(Received for publication, October 1, 1996, and in revised form, December 30, 1996)
From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
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.
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 and
(3).
A large number of genes encode the different
pore-forming subunits
(18 genes cloned in mammals (4)), and an increasing number of
-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
- and
-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 -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
-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
inact ~30 ms for a very fast inactivating channel,
such as (Kv1.4) (15), to
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
-subunits (20-22).
Although the Kv5.1 and Kv6.1 proteins apparently have the structural
hallmarks of functional K+ channel -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.
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 CellsVoltage-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 MicroscopyThe 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.
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 Kv8.1, 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 OocytesPreparation 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.
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).
To verify that the COS cells were able to correctly express well known
functional K+ channel -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 -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 CharacteristicsThe
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 -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 (Kv1.5
) (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/Kv1.5
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, Kv8.1
, 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 Kv8.1
plasmids (1:5
ratio) showed no detectable K+ current (Fig.
2A).
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 Kv1.5 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
-galactosidase or Kv1.5
.
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.
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 (
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).
|
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
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).
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 (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
(
act 10.9 ms) to a 1.5-fold decrease for Kv2.1M1
(
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).
|
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).
We recently reported the cloning of Kv8.1, a brain-expressed
K+ channel -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 -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.
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.