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The Journal of Neuroscience, December 1, 1998, 18(23):9585-9593
Heteromultimeric Potassium Channels Formed by Members of the Kv2
Subfamily
Judith T.
Blaine and
Angeles B.
Ribera
Department of Physiology and Biophysics, University of Colorado
Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
Four  -subunits are thought to coassemble and form a
voltage-dependent potassium (Kv) channel.
Kv -subunits belong to one of four major subfamilies
(Kv1, Kv2, Kv3, Kv4). Within a subfamily up to eight different genetic
isotypes exist (e.g., Kv1.1, Kv1.2). Different isotypes within the Kv1
or Kv3 subfamily coassemble. It is not known, however, whether the only
two members of the vertebrate Kv2 subfamily identified thus far, Kv2.1
and Kv2.2, heteromultimerize. This might account for the lack of
detection of heteromultimeric Kv2 channels in situ
despite the coexpression of Kv2.1 and Kv2.2 mRNAs within the same cell.
To probe whether Kv2 isotypes can form heteromultimers, we developed a
dominant-negative mutant Kv2.2 subunit to act as a molecular poison of
Kv2 subunit-containing channels. The dominant-negative Kv2.2 suppresses
formation of functional channels when it is coexpressed in oocytes with
either wild-type Kv2.2 or Kv2.1 subunits. These results indicate that Kv2.1 and Kv2.2 subunits are capable of heteromultimerization. Thus, in
native cells either Kv2.1 and Kv2.2 subunits are targeted at an early
stage to different biosynthetic compartments or heteromultimerization otherwise is inhibited.
Key words:
potassium channels; heteromultimers; coassembly; Kv2; dominant-negative mutant; Xenopus
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INTRODUCTION |
Voltage-dependent potassium
(Kv) channels play many crucial roles in neuronal
function. Functional Kv channels are believed to contain
four pore-forming -subunits (MacKinnon, 1991 ). The genes encoding
Kv -subunits are classified into four major subfamilies: Kv1, Kv2, Kv3, and Kv4 (Chandy, 1991). Each subfamily contains different genetic isotypes that may encode functionally distinct channels, augmenting potassium channel diversity.
Potassium channel heterogeneity is increased further by the possibility
of forming homo- as well as heteromultimeric channels. Isotypes within
the same subfamily form heterotetramers, indicating a barrier to
subunit coassembly across subfamilies (Covarrubias et al., 1991).
However, in a few cases, subunits encoded by members of different
subfamilies appear to coassemble (Shahidullah et al., 1995a,b; Chen et
al., 1996; Hugnot et al., 1996; Post et al., 1996; Patel et al., 1997;
Salinas et al., 1997a,b). Heteromultimerization of either Kv1 or Kv3
subfamily members has been demonstrated directly in the oocyte
(Christie et al., 1990; Isacoff et al., 1990; Ruppersberg et al., 1990;
Weiser et al., 1994), and Kv1 subfamily heteromultimers have been
detected immunohistochemically in situ (Sheng et al., 1993;
Wang et al., 1993; Veh et al., 1995). Furthermore, transcripts encoded
by different members of either the Kv2 or Kv4 subfamilies have been
detected within the same neurons (Hwang et al., 1992; Song et al.,
1998), allowing for possible heteromultimer formation. However,
immunocytochemical studies indicate that Kv2.1 and Kv2.2 subunits do
not colocalize, although in certain neurons their encoded proteins are
detectable within the same cell (Hwang et al., 1993).
Because immunocytochemical data raise the possibility that Kv2
subfamily members do not heteromultimerize, this study investigates directly whether Kv2.1 and Kv2.2 subunits can heteromultimerize with
each other. When coexpressed in the oocyte, wild-type Kv2.1 and Kv2.2
subunits induce currents that are functionally very similar (Burger and
Ribera, 1996) (Fig. 1). Thus,
heteromultimerization would not be revealed by unique functional
properties of a resultant channel, a strategy used successfully to
detect Kv1 and Kv3 heteromultimers (Christie et al., 1990; Isacoff et
al., 1990; Ruppersberg et al., 1990; Weiser et al., 1994). To probe
whether Kv2.1 and Kv2.2 subunits heteromultimerize, we developed a
mutant Kv2.2 subunit that serves as a molecular poison of Kv2.2
channels. Coexpression of mutant and wild-type Kv2.2 subunits leads to
a reduction in the currents induced, indicating that the inclusion of
the mutant subunit in a Kv2.2 tetramer eliminates function. Further,
coexpression of dominant-negative Kv2.2 and wild-type Kv2.1 subunits
also decreases functional Kv2 channel expression. These results
indicate that the mutant Kv2.2 subunit poisons Kv2.1- as well as
Kv2.2-containing channels, providing evidence that Kv2.1 and Kv2.2
subunits are capable of heteromultimerization. Thus, the failure to
detect Kv2.1/Kv2.2 heteromultimers in situ suggests the
existence of either modifications of native Kv2.1 and Kv2.2 subunits
that prevent their coassembly or neuronal sorting mechanisms that
target each subunit to distinct biosynthetic compartments.
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Figure 1.
Xenopus Kv2.1 and Kv2.2
transcripts induce the expression of functionally similar currents.
A, Oocytes were injected with 0.2 ng of wild-type Kv2.1
RNA (left), 1.8 ng of wild-type Kv2.2 RNA
(center), or 0.2 ng of Kv2.1 RNA plus 1.8 ng of Kv2.2
RNA (right). The injection of a mixture of wild-type
Kv2.1 and Kv2.2 transcripts induces larger currents than either Kv2.1
or Kv2.2 RNA injected alone. Currents were generated in response to 160 msec voltage steps to potentials ranging from  50 to +100 mV from a
holding potential of 80 mV; leak-subtracted currents are shown (see
Materials and Methods). Calibration: 1.9 µA, 55 msec.
B, The voltage-dependent properties of activation for
the currents induced by the expression of wild-type Kv2.1 alone
(n = 39) or a functionally equal amount of
wild-type Kv2.1 and Kv2.2 RNA (n = 34) are shown on
the left. Similar graphs for wild-type Kv2.2 currents
(n = 31) and those induced by a functionally equal
amount of wild-type Kv2.1 and Kv2.2 transcripts are shown on the
right. Symbols are mean values; error
bars indicate SD.
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MATERIALS AND METHODS |
RNA synthesis. The entire coding region of the
Xenopus Kv2.1 gene surrounded by the 5' and 3' untranslated
sequences of Xenopus  -globin [which confer stability and
allow efficient translation of in vitro transcribed mRNAs
(Liman et al., 1992)] was excised from the pGEMHEX9 construct (Burger
and Ribera, 1996) and cloned into the pALTER vector (Promega, Madison,
WI), creating the recombinant pA2.1 clone. The entire coding region of
the Xenopus Kv2.2 gene flanked by the 5' -globin
untranslated region (UTR) likewise was removed from the pGEMHEX12-2
construct (Burger and Ribera, 1996) and inserted into the pALTER vector
to generate the recombinant pA2.2 clone. Although pGEMHEX12-2 and pA2.2
lack the 3' -globin UTR, high expression levels are still obtained
from these clones (Burger and Ribera, 1996; this study). pA2.1 and
pA2.2 both were linearized with SphI. Capped, sense RNA was
transcribed in vitro by using T7 RNA polymerase in the
presence of ribonucleotide triphosphates (Pharmacia, Piscataway, NJ)
and cap analog (Boehringer Mannheim, Indianapolis, IN). RNA
concentrations and integrity were determined spectrophotometrically and
by electrophoresis in agarose-formaldehyde gels in combination with
ethidium bromide staining.
Site-directed mutagenesis. The tryptophan-to-phenylalanine
(W F) substitution in the pore region was achieved by using the pALTER in vitro mutagenesis system according to the
manufacturer's instructions (Promega). The tryptophan-to-cysteine
(WC) and tyrosine-to-threonine (YT) mutations were made with
PCR cassette mutagenesis in a procedure modified from Hollmann
et al. (1994), as follows. The generation of each mutation involved the
use of four primers in two sequential PCR reactions. Two of the primers
(Universal Forward, Universal Reverse) flank the pore region. Each
contains a unique native restriction site (SalI or
NcoI) used for the subsequent replacement of a PCR-generated
pore cassette into the Kv2.2 gene. The two other primers (Pore Sense,
Pore Antisense) recognize a sequence within the pore, are complimentary
to each other, and encode the desired mutation (WC or YT) as well
as an engineered silent restriction site to provide an initial means of
screening resultant clones. The first round of PCR involved two
separate reactions, using as primers either (1) Universal Forward and
Pore Antisense or (2) Universal Reverse and Pore Sense. Depending on
the mutant to be generated, the template in these reactions was either
wild-type Kv2.2 DNA (for the WC mutant) or WF Kv2.2 or WC
Kv2.2 DNA (for the WF-YT Kv2.2 and WC-YT Kv2.2 clones,
respectively). After gel purification of the desired round one
fragments, the second round of PCR used Universal Forward and Universal
Reverse as primers and the PCR products from the two round one
reactions as both template and primers. In the initial cycle of the
second round of PCR, the region of overlap between the two first round
fragments was extended. During subsequent cycles these extended
fragments were amplified by the Universal Forward and Reverse Primers.
The final PCR product was cut with SalI and NcoI,
gel-purified, and ligated into the pA2.2 construct that previously had
been digested with SalI and NcoI. Clones
identified as ones containing the desired mutations by restriction
enzyme analysis were sequenced to confirm that (1) the correct
substitutions had been made in the pore region, (2) no other mutations
were introduced into the cassette during PCR amplification, and (3)
introduction of the SalI-NcoI pore cassette
occurred without frame shifts. In addition, wild-type Kv2.2 and each
mutant clone was cut with 7-11 different restriction enzymes. The
restriction digest patterns were compared in each case to ensure that
unexpected alterations had not taken place elsewhere in each mutant.
RNA synthesis from the mutant clones was as described above for
wild-type Kv2.1 and Kv2.2 clones.
Oocyte injection and recording. Fifty nanoliters of solution
containing varying ratios of wild-type/mutant potassium channel RNA
(0.002-0.3 mg/ml) were injected into stage VI defolliculated Xenopus oocytes prepared as described previously (Burger and
Ribera, 1996). Oocytes were incubated at 18°C, and voltage-activated
potassium currents were recorded with standard two-electrode voltage
clamp procedures (Axoclamp 2A amplifier, Axon Instruments, Foster City, CA) 1-2 d after injection. Voltage protocols and data analysis were
accomplished with the pCLAMP suite of programs (Axon Instruments). Currents were sampled at 200 µsec and leak-subtracted by a P/4 protocol. The electrode solution consisted of 3 M KCl and
10 mM HEPES, pH 7.4, whereas the bath contained Barth's
solution [(in mM) 88 NaCl, 1 KCl, 0.33 Ca(NO3)2, 2.4 NaHCO3, 0.82 MgSO4, and 5 Na-HEPES, pH 7.4]. Electrode resistance ranged between 0.1 and 0.5 M . Oocytes were not used if the holding current exceeded 200 nA at
80 mV.
Data analysis. The binomial equation was used to calculate
the fraction (Fi) of channels of a
particular type i:
where FWT and FMUT
are the fractions of wild-type and mutant subunits. Discussion reviews
the assumptions made in using the binomial equation (MacKinnon,
1991).
 2 analysis (Bevington, 1969) was used to evaluate
statistically the number of WC-YT Kv2.2 mutant subunits required
for a block that gave the best fit to the data. A reduced form of the 2 statistic was used such that smaller 2
values indicate better fits and 2 < 1 indicates a fit
to the data within 1 SD. 2 analysis was performed only
for wild-type Kv2.2/WC-YT mixtures (i.e., for Table 3 and the
limited data of Table 4 referring to the Kv2.2 mixtures), because the
binomial equation cannot be used to predict the number of WC-YT
Kv2.2 subunits required to block Kv2.1 channels (wild-type
Xenopus Kv2.1 and Kv2.2 may have different single-channel
conductances). For the case of two WC-YT Kv2.2 mutant subunits
required for a block, 2 values were calculated without
taking the order of mutant subunits in the tetramer into account (i.e.,
a channel with two mutant subunits side by side was considered
equivalent to one with alternating wild-type and mutant subunits).
Consideration of the order of mutant subunits in the heterotetramer
gave a worse fit to the data, as demonstrated by the following. For two
adjacent WC-YT mutant Kv2.2 subunits required for a block, the
2 values are 1.34 and 5.53 for Tables 3 and 4,
respectively, whereas for alternating mutant subunits required for a
block, the 2 values are 4.0 and 10.81 for Tables 3 and
4, respectively. These compare with 2 values of 0.26 and
2.07 for Tables 3 and 4 in the case of two mutant subunits required for
a block regardless of order.
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RESULTS |
Creation of a dominant-negative mutant Kv2.2 subunit
The major goal of this study was to determine whether
heteromultimerization of different isotypes occurs within the Kv2
family. Accordingly, we constructed a dominant-negative Kv2 subunit
with the following essential properties: (1) homomultimeric channels assembled from mutant subunits are not functional (negative), and (2)
inclusion of less than three mutant subunits in a heterotetramer yields
a nonfunctional channel (dominant). Previous work has shown that
targeting the pore region is an effective strategy for generation of a
Kv1 dominant-negative subunit (Taglialatela et al., 1994; Ribera, 1996;
Ribera et al., 1996; Tinker et al., 1996). Mutations in the
Xenopus Kv2.2 channel therefore were confined to the pore region. An advantage of this approach is that the pore has not been
implicated as a region required for the coassembly of subunits.
The initial mutation made was a tryptophan-to-phenylalanine (WF)
substitution in the putative pore region of Kv2.2 (Table 1). The analogous alteration in fly Kv1
results in a nonconducting potassium channel that still undergoes
normal gating transitions (Perozo et al., 1993). In Xenopus
Kv1.1 the tryptophan-to-phenylalanine mutation gives rise to an
efficient dominant-negative subunit (Ribera, 1996). Introduction of the
WF mutation into Xenopus Kv2.2 (WF Kv2.2), however,
results in a subunit that, when injected into the oocyte, still induces
ionic current although the current amplitude is reduced as compared
with wild-type Kv2.2 expressed alone (Fig.
2C). Further, coinjection of
wild-type Kv2.2 and WF Kv2.2 subunits into the oocyte does not lead
to a reduction in current as compared with wild-type Kv2.2 expressed
alone (Fig. 3B), indicating
that the WF Kv2.2 mutant subunit is neither negative or
dominant.
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Figure 2.
Amino acid substitutions in the pore region create
nonfunctional Kv2.2 subunits. Currents are induced in the oocyte by the
injection of 6.25 ng of wild-type Kv2.2 RNA (A)
or 6.25 ng of each of the Kv2.2 pore mutants
(C-F). Homomultimeric expression of WC Kv2.2,
WF-YT Kv2.2, and WC-YT Kv2.2 transcripts does not result in
the formation of functional channels (D-F),
whereas injection of WF Kv2.2 RNA (C) induces
currents with reduced amplitude as compared with wild-type Kv2.2. The
injection of RNase-free water into the oocyte does not induce currents
(B). Currents were generated in response to 60 msec voltage steps to potentials ranging from 50 to +100 mV from a
holding potential of 80 mV; leak-subtracted currents are shown (see
Materials and Methods). Calibration: 3.5 µA, 20 msec.
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Figure 3.
Double mutations in the Kv2.2 pore region are
required for the construction of an efficient inhibitory subunit.
Currents were recorded from oocytes injected with wild-type Kv2.2
transcripts alone (A) or wild-type Kv2.2 RNA
mixed with each pore mutant in a 1:1 ratio
(B-E). The amount of wild-type Kv2.2 RNA in each
injection solution was kept constant. Coinjection of the WF Kv2.2
subunit caused no reduction in current amplitude as compared with
wild-type Kv2.2 currents (B); coexpression of
WC Kv2.2 and WF-YT Kv2.2 with wild-type Kv2.2 led to an
intermediate reduction in current amplitude (C,
D), and coinjection of WC-YT Kv2.2 transcripts
caused the greatest decrease in current size (E).
Currents were generated in response to 60 msec voltage steps to
potentials ranging from 50 to +100 mV from a holding potential of
80 mV; leak-subtracted currents are shown (see Materials and
Methods). Calibration: 3.5 µA, 20 msec.
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On the basis of the suggestion of Dr. Rolf Joho (Baylor College of
Medicine, Houston, Texas), we next mutated this tryptophan (Trp369) to a cysteine (Table 1), because this
alteration in rat Kv2.1 leads to the creation of a subunit that does
not form functional channels (Kurz et al., 1995). When expressed in the
oocyte, homomultimeric WC Kv2.2 mutant channels are also
nonfunctional (Table 1; Fig. 2D).
An initial assessment of the ability of the WC Kv2.2 mutant to act
as a dominant-negative subunit is provided by coinjection with
wild-type Kv2.2 RNA in a 1:1 ratio. We define the potency of a
dominant-negative -subunit as the number of -subunits in a
channel (i.e., 4) (MacKinnon, 1991; Liman et al., 1992) divided by the
number of mutant subunits required to render a heteromultimer nonfunctional. If one, two, three, or four mutant subunits are required, the potencies are 4, 2, 1.33, and 1, respectively. We desire
a potency of 2 or greater.
In our experiments the absolute amount of wild-type Kv2.2 RNA in each
injection solution is kept constant by dilution either with RNase-free
water or the appropriate mutant Kv2.2 RNA. A 1:1 mixture of wild-type
and mutant transcripts injected into the oocyte results in the
induction of smaller currents (Fig. 3C). Assuming that
potassium channels are tetrameric, coexpression of wild-type and mutant
subunits should lead to the formation of five channel classes:
wild-type and mutant homomultimers and heteromultimeric channels
containing one, two, or three mutant subunits. The binomial equation is
used to predict the percentage of channels expected in each class for
various ratios of wild-type/mutant RNA (MacKinnon, 1991) (see equation
in Materials and Methods). In the case of a 1:1 mixture of wild-type
and mutant subunits, if two mutant subunits suffice to render
heteromultimeric channels nonfunctional, 31.25% of the channels formed
will conduct ionic current. Further, oocytes injected with the 1:1
mixture contain twice as much RNA and presumably twice the number of
channels as those receiving wild-type transcripts alone. Accordingly,
the current amplitude recorded from oocytes injected with the 1:1 mixture of wild-type and WC Kv2.2 subunits should be 63% of that recorded from oocytes injected only with wild-type Kv2.2 mRNA. The
results of the mixing experiment (Table
2; Fig. 3) indicate, however, that more
than two WC Kv2.2 subunits are required to suppress function in the
heteromultimer. The WC Kv2.2 mutant thus has a potency of
<2.
Further mutagenesis directed at the creation of a dominant-negative
subunit was guided by comparison of the pore regions of the fly Kv1 and
Xenopus Kv2.2 channels. In the Drosophila Kv1 gene in which the original tryptophan-to-phenylalanine mutation was
made, the amino acid at position 449 is a threonine, whereas in
Xenopus Kv2.2 the corresponding residue is a tyrosine
(Tyr388). So that the Xenopus mutant
Kv2.2 clones would resemble more closely the fly Shaker H4
W-to-F mutant, Tyr388 was altered to threonine
(following the suggestion of Dr. Rod MacKinnon, Rockefeller
Institute, New York), generating the double mutants WF-YT Kv2.2
and WC-YT Kv2.2 (Table 1). When injected into the oocyte, neither
WF-YT Kv2.2 nor WC-YT Kv2.2 subunits induce the expression
of ionic currents (see Fig. 2E,F). They thus
qualify as negative subunits. To provide an initial measure of the
potency of each mutant clone, we coinjected wild-type Kv2.2 transcripts
and each double mutant in a 1:1 ratio. Under these conditions, both
WF-YT Kv2.2 and WC-YT Kv2.2 subunits cause a reduction in
current amplitude as compared with wild-type Kv2.2 RNA injected alone
(Fig. 3D,E). However, the WC-YT Kv2.2 mutant subunit
induces a greater reduction in functional expression (Table 2). This
experiment suggests that the potency of the WF-YT Kv2.2 mutant is
less than that of the WC-YT mutant.
The potency of the WC-YT Kv2.2 subunit is examined further by
coinjecting different ratios of wild-type Kv2.2/WC-YT mutant Kv2.2 transcripts into the oocyte. Adding increasing amounts of WC-YT Kv2.2 RNA to the injection solution leads to a progressive decrease in the current amplitude as compared with that recorded from
oocytes expressing wild-type Kv2.2 RNA alone (Fig.
4). Close to two mutant subunits render
heteromultimeric channels nonfunctional (Table
3), indicating that the negative
phenotype of the WC-YT Kv2.2 subunit is dominant and has a
potency of 2, as desired.
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Figure 4.
WC-YT Kv2.2 behaves as expected for a
dominant-negative subunit. Oocytes were injected with wild-type Kv2.2
RNA (A) or two different ratios of wild-type and
WC-YT mutant transcripts (B, C).
The amount of wild-type RNA in each injection solution was kept
constant. Increasing the ratio of mutant to wild-type RNA led to the
induction of smaller currents despite the increasing amount of total
RNA injected into the oocyte. Currents were generated in response to 60 msec voltage steps to potentials ranging from 50 to +100 mV from a
holding potential of 80 mV; leak-subtracted currents are shown (see
Materials and Methods). Calibration: 2.7 µA, 20 msec.
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The amplitude of the current expressed by oocytes injected with a
mixture of wild-type Kv2.2 and WC-YT mutant transcripts as
compared with that induced in oocytes containing wild-type Kv2.2 RNA
alone was measured at a single voltage. Therefore, changes in the
conductance-voltage relationships for currents recorded from oocytes
injected with wild-type Kv2.2 RNA versus those injected with wild-type
plus dominant-negative mutant transcripts could complicate
interpretation of the data. To ensure that the decrease in current
amplitude recorded from coinjected oocytes is not attributable to an
alteration in voltage-dependent properties of activation, we compared
the conductance-voltage relationship for wild-type currents and those
recorded from oocytes expressing a wild-type/mutant mixture. The
voltage-dependent properties of activation are similar for oocytes
injected with either wild-type or wild-type/WC-YT transcripts
(see Fig. 6C,D). This suggests that channels containing mutant Kv2.2 subunits do not demonstrate altered voltage-sensing properties.
Coexpression of wild-type Kv2.1 and dominant-negative
Kv2.2 subunits
If Kv2.1 and Kv2.2 subunits coassemble, inclusion of the
WC-YT mutant Kv2.2 subunit in Kv2.1-containing channels should result in a reduction in current as compared with that induced by
homomultimeric Kv2.1 channels. Thus, wild-type Kv2.1 and
dominant-negative Kv2.2 subunits were coexpressed in the oocyte.
Currents induced in the oocyte by injection of wild-type Kv2.1
transcripts are much larger, however, than those induced by an equal
amount of wild-type Kv2.2 RNA (data not shown). An appropriate normalization thus was required for examination of the formation of
Kv2.1 homomultimers, Kv2.2 homomultimers, and possible Kv2.1/Kv2.2 heteromultimeric channels. For these experiments, normalization was
achieved on the basis of functional expression rather than on the
amount of RNA injected. The amount of wild-type Kv2.1 injected into the
oocyte was an order of magnitude less than that of wild-type Kv2.2 or
the WC-YT Kv2.2 mutant so as to obtain equivalent functional expression of the two channel isotypes (Fig.
5A1,B1). The expectation of
equivalent functional expression was verified by normalizing the
current amplitudes obtained to both wild-type Kv2.1 alone and wild-type
Kv2.2 alone (Table 4). Coexpression of
wild-type Kv2.1 and dominant-negative mutant Kv2.2 transcripts in
either a 1:1 or a 1:4 ratio results in a reduction in current amplitude as compared with wild-type Kv2.1 or wild-type Kv2.2 RNA injected alone
(Table 4; Fig. 5C2,C3), implying the formation of
heteromultimeric channels in which the Kv2.2 mutant suppresses
wild-type Kv2.1 subunit function.
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Figure 5.
Kv2.1 and Kv2.2 subunits coassemble. Oocytes were
injected with wild-type Kv2.1 alone (A1), wild-type
Kv2.2 alone (B1), a functionally equal amount of
wild-type Kv2.1 and Kv2.2 RNA (A2), four times the
functional amount of wild-type Kv2.2 as Kv2.1 RNA (A3),
an equal amount of wild-type Kv2.2 and dominant-negative Kv2.2 RNA
(B2), four times the amount of dominant-negative Kv2.2
RNA as wild-type Kv2.2 transcripts (B3), a functionally
equal amount of wild-type Kv2.1 RNA and dominant-negative Kv2.2 RNA
(C2), or four times the functionally equivalent amount
of dominant-negative Kv2.2 transcripts as wild-type Kv2.2 RNA
(C3). The dominant-negative Kv2.2 subunit causes a
similar reduction in current amplitude when it is coexpressed with
either wild-type Kv2.1 or wild-type Kv2.2 subunits (compare
B2 and C2, B3 and
C3) despite the increased amount of total RNA injected
into the oocyte. Coexpression of wild-type Kv2.1 and Kv2.2 subunits
leads to the induction of larger currents (A2,
A3). In the diagram (C1), open
circles represent wild-type Kv2.1 subunits, and filled
circles represent dominant-negative Kv2.2 subunits. The
heteromultimeric Kv2.1/dominant-negative Kv2.2 channels represented in
brackets indicate two possible arrangements for a
heterotetramer containing two mutant subunits. Currents were generated
in response to 160 msec voltage steps to potentials ranging from 50
to +100 mV from a holding potential of 80 mV; leak-subtracted
currents are shown (see Materials and Methods). Calibration: 3.6 µA,
80 msec.
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Although the results are consistent with the dominant-negative Kv2.2
subunit poisoning Kv2.1 as well as Kv2.2-containing channels, other
explanations exist. For example, the decrease in current size recorded
from coinjected (wild-type/mutant) oocytes could be attributable to
saturation of the translation machinery of the oocyte with higher RNA
doses. However, larger currents are induced in cells expressing a
mixture of wild-type Kv2.1 and Kv2.2 transcripts as compared with those
recorded from oocytes expressing only a single RNA species (Table 4;
Fig. 5A2,A3). Alternatively, the conductance-voltage
relationships for wild-type Kv2.1 currents and those recorded from
oocytes expressing a 1:1 ratio of wild-type Kv2.1/WC-YT Kv2.2
subunits could be altered. However, these are similar (Fig.
6A,B). Thus, the
reduction in current size seen in coinjected oocytes is not
attributable to any difference in the voltage-dependent properties of
activation for wild-type Kv2.1 or wild-type Kv2.1/WC-YT Kv2.2
currents. Further, normalization was according to current amplitude and
not the number of channels; the single-channel conductances of
Xenopus Kv2.1 and Kv2.2 heteromultimers are not known and
might be different. However, similar results were obtained whether the
normalization was done for Kv2.1 or Kv2.2 current amplitudes (Table
4).
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Figure 6.
The conductance-voltage relations for wild-type
Kv2.1 or wild-type Kv2.2 subunits expressed alone or in combination
with WC-YT mutant or wild-type Kv2.1 and Kv2.2 subunits are
similar. Conductance-voltage relations were plotted for wild-type
Kv2.1 subunits alone or in combination with different ratios of
dominant-negative mutant Kv2.2 subunits (A,
B). Similar graphs were derived for wild-type Kv2.2
subunits injected into the oocyte alone or with different ratios of
WC-YT Kv2.2 mutant subunits (C, D).
The steady-state activation properties of the wild-type Kv2.1 or Kv2.2
current and the current remaining with the coexpression of either
wild-type Kv2.1 or wild-type Kv2.2 and different ratios of the mutant
Kv2.2 subunit are similar. This is the result expected if the subunit
acts in a dominant-negative manner or does not demonstrate altered
voltage-sensing properties. Symbols are mean values;
error bars indicate SD. The SD bars are large for wild-type/WC-YT
Kv2.2 mixtures (e.g., A, B). In these
cases the current amplitudes are small, and the endogenous currents of
the oocyte presumably contribute substantially to the overall current.
The numbers of oocytes range from 17 to 39.
|
|
The dominant-negative Kv2.2 subunit differs from wild-type Kv2.2 at
residues located in the pore region rather than regions implicated in
heteromultimerization. Thus, we conclude that Kv2.1 and Kv2.2 subunits
are able to form heteromultimeric channels.
| |
DISCUSSION |
The dominant-negative WC-YT Kv2.2 subunit, which differs
from wild-type Kv2.2 at only two pore residues, was used to probe Kv2.1/Kv2.2 subunit heteromultimerization. We established the potency
of the WC-YT subunit by coexpressing various ratios of wild-type
and dominant-negative mutant Kv2.2 transcripts in oocytes and measuring
the amplitudes of residual currents as compared with those induced by
wild-type Kv2.2 transcripts expressed alone. The results indicate that
Xenopus Kv2.1 and Kv2.2 subunits can form heteromultimeric
channels in a heterologous expression system.
The binomial equation was used to calculate the percentage of channels
in each class expected for a particular ratio of
wild-type/dominant-negative mutant RNA and thus the current amplitude
predicted for a mutant subunit of a given potency. Use of the binomial
distribution in this manner involves the following five assumptions:
(1) each channel consists of four -subunits and no auxiliary
subunits, (2) for each RNA species the number of subunits synthesized
and channels formed is linearly dependent on the amount of RNA
injected, (3) there is no preferential coassembly of wild-type and
mutant subunits into either homo- or heteromultimeric channels, (4)
when coassembled into heteromultimeric channels, mutant subunits either have no effect on conductance or suppress function completely, and (5)
the order of subunits in a heterotetrameric channel is not relevant
(see Fig. 5C1 and Materials and Methods). The first assumption has been tested in other studies (MacKinnon, 1991; Liman et
al., 1992). To maximize the validity of the second assumption, we kept
the final concentrations of all RNAs (0.002-0.3 mg/ml) in a range that
should not saturate the translation machinery of the oocyte. However,
even with these efforts the system showed nonlinearity (e.g., Table 4).
The third assumption is reasonable, given that all mutations were
targeted to the pore region and that assembly domains have been located
elsewhere (Li et al., 1992; Shen et al., 1993; Babila et al., 1994;
Hopkins et al., 1994; Lee et al., 1994; Shen and Pfaffinger, 1995). The
fourth assumption has been examined previously at the single-channel level and found to be valid for dominant-negative Kv1.1 subunits (Ribera et al., 1996).
Although the assumptions that have been made are reasonable, alternate
tests of the potency of the dominant-negative Kv2.2 subunit would be
useful and were sought. We attempted to engineer either a
pharmacological tag (sensitivity to Agitoxin2; Gross and MacKinnon, 1994) or a functional tag (fast inactivation; Lu and
Miller, 1995) into the mutant subunit. However, neither approach yielded a subunit that was either functional or distinguishable on the
basis of the desired engineered tag.
Several different mechanisms can account for the dominant-negative
phenotype of the WC-YT Kv2.2 mutant. For example, channels containing a WC-YT Kv2.2 subunit could be locked permanently in
an inactivated state, as is the case for the fly Kv1.1
tryptophan-to-phenylalanine mutant (Young et al., 1997). Alternatively,
conduction through the pore of WC-YT Kv2.2-containing channels
could be blocked, or the dominant-negative Kv2.2 mutant could prevent
wild-type Kv2.2 subunits from reaching the surface. Our data, however,
do not allow us to distinguish between these possibilities.
Although the structural basis of Kv2 subunit coassembly has not yet
been defined, studies that use Kv1 isotypes have implicated N-terminal
regions (Li et al., 1992; Shen et al., 1993; Babila et al., 1994;
Hopkins et al., 1994; Lee et al., 1994; Shen and Pfaffinger, 1995).
Similarly, Xu et al. (1995) have demonstrated an association between
the N-terminal portions of the fly Shab and rat Kv2.1 genes.
VanDongen et al. (1990), however, reported that a truncated rat Kv2.1
subunit, missing 139 amino acids from the N terminus and 318 amino
acids from the C terminus, forms homomultimers for which the functional
properties resemble those of the wild-type rat Kv2.1 channel. Thus, for
Kv2 channels, regions other than the N and C termini may contribute to
subunit coassembly. In addition, Kv2.1 subunits appear to coassemble
with members of the Kv6, Kv8, and Kv9 subfamilies (Hugnot et al., 1996;
Post et al., 1996; Patel et al., 1997; Salinas et al., 1997a,b) as well
as the Kv2.3r subunit (Castellano et al., 1997).
Our work showing that Xenopus Kv2.1 and Kv2.2 subunits form
heteromultimeric channels in the oocyte is intriguing, given that rat
Kv2.1 and Kv2.2 channels localize to distinct areas within a neuron
(Trimmer, 1991; Hwang et al., 1993; Maletic-Savatic et al., 1995;
Rhodes et al., 1995). The apparent discrepancy between the results
obtained in the two systems may be attributable to differences between
the Xenopus and rat Kv2 clones. However, Scannevin et al.
(1996) have characterized a domain within the rat Kv2.1 polypeptide
that is necessary for the correct localization of the Kv2.1 channel in
polarized Madin-Darby canine kidney (MDCK) cells, a cell line used to
model membrane protein targeting. In MDCK cells, rat Kv2.1 localizes to
the lateral cell membrane, which correlates with its somatodendritic
disposition in neurons. Although the region identified by Scannevin et
al. (1996) is overall only 46% identical between rat and
Xenopus Kv2.1 (157 amino acid identity over 341 residues),
there are 14 stretches within this region in which the percentage of
identity is 75% or greater over at least four consecutive residues.
Further, preliminary data suggest that localization of
Xenopus Kv2.1 in MDCK cells is similar to that of the rat
Kv2.1 channel (R. Scannevin, personal communication). These
results suggest that Xenopus Kv2.1 polypeptides also would be localized selectively in situ.
Taken together with our demonstration of Kv2.1/Kv2.2 coassembly in the
oocyte, the available evidence suggests that other mechanisms account
for the lack of detection of heteromultimeric Kv2 channels in
vivo. For example, Kv2.1/Kv2.2 heteromultimers might be present at
very low, and thus undetectable, levels in the neuronal membrane.
Another reason for the lack of in situ heteromultimer
detection might be restricted spatial and temporal expression of the
Kv2.1 and Kv2.2 channel subunits. Maletic-Savatic et al. (1995) have
found that, in the developing rat hippocampus, Kv2.1 polypeptides are
expressed first, followed 4 d later by Kv2.2. Kv2.1 and Kv2.2
channels also might interact with different cytoskeletal elements,
allowing for discrete potassium channel localization. In rat brain, for
instance, Kv2.1 polypeptides are found in tight clusters in the
neuronal membrane, whereas Kv2.2 proteins appear in a more diffuse
pattern (Trimmer et al., 1991; Hwang et al., 1993; Maletic-Savatic et
al., 1995; Rhodes et al., 1995). The localization domain identified by
Scannevin et al. (1996), which targets rat Kv2.1 to the lateral
membrane in MDCK cells, also appears sufficient to induce Kv2.1
clustering in this cell line, suggesting its requirement for this
distinctive localization pattern. Potential differential modifications
of Kv2.1 and Kv2.2 subunits in situ also might prevent
subunit coassembly between different members of the Kv2 subfamily. For
example, Patton et al. (1997) have shown that transcripts encoded by
the squid Kv2 channel are modified by RNA editing. Similarly,
post-transcriptional and/or post-translational modification of subunits
might influence the heteromultimerization of different Kv2 subunits.
What are the functional implications of the failure to detect
Kv2.1/Kv2.2 heteromultimers in situ? First, each channel
type could demonstrate distinctive, labile functional properties. In heterologous expression systems, both Kv2.1 and Kv2.2 homomeric channels generate sustained delayed rectifier-type currents that appear
functionally similar (Hwang et al., 1992; Shi et al., 1994; Burger and
Ribera, 1996; this study). Further, under the conditions in which
Kv2.1/Kv2.2 heteromultimers form, only minor functional differences are
noted between the homo- and heteromultimeric Kv2 channels (see, for
example, Fig. 1). Accordingly, the expression of only homomultimeric
functionally similar channel complexes in vivo may allow for
the subunit-specific modulation of Kv2.1 versus Kv2.2 channels by
either additional subunits or enzymes capable of interaction with one,
but not the other, homotetramer. In the Xenopus Kv2
subfamily, for instance, Kv2.2 has a unique cAMP phosphorylation site
at amino acid 14 and a unique tyrosine phosphorylation site at amino
acid 512. Second, inclusion of Kv2.2 subunits in Kv2.1-containing
complexes might disrupt the characteristic clustering of Kv2.1 channels
seen in situ (Trimmer et al., 1991; Hwang et al., 1993;
Maletic-Savatic et al., 1995; Rhodes et al., 1995). These Kv2.1 channel
clusters might be crucial for correct neuronal function.
Future work will elucidate both the domains required for the coassembly
of subunits encoded by members of the Kv2 subfamily and the mechanisms
involved in the segregated localization of Kv2 polypeptides. These
studies will allow for a greater understanding of the mechanisms that
allow voltage-gated potassium channels to play multiple roles in the
acquisition and maintenance of electrical excitability in specific
neuronal compartments.
| |
FOOTNOTES |
Received June 29, 1998; revised Sept. 3, 1998; accepted Sept. 10, 1998.
This work was supported by National Institutes of Health Grant NIH
5T32NS07083, National Research Service Award Fellowship 5 F30
MH11349-03 to J.T.B., and National Institutes of Health Grant
RO1-NS25217 to A.B.R. We thank Drs. R. Joho and R. MacKinnon for their
suggestions regarding mutagenesis, Dr. J. Karpen for assistance with
statistical analysis, and an anonymous reviewer for insightful comments
on this manuscript. We also thank H. Chouinard, A. Hofmann, Dr. M. Lazaroff, A. Linares, Dr. T. Nick, Dr. R. Scannevin, and Dr. J. Trimmer
for helpful discussions.
Correspondence should be addressed to Judith T. Blaine, Department of
Physiology and Biophysics, C-240, University of Colorado Health
Sciences Center, Denver, CO 80262.
| |
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Top
Abstract
Introduction
