2/Kv8.1 currents show significant changes compared with original Kv2.1 or Kv2.2 currents. Midpoints of activation display modest Kv8.1-induced shifts for Kv2.1 (+1.4 mV) and Kv2.2 (-4.7 mV) respectively, but the activation time constants (tau act) are increased by approximately a factor of 1.5 for both currents in the presence of Kv8.1. The most spectacular effects are seen on inactivation. For both currents, the voltage dependence of the steady-state inactivation curves shows a -42 mV-shift (Fig. 3, E and F) associated with an important slowing down of the inactivation process. For example, during a 10-s depolarization to +30 mV, only 29% of the Kv2.1/Kv8.1 and 23% of the Kv2.2/Kv8.1 currents are inactivated, whereas under the same conditions Kv2.1 and Kv2.2 currents display ~60% inactivation (Table I).

Table I.

Parameters of activation and inactivation for Kv2.1, Kv2.2, and Kv3.4 expressed alone or coexpressed with the Kv8.1 subunit in Xenopus oocyte

Molecular ratios of injected plasmids are those indicated in Fig. 3. For Kv2.1/(NtH5Kv8)-Kv1.3 coinjection with an 0.1 ratio was used, corresponding to a 50% inhibition of Kv2.1 current. Activation parameters were elicited by 500-ms depolarizing pulses from -80 mV to +60 mV in 10-mV increments with a holding potential at -80 mV. The current (I) was recorded at the end of the pulse and plotted against membrane potential (Vm). The membrane conductance (G) was calculated for a given command voltage (Vm) and peak current responses (Ipeak) from the expression G = Ipeak/(Vm - EK), where EK is the K+ reversal potential. G was normalized versus a maximum (Gmax) and plotted against Vm. These plots were fitted to a Boltzmann distribution of the form, G/Gmax = 1/1 + exp {(E0.5(act) - Vm)/kact}. E0.5(act) is the midpoint of activation, and kact is the slope factor. tau act corresponds to the time constant of activation for a monoexponential fitting of the +40-mV activation curve. Inactivation parameters were determined by 500-ms step depolarizations at +30 mV preceded by 10-s prepulses from -120 mV to +30 mV. Current (I) was recorded at the end of 500-ms pulses, normalized by a maximum (Imax), and plotted against prepulse potential (Vm). This representation is the inactivation curve that can be fitted with a form of the Boltzmann equation, I/Imax = 1/1 + exp {(E0.5(inact) - Vm)/kinact}. E0.5(inact) is the midpoint of inactivation, and kinact is the slope factor. The percent of inactivated current was calculated from the ratio of measured currents at 10 s and at 0.1 s from pulses at +30 mV. Values are the mean ± S.E. for five oocytes measured.


Activation
Inactivation
 tau act E0.5(act) % inactivated current at 10 s E0.5(inact)

ms mV mV
Kv2.1 18.5  ± 2.8 8.8  ± 0.6 61  ± 2  -21.5  ± 0.8
Kv2.1 + Kv8.1 27.2  ± 2.5 10.2  ± 0.9 29  ± 1  -62.9  ± 0.8
Kv2.1 + (NtH5Kv8)-Kv1.3 23.4  ± 2.1 8.5  ± 0.9 70  ± 1  -19  ± 1.0
Kv2.2 32.2  ± 2.1 13.2  ± 0.5 60  ± 1  -13.5  ± 0.8
Kv2.2 + Kv8.1 45.4  ± 3.6 8.5  ± 1.4 23  ± 1  -55.9  ± 1.8
Kv3.4 NDa 27.5  ± 0.6 85  ± 3  -20.8  ± 0.8
Kv3.4 + Kv8.1 ND 25.5  ± 0.9 89  ± 3  -20.4  ± 1.2

a ND, not determined.

The Modification of the Inactivation Process of Kv2 Currents Is Mediated by the S6 Segment of Kv8.1

The slow inactivation process of other types of K+ channels appears to involve a constriction of the extracellular mouth of the channel (20-22, 34), and critical amino acids controlling this type of inactivation have been localized in the pore domain and in the S6 domain. A first experiment to test the possible implication of the S6 segment of Kv8.1 in the modification of the inactivation process of the Kv2 current was made by coexpressing the Kv2.1 subunit in oocytes with a chimeric protein (designated as (NtH5Kv8)-Kv1), which contains the Kv8.1 sequence from the NH2 terminus to the end of the pore domain followed by the S6 and COOH-terminal regions of the Kv1.3 subunit. This chimera which is inactive by itself is known to completely inhibit Kv2.1 current (1). A 50% inhibition of Kv2.1 currents occurred upon injection of the (NtH5Kv8)-Kv1/Kv2.1 mixture in a 0.1 ratio (data not shown). The steady-state activation and tau act of the remaining current are the same as those of the Kv2.1 current, and the properties of inactivation are only slightly modified as compared with Kv2.1 (Table I). Therefore the COOH-terminal end of Kv8.1 which contains the S6 segment seems to be important for the modifications of the kinetic properties of Kv2.1

The alignment of the sequence of the S6 segment of Kv8.1 with S6 segments of other functional Kv subunits is presented in Fig. 4A. Kv8.1 is atypical in 4 positions in this segment: (i) an alanine is localized at position 412 instead of a glycine in all other functional Kv subunits (M1, Fig. 4A); (ii) an isoleucine at 420 replaces a conserved valine residue (M2); (iii) an alanine is found at 428 instead of a proline (M3) in other functional Kv subunits; (iv) at position 433, an arginine is found instead of a very conserved asparagine (M4).


Fig. 4. Expression of mutated Kv2.1 subunits in Xenopus oocytes and mutated Kv8.1 subunits in COSm6. A, alignment of Kv S6 transmembrane segments. Identical amino acids are indicated by negative print, and homologous residues are shaded. Kv2.1M1 to Kv2.1M8 indicate the Kv8.1 residues introduced in the Kv2.1 subunit. B, current traces recorded 2 days after oocytes injection of indicated cRNAs. The holding potential is -80 mV and -120 mV for Kv2.1M1 and Kv2.1M7 mutations. Outward currents are evoked by +10-mV increments from holding potential to +30 mV. C, an estimation of inactivation kinetics of Kv2.2-generated K+ currents in COSm6 cells was made by taking the ratio of the current inactivated during a 10-s pulse to +30 mV over the peak current. Co-transfection of Kv2.2 with Kv8.1 decreases the percentage of inactivation from 42 ± 5% to 16 ± 2% (p < 0.01%). Introduction of the M3M7 mutations (A428P/I416C) in Kv8.1 (mutant Kv8.1M3M7) largely restores the inactivation properties of Kv2.2 (35 ± 4% of inactivation, p < 0.1%). The inset shows two representative current traces.
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To explore the role of these amino acids in K+ channel properties, the Kv2.1 subunit itself was mutated to introduce these single residues peculiar to the Kv8.1 sequence (mutants Kv2.1M1 to Kv2.1M4, Fig. 4, A and B). Four additional mutations were carried out corresponding to other amino acids specific to the Kv8.1 sequence, but not situated at necessarily conserved positions in other sequences. These mutations are L384T/L385T (Kv2.1M5, Fig. 4A), G391F/L392M (Kv2.1M6), C394I (Kv2.1M7), and I401L (Kv2.1M8).

Current traces recorded after expression of mutated Kv2.1 proteins in Xenopus oocytes are shown in Fig. 4B. Corresponding electrophysiological parameters are reported in Table II. All the mutations significantly alter current properties. Channel opening is most dramatically affected by mutation Kv2.1M3 (Pro replaced by Ala). This mutation produces a 30-fold decrease of the rate of activation (tau act 501.5 ms versus 17.7 ms for wild type Kv2.1) together with a +17.6 mV shift of the activation midpoint (+5.4 mV versus -10.5 mV for wild type). Other mutations induce smaller modifications of the activation process ranging from a 1.6-fold acceleration for Kv2.1M2 (tau act 10.9 ms) to a 1.5-fold decrease for Kv2.1M1 (tau act 26.3 ms). Changes in activation midpoints ranged from a -13.7 mV shift for Kv2.1M7 (E0.5(act) = -4.7 mV) to a +17.6 mV change for Kv2.1M3 (E0.5(act) = +26.8 mV).

Table II.

Current characteristics of native and mutated Kv2.1 subunits

Oocyte currents were analyzed as described in TABLE I. Activation parameters were elicited by 500-ms pulses (3 s for M3) from -80 mV to +60 mV in 10-mV increments with a holding potential at -80 mV (-120 mV for M1 and M7). E0.5(act) is the midpoint of activation, and kact is the slope factor. The time constants for activation (tau act) are determined at +40 mV. Inactivation parameters were determined by 500-ms step depolarizations from -80 mV to +30 mV preceded by 20-s prepulses from -80 mV (-120 mV for M1 and M7) to +30 mV (+70 mV for M3). E0.5(inact)) is the midpoint of inactivation, and kinact is the slope factor. The percent of inactivated current has been calculated for a pulse at +30 mV at 10 s after the peak current. Values are the mean ± S.D. for 6 to 11 oocytes measured.


Activation
Inactivation
 tau act E0.5(act) kact % inactivated current at 10 s E0.5(inact) kinact

ms mV mV mV mV
Kv2.1 17.7  ± 1.6 9.2  ± 1.2 11.3  ± 0.3 67  ± 7  -24.6  ± 2.4  -7.4  ± 0.9
Kv2.1M1 26.3  ± 8.2 13.6  ± 2.5 18.4  ± 1.0 41  ± 6  -58.5  ± 4.1  -13.4  ± 1.8
Kv2.1M2 10.9  ± 1.4  -2.4  ± 2.8 10.6  ± 0.8 19  ± 2  -40.5  ± 2.8  -7.9  ± 0.6
Kv2.1M3 501.5  ± 85.8 26.8  ± 2.0 12.1  ± 0.6 10  ± 3 +31.4  ± 7.2  -15.8  ± 0.7
Kv2.1M4 11.6  ± 1.1 14.3  ± 1.2 13.0  ± 0.7 57  ± 2 +1.0  ± 1.9  -13.9  ± 1.2
Kv2.1M5 15.2  ± 0.8 6.8  ± 0.9 11.4  ± 0.4 76  ± 6  -30.8  ± 2.0  -9.8  ± 1.7
Kv2.1M6 20.1  ± 1.4 12.6  ± 1.9 13.8  ± 1.5 14  ± 3  -37.0  ± 3.4 12.5  ± 1.0
Kv2.1M7 11.1  ± 1.2  -4.7  ± 5.3 20.0  ± 1.6 2  ± 1  -66.1  ± 4.4 13.3  ± 1.0
Kv2.1M8 12.8  ± 1.0 3.0  ± 5.3 9.8  ± 1.6 26  ± 4  -24.2  ± 2.7  -9.6  ± 2.3

Except for the Kv2.1M5 mutation, all mutations produced a reduction of the inactivation process of the Kv2.1 channel. The most spectacular modifications are observed for K+ currents produced by Kv2.1M3 (P406A) and Kv2.1M7 (C394I) mutants that inactivate only by 2-10% during a 10-s depolarizing pulse to +30 mV, while the control Kv2.1 current inactivates by 67% in the same conditions (Fig. 4B). Both mutants also show a considerable shift of the inactivation midpoint toward positive (Kv2.1M3, +56 mV shift) or negative potentials (Kv2.1M7, -42 mV shift).

Since the Kv2.1M3 (P406A) and Kv2.1M7 (C394I) mutations strongly altered the Kv2.1 current, the influence of these positions on the effect of Kv8.1 on Kv2 expression was also examined. Three mutated Kv8.1 subunits were prepared, Kv8.1M3 (A428P), Kv8.1M7 (I416C), Kv8.1M3M7 (A428P, I416C), and their effect on Kv2.2 current was tested in COS cells. None of these mutants produced a K+ current by itself. The Kv8.1M3 as well as the Kv8.1M7 mutants influenced the Kv2.2 current exactly as the wild type Kv8.1. Particularly, the effect of these Kv8.1 mutants on the inactivation of Kv2.2 currents was not significantly different from the effect of Kv8.1 itself on Kv2.2 current inactivation (Fig. 4C). The Kv8.1 subunit carrying both A428P and I416C mutations (Kv8.1M3M7) behaved differently. Activation kinetics for Kv8.1M3M7/Kv2.2 currents were intermediate between those of Kv2.2 and Kv8.1/Kv2.2 currents (not shown), the E0.5 of inactivation was similar to that of the Kv2.2 current (Kv2.2, -37.1 mV; Kv2.2/Kv8.1, -48.1 mV; Kv2.2/Kv8.1M3M7, -36.0 mV), and K+ current inactivation was more pronounced than for the Kv8.1/Kv2.2 current (Fig. 4C).


DISCUSSION

We recently reported the cloning of Kv8.1, a brain-expressed K+ channel alpha -subunit sharing only 40% identity with other subunits of the Kv channel family (1). Kv8.1 cannot by itself generate currents in Xenopus oocytes. However, Kv8.1 can specifically inhibit currents mediated by Kv2 (Kv2.1 and Kv2.2) and Kv3 (Kv3.4) subunits, and immunoprecipitation studies have shown that this is due to the formation of Kv8.1/Kv2 heteropolymers.

This paper first analyzes the properties of expression of Kv8.1 in the mammalian COS cell line. Again, no K+ current could be recorded from these cells when they expressed only Kv8.1. One important reason for this lack of expression is an inadequate subcellular localization of Kv8.1. Identification of the Kv8.1 protein with antibodies indicates that it remains in the endoplasmic reticulum and is apparently unable to reach the surface membrane (Fig. 1, A, B, and C). This is unlike other Kv subunits that generate K+ currents in these COS cells such as Kv1.5 and Kv2.2 which, as expected, are found to be localized at the plasma membrane (Fig. 1, D and E). Intracellular accumulation of Kv8.1 corresponds to an intrinsic property of this particular Kv channel subunit. However, Kv8.1 is transferred to the plasma membrane, if it is coexpressed with Kv2.2 (Fig. 1, F and G), a subunit which interacts with Kv8.1 (1), and which, when expressed alone, forms functional K+ channels at the surface membrane. Similar situations where a channel protein is not able by itself to reach the plasma membrane and requires the association with other subunits for proper movement to the surface are not unusual. One similar example concerns subunits of the amiloride-sensitive Na+ channel (35). Since no known endoplasmic reticulum localization sequence is found in the Kv8.1 subunit, it is probable that endoplasmic reticulum retention arises from protein misfolding and/or inefficient assembly. Association with the Kv2.2 subunit, which has access by itself to the plasma membrane, corrects the defect and facilitates Kv8.1 transport. The Kv2.2/Kv8.1 complex, once it has reached the surface membrane, forms a functional K+ channel with activation and inactivation properties that are different from those of the Kv2.2 channel.

Coexpression of Kv8.1 and Kv2.2 proteins in COS cells does not lead to a specific K+ current reduction as compared with expression of Kv2.2 alone. Only large Kv8.1/Kv2.2 plasmid ratios (50:1) lead to smaller K+ currents. However, the latter effects are not specific since they are also observed in Kv2.2-expressing cells transfected with a high beta -galactosidase/Kv2.2. The reduction of the K+ current magnitude is then due to a decrease of Kv2.2 expression due to competition between the two plasmids at the transfection, transcription, and/or translation levels. Conversely, in Xenopus oocytes, dramatic K+ current reductions eventually leading to complete inhibition of Kv2.1, Kv2.2, and Kv3.4 currents were observed upon injection of Kv8.1 with sufficiently high Kv8.1/Kv2.1, Kv8.1/Kv2.2, or Kv8.1/Kv3.4 cRNA ratios (see Fig. 5 in Ref. 1). In that case, inhibitions of K+ current correspond to a very specific process and are linked to the heteropolymeric interaction of the Kv8.1 subunit with Kv2 and Kv3 subunits (1). It is probable that, as in COS cells, Kv8.1 normally stays in intracellular compartments in Xenopus oocytes, and that when expressed at a high level Kv8.1 acts as a dominant trapping subunit that retains Kv2 and Kv3 subunits in intracellular membranes and prevents them from reaching the surface membrane.

The major effect of the coexpression of Kv8.1 with Kv2.2 in COS cells concerns the inactivation process. Its voltage-dependence is shifted toward negative potentials, and its rate of inactivation is considerably slowed down. Clearly then, the Kv2.2/Kv8.1 heteromultimer has kinetic properties different from Kv2.2 homomultimers. Interestingly enough, when low concentrations of Kv8.1 are expressed in Xenopus oocytes to produce only a partial inhibition of Kv2.1 or Kv2.2 channels, the remaining K+ channel activity is unlike the activity observed for Kv2.1 or Kv2.2 channels expressed in the absence of Kv8.1. The kinetics are drastically altered indicating that new types of channels are formed corresponding to Kv2.1/Kv8.1 or Kv2.2/Kv8.1 heteromultimers. The characteristics of these new channels are similar to those observed upon expression of Kv2.2 and Kv8.1 in COS cells. They have a changed voltage dependence of their inactivation (a shift as large as -41/-42 mV for both the Kv2.1/Kv8.1 and Kv2.2/Kv8.1 combinations), and they have a largely decreased rate of inactivation. Even if results obtained in oocytes and in COS cells are not absolutely identical, they are qualitatively and strikingly similar. The differences between the two systems, for example in the voltage shift of the inactivation process, might be due to factors such as a different lipid composition of the membrane, different associations with the cytoskeleton elements, different phosphorylation processes, etc. Altogether, these results indicate that when expressed in relatively low concentrations Kv8.1, even in Xenopus oocytes, can reach the surface membrane via its association with Kv2.1 or Kv2.2 and form there new types of channels with new kinetics properties. One reasonable possibility is that heterotetramers containing a dominant number of Kv8.1 subunits versus Kv2.1, Kv2.2, or Kv3.4 subunits are unable to reach the plasma membrane, whereas tetramers containing a dominant proportion of Kv2.1, Kv2.2, or Kv3.4 subunits reach the plasma membrane and are expressed there with new kinetic properties. It is known that a single subunit in a heterologous tetramer can impose new kinetics (36-38).

Several observations strongly suggest that the S6 segment in Kv8.1 is a key element in the Kv2 current modification. First, coexpression of Kv2.1 with a chimeric subunit in which the S6 COOH terminus domain of Kv8.1 has been replaced by the corresponding region of Kv1.3 ((NtH5Kv8)-Kv1 (Table I)) suppresses the induction of a slow inactivation rate. Moreover the shift of the voltage dependence of the steady-state inactivation observed for Kv8.1/Kv2.1 currents is absent for the (NtH5Kv8)-Kv1/Kv2.1 currents. The second indication comes from the fact that following coexpression of Kv3.4 with Kv8.1, the inactivation of the Kv3.4 current which is of the N-type ball and chain mechanism involving the NH2-terminal part of the protein (39), is not significantly modified. Since the S6 domain of Kv8.1 appears to play a central role in imposing new inactivation characteristics to the Kv2 current, identification of important amino acids for this phenomenon was achieved by a mutagenesis approach of Kv2.1. The strategy was to introduce singular Kv8.1 residues not present in the S6 region of other expressing Kv channels into the Kv2.1 S6 segment. This led to K+ currents with new characteristics. Particularly spectacular effects on inactivation were observed when Pro-406 was replaced by an alanine (Ala-428 in the Kv8.1 sequence, mutant Kv2.1M3) and Cys-394 was replaced by an isoleucine (Ile-416 in the Kv8.1 sequence, mutant Kv2.1M7). These 2 mutants of Kv2.1 generate nearly non-inactivating K+ channels. Clearly then, the S6 segment of Kv8.1 appears to contain structural elements, especially Ile-416 and Ala-428, that change the kinetics of inactivation of the Kv2 channels when it forms multimers with the Kv8.1 subunit. Finally, a last indication for the involvement of S6 residues is provided by mutations of the S6 segment of Kv8.1. A mutant of Kv8.1, in which Ile-416 and Ala-428 have been replaced by a cysteine and a proline, respectively (mutant Kv8.1M3M7), essentially loses its properties to modify the inactivation kinetics of Kv2.2.

The inactivation process of voltage-gated K+ channels is important for defining the shape and for the integration of electrical signals and can occur over a wide range of time scales. It is probable that in the regions of the mammalian brain that express Kv8.1 and Kv2 subunits at high levels (40), an interaction of Kv8.1 with Kv2 subunits will result in the generation of K+ currents similar to those recorded in both the Xenopus oocyte and COS cells. In fact, a current (IK(slow)) with properties similar to those of the Kv8.1/Kv2 assembly in COS cells has been described recently in CA3 pyramidal cells of rat hippocampus, where its role would be to control discharge onset after a period of membrane hyperpolarization (41).

The interesting question is whether one can observe in the functioning nervous system both the inactivation effect of Kv8.1, which occurs in COS cells and in Xenopus oocytes, and the total suppression of the activity of both Kv2 (Kv2.1 and Kv2.2) and Kv3 channels observed in Xenopus oocytes expressing large amounts of Kv8.1. If it were the case, adequate modulations of Kv8.1 expressions in the nervous system could produce drastic changes in the electrophysiological identity of neurons, i.e. slowing K+ channel inactivation in some and totally inhibiting K+ channels in others. One can even suspect that the two types of modulations could take place in a single neuron in different localizations (soma, dendrites, synapses). The effects described in this paper might be associated with drastic long term modifications of K+ channel expression, which would lead to drastic changes in the function of the neurons carrying the Kv8.1 channels together with its partners of the Kv2 and Kv3 families. Since the Kv8.1 subunit is abundantly expressed in the hippocampus, its capacity to produce long term changes in electrical signals might have a role in long term potentiation and memory processes.


FOOTNOTES

*   This work was supported by the CNRS, the Association Française contre les Myopathies (AFM), the Ministère de l'Enseignement Supérieur et de la Recherche under Contract MESR ACC SV9 9509 113, and the Commission of the European Communities under Contract CHRX-CT93-0167.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 33-04-93-95-77-00 or -02; Fax: 33-04-93-95-77-04; E-mail: ipmc{at}unice.fr.
1   The abbreviation used is: PBS, phosphate-buffered saline.
2   F. Lesage, unpublished data.

ACKNOWLEDGEMENTS

We are very grateful to Dr. Hwang and Dr. Li (The John Hopkins University, Baltimore) for the gift of Kv2.2 clones, to Dr. Joho (Baylor College of Medicine, Houston) for the gift of Kv2.1. We gratefully thank Y. Benhamou, D. Doume, C. Le Calvez, M. Jodar, and G. Jarretou for expert technical assistance and F. Lesage for the gift of polyclonal antibodies against Kv1.5.


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