(Received for publication, November 13, 1995; and in revised form, January 8, 1996)
From the
To begin to study the molecular bases that determine the
selective interaction of the -subunits of voltage-gated
K
channels with
-subunits observed in
situ, we have expressed these polypeptides in transfected
mammalian cells. Analysis of the specificity of
/
-subunit
interaction indicates that both the Kv
1 and Kv
2
-subunits display robust and selective interaction with the five
members of the Shaker-related (Kv1)
-subunit subfamily
tested. The interaction of these
-subunits with Kv1
-subunits
does not require the
-subunit N-terminal domains. Thus, the
previously observed failure of N-terminal mutants of Kv
1 to
modulate inactivation kinetics of Kv1 family members is not simply due
to a lack of subunit interaction. Interaction of these
-subunits
with members of two other subfamilies (Shab- and Shaw-related) could not be detected. Somewhat surprisingly, a
member of the Shal-related subfamily was found to interact
with
-subunits; however, this interaction had biochemical
characteristics distinct from the
-subunit interaction with Kv1
family members. In all cases, Kv
1 and Kv
2 exhibited
indistinguishable
-subunit selectivity. These studies point to a
selective interaction between K
channel
- and
-subunits mediated through conserved domains in the respective
subunits.
Voltage-dependent K channels are fundamental
and diverse components of neuronal activity. Molecular cloning studies
have identified over a dozen distinct K
channel genes
and shown that the encoded pore-forming
-subunits are members of a
large, multigene superfamily that includes Na
and
Ca
channel
-subunits(1) . Although
expression of these individual
-subunits alone is sufficient to
generate voltage-gated channels exhibiting many features of the
corresponding channels in situ, studies on native
Na
and Ca
channels in neurons and
other excitable cells have confirmed the existence of auxiliary
polypeptides in tight association with
-subunits(2) .
Cloning of these auxiliary subunits and their subsequent co-expression
with
-subunits has shown that the expression level, gating, and
conductance properties of expressed channels are profoundly influenced
by the presence of auxiliary subunits(2) .
Recently, it has
been discovered that K channels also have auxiliary
(
) subunits. A cDNA encoding a
-subunit copurifying with the
bovine brain DTX acceptor complex was recently isolated(3) .
Subsequently, cDNAs encoding three highly related yet distinct
-subunit isoforms were isolated from rat brain (Kv
1 and
Kv
2, (4) ) and from ferret (Kv
3, (5) ) and
human (hKv
3, Refs. 6 and 7) heart. Although dissimilar in their
primary structures,
-subunits of K
and
Ca
channels exhibit general structural similarity in
that they are basic (pI
9.5), hydrophilic, and presumably
peripheral membrane proteins present at the cytoplasmic face of the
plasma membrane (2) .
Co-expression of Kv1 was found to
greatly accelerate the rate of inactivation of K
currents expressed from the Kv1.1 or Kv1.4
-subunit cDNAs in Xenopus oocytes(4) . Kv
3, which is an
alternatively spliced product of the Kv
1 gene, accelerates the
rate of inactivation of K
currents expressed from
Kv1.4 or Kv1.5 but not from Kv1.1, Kv1.2, or Kv2.1
cDNAs(5, 6, 7) . These results suggest that
-subunit modulation of
-subunit gating can contribute
additional functional diversity to K
channels in
excitable cells. Surprisingly, co-expression of the highly related
Kv
2 had no effect on inactivation, apparently due to the lack of
the N-terminal ``ball'' domain present in Kv
1 that is
both necessary and sufficient for the observed modulation of
inactivation(4) . However, from the published
electrophysiological analysis of
/
-subunit interaction
presented, it was also possible that the lack of observed Kv
2
effects was simply due to a lack of Kv
2 interaction with the
co-expressed
-subunits.
We previously used an antibody raised
against the C terminus of the bovine -subunit, predicted to
recognize both Kv
1 and Kv
2 in rat brain, to investigate the
expression of these
-subunits in situ(8) . A
major 38-kDa polypeptide and a minor 41-kDa polypeptide were detected
in rat brain membrane fractions by immunoblot analysis. These two bands
correspond closely to the predicted sizes of Kv
2 and Kv
1,
respectively. Immunoprecipitation experiments showed that the major
38-kDa polypeptide is associated and colocalizes with Kv1.2 and Kv1.4,
but not Kv2.1, in rat brain(8) , suggesting the selective
interaction of K
channel
- and
-subunits.
As a first step toward understanding the molecular mechanisms that
determine subunit composition of K channels, rat
Kv
1 and Kv
2 were transfected either alone or together with
K
channel
-subunits into mammalian cells lacking
these proteins. Kv
1 and Kv
2 exhibited selective interactions
with Shaker- and Shal-related
-subunits but did
not interact with Shab- and Shaw-related
-subunits. The interaction with
-subunits did not require the
N-terminal domain necessary for the previously observed effects of some
-subunits on
-subunit inactivation.
As a
result of this cDNA screening, we also obtained several Kv2
clones. However, none of these clones contained full coding sequences.
We again employed reverse transcriptase-PCR using the following primer
set (5`-primer, B2-5 5`-CTGATCTAGATAAGTGAGGC-3`; 3`-primer,
B2-3 5`-CTATCGATGACTTAGGATCTATAGTCC-3`), flanking the entire
1101-bp coding region of Kv
2, to obtain the coding region of
Kv
2. After 25 rounds of PCR, a specific amplification product of
the predicted size was obtained and subcloned into pRBG4 at the XbaI and ClaI sites. Eight cDNAs corresponding to the
predicted size were identified by restriction mapping. One of these
clones was analyzed by sequencing and was used as Kv
2/RBG4.
A 1.7-kbp EcoRI fragment of pKB16, which contains the
entire coding region, was isolated and ligated into pRBG4 to generate
Kv1/RBG4. Expression vectors containing
-subunit cDNAs were
constructed by cloning the respective coding regions into pRBG4 as
follows. Kv1.1/RBG4 was generated by digesting Kv1.1/pS- (9) with PstI and HindIII, followed by
ligation with PstI/HindIII-digested pRBG4. To
generate Kv1.2 (rat RAK)/RBG4, a BglII fragment containing a
coding region was isolated from rat RAK/pSP64T (15) and cloned
into Bluescript SK
BglII, a vector in the EcoRI site was changed to BglII, (
)which
was then digested with XbaI and HindIII, and the
fragment was cloned into pRBG4. Kv1.3/RBG4 was constructed by digesting
D541/pGemA (10) with EcoRI, followed by ligation into EcoRI-digested pRBG4. Kv1.5/RBG4 was generated by digesting
clone D469 (10) with BstEII, followed by blunting with
Klenow and ligation into EcoRV-digested pRBG4. Kv1.6/RBG4 was
generated by digesting Kv2/pGEMA (10) with NotI,
followed by blunting with Klenow, digestion with EcoRI, and
ligation with EcoRV/EcoRI-digested pRBG4. The
construction of Kv2.1/RBG4 was described previously(19) .
Kv4.2/RBG4 was generated by digesting ratShal1/SK- with HincII and ligating the resultant fragment into EcoRV-digested pRBG4. The mammalian expression plasmid for
Kv3.1 (in pRc/CMV) was obtained from Dr. T. Perney (Rutgers
University).
Figure 1:
Immunoblot analysis of
-subunits in rat brain membranes and in transfected COS-1 cells.
Crude rat brain membranes (75 µg, lane 1) and the
detergent extracts of COS-1 cells transfected with Kv
1/RBG4 (lane 2), Kv
2/RBG4 (lane 3), or mock-transfected
cells (lane 4) were fractionated on a 9% SDS gel, transferred
to nitrocellulose, and the resultant immunoblot probed with anti-
subunit antibody. Signals were visualized by autofluorography using
ECL. Numbers on right refer to mobility of prestained
molecular weight standards.
Figure 2:
Association of Kv1 and Kv
2 with
Kv1.2 in co-expressing COS-1 cells. Kv1.2 and
-subunits were
expressed either alone or co-expressed with one another. Cells were
labeled with [
S]methionine for 2 h, harvested in
lysis buffer, and the lysates were subjected to immunoprecipitation
with anti-Kv1.2 (``
'' lanes) or anti-
(``
'' lanes) antibody. The combinations of
Kv1.2 and
-subunit cDNAs are shown above the lanes. Numbers on left refer to mobility of
prestained molecular weight standards.
Addition of a denaturing
agent, such as the detergents SDS and deoxycholate, should affect
polypeptide folding and disrupt the noncovalent protein-protein
interactions typical of most multi-subunit membrane protein
complexes(20) . To test if K channel subunit
association was through similar noncovalent interactions,
immunoprecipitation reactions were performed in the presence of such
denaturing agents. The coprecipitation of Kv1.2 with Kv
2 could be
disrupted by the addition of 0.2% SDS and 0.5% sodium deoxycholate
during the immunoprecipitation reactions; this treatment has no effect
on the direct immunoprecipitation of the subunits themselves (Fig. 3A). Similar results were obtained for
Kv1.2-Kv
1 interaction (not shown). Thus, K
channel
/
-subunit interaction has similar sensitivity
to denaturing detergents as exhibited for other multisubunit membrane
protein complexes(20) .
Figure 3:
A, effect of SDS addition to
immunoprecipitation reactions. Cells were transfected with Kv1.2 and
Kv2. Cells were labeled with [
S]methionine
for 2 h, harvested in lysis buffer, and the lysates were subjected to
immunoprecipitation with anti-Kv1.2 (``
'' lanes) or anti-
(``
'' lanes)
antibody or without antibody (``-'' lanes).
Immunoprecipitation reactions were carried out under the presence (right panel) or absence (left panel) of 0.2% SDS and
0.5% sodium deoxycholate. Numbers on left refer to
mobility of prestained molecular weight standards. B,
immunoprecipitation from the mixed-cell lysate of individually
transfected dishes of cells. COS-1 cells individually transfected with
Kv1.2, Kv
1, or Kv
2 were mixed and lysed, and the lysates were
subjected to immunoprecipitation. The combinations of the singly
transfected cells used for immunoprecipitation are indicated at the top of the lanes labeled ``mix''. Lanes labeled ``co'' show the results
obtained from cotransfected cells expressing the same
/
-subunit combination. The immunoprecipitation reactions were
performed with anti-Kv1.2 (``
'' lanes) or
anti-
(``
'' lanes) or without antibody
(``-'' lanes). Numbers on left refer
to mobility of prestained molecular weight
standards.
To test whether co-expression within
the same cell is necessary for subunit interaction, individually
transfected dishes of COS-1 cells expressing either Kv1.2 or Kv2
were harvested. The cells were then pooled, and the pooled mixture of
cells was extracted under standard conditions. The resultant lysates
were then subjected to immunoprecipitation with subunit-specific
antibodies. These experiments yielded no co-immunoprecipitation of
- and
-subunits above background (no antibody lanes), showing
that co-expression within the same cell is necessary for subunit
interaction (Fig. 3B).
Previous studies had shown
that deletion of the N terminus of Kv1 destroyed its ability to
modulate inactivation(4) . To test whether this was simply due
to a lack of interaction, an N-terminal truncation mutant,
Kv
1
N70, which lacks amino acids 1-70, was co-expressed
with Kv1.2. As shown in Fig. 4A, Kv
1
N70 can
be efficiently co-immunoprecipitated with anti-Kv1.2 antibody and vice-versa. Thus, removal of the domain necessary for
Kv
1-mediated modulation of inactivation does not disrupt
/
-subunit interaction, showing that the loss of the ability
of such mutants to modulate inactivation is not due to an inability to
interact with
-subunits. A similar N-terminal deletion of Kv
2
(Kv
2
N22) also exhibited interaction with Kv1.2 that was
indistinguishable from wild-type Kv
2 (Fig. 4B).
These data indicate that the N-terminal domains of
-subunits are
not necessary for the interaction with
-subunits and that the
interaction domain lies somewhere else in the
-subunit sequence.
Figure 4:
Association of N-terminal truncation
mutants of Kv1 and Kv
2 with Kv1.2. A, Kv1.2 and
Kv
1
N70 were co-expressed in COS-1 cells, labeled with
[
S]methionine for 2 h, and the lysate was
examined by immunoprecipitation with anti-
(``
'' lane) or anti-Kv1.2 (``
'' lane)
antibody. B, Kv1.2 and Kv
2
N22 were co-expressed in
COS-1 cells, labeled with [
S]methionine for 2 h,
and the lysate was examined by immunoprecipitation with anti-
(``
'' lane) or anti-Kv1.2 (``
'' lane) antibody. Numbers on left refer to
mobility of prestained molecular weight
standards.
Figure 5:
Association of Kv1 and Kv
2 with Shaker-related
-subunits. Interactions between various
- and
-subunits were examined by immunoprecipitation. Panels show the results of co-immunoprecipitation reactions
obtained from COS-1 cells co-expressing Kv
1 and Kv
2 with
Kv1.1, Kv1.3, Kv1.5, or Kv1.6
-subunits. The combinations of
- and
-subunit cDNAs are shown above the lanes. Cells were labeled with
[
S]methionine for 2 h, and the lysates were
examined by immunoprecipitation with anti-
(``
'' lanes) or antibody specific to the
-subunit shown above the each panel (``
'' lanes). Numbers on left refer to mobility of
prestained molecular weight standards.
Figure 6:
Association of Kv1 and Kv
2 with Shab- and Shaw-related
-subunits. Panels show the results of co-immunoprecipitation reactions obtained from
COS-1 cells co-expressing Kv
1 and Kv
2 with Shab-related (Kv2.1, panel A) or Shaw-related (Kv3.1, panel C)
-subunits. In panel B, lysates from COS-1 cells transfected with Kv2.1 and
Kv
2 were subjected to immunoprecipitation under the presence (right panel) or absence (left panel) of 0.2% SDS and
0.5% sodium deoxycholate. Cells were labeled with
[
S]methionine for 2 h, and the lysates were
examined by immunoprecipitation with anti-
(``
'' lanes), anti-Kv2.1 (pGEX-drk1, ``
'' lanes in A and B), or anti-Kv3.1 (``
'' lanes in C). Numbers on left refer
to mobility of prestained molecular weight
standards.
Figure 7:
A, association of Kv1 and Kv
2
with Shal-related
-subunit. Kv4.2 and Kv
1
(+
1) or Kv
2 (+
2) were co-expressed in COS-1
cells. Cells were labeled with [
S]methionine for
2 h, and the lysate was examined by immunoprecipitation with anti-
(``
'' lanes) or anti-Kv4.2 (``
'' lanes) antibody. Numbers on left refer to
mobility of prestained molecular weight standards. B, effect
of SDS addition on the co-immunoprecipitation of Kv1.2/Kv
2 and
Kv4.2/Kv
2. COS-1 cells were transfected with Kv1.2 and Kv
2 or
Kv4.2 and Kv
2 and labeled with
[
S]methionine for 2 h. The immunoprecipitation
reactions were carried out under the presence of various concentrations
of SDS indicated above the lanes. Anti-
antibody
was used for precipitation. Numbers on left refer to
mobility of prestained molecular weight
standards.
Our previous study using an antibody against a sequence
conserved in both Kv1 and Kv
2 revealed the existence of
multiple immunoreactive
-subunits in rat brain(8) . Here,
analysis of transfected cells expressing recombinant Kv
2 and
Kv
1 reveals that a minor 44-kDa rat brain
-subunit comigrates
with Kv
1, while the major 38-kDa
-subunit comigrates with
Kv
2. The other immunoreactive
-subunit at 41 kDa, which is
recognized by the
-subunit antibody, is apparently neither
Kv
1 nor Kv
2 and suggests the existence of an additional, as
yet uncharacterized member of the
-subunit gene family in rat
brain. Recent cloning of a partial cDNA for a rat Kv
3
-subunit, which shares the same nucleotide sequence with Kv
1
except for its unique N-terminal region and is predicted to encode a
polypeptide of 45 kDa, strongly suggests the presence of at least one
alternatively spliced product of the Kv
1 gene(5) . Studies
with subtype-specific antibodies will allow for the eventual
unequivocal identification and localization of each of the individual
components of the
-subunit pool in brain. Moreover, comprehensive
molecular analysis of the
-subunit gene family will lead to the
identification of other
-subunits, for instance, those associated
with Shab- (see (12) ), Shaw-, and Shal- (21) related K
channels.
We
found that Kv1 and Kv
2 expressed in COS-1 cells could
associate with all five of the Shaker-related subfamily
members tested (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6), as well as with the Shal-related Kv4.2. The ratio of the amount of
co-immunoprecipitation seen on anti-
and anti-
lanes
exhibited some variation among the different
/
pairwise
combinations. This discrepancy in the extent of reciprocity of
co-immunoprecipitation could be due to the relatively low efficiency of
immunoprecipitation with anti-
antibody due to the high expression
level of
-subunit in the cotransfected cells. Kv2.1 and Kv3.1
exhibited no detectable co-immunoprecipitation, suggesting these two
-subunits are unable to interact with Kv
1 and Kv
2.
Subcellular localization of Kv2.1 and
-subunits in transfected
cells is consistent with this model in that immunofluorescence staining
of cells co-expressing Kv2.1 and Kv
2 shows no overlap of
-
and
-subunit staining, while cells co-expressing Kv1.2 and
Kv
2 with Kv
2 show extensive overlap throughout the cells. (
)
Our results provide direct biochemical evidence for
selective interaction of K channel
-subunits with
only a subset of the
-subunit gene family, have greatly expanded
the initial observations of Rettig et al.(4) who
showed that Kv1.1 and Kv1.4 interact functionally with Kv
1 in
oocytes(4) , and provide the evidence for a direct, noncovalent
interaction between
- and
-subunits. These results also
confirm and extend our previous studies of rat brain
/
-subunit association in situ, where we found that
neuronal
-subunits could be coprecipitated with rat brain Kv1.2
and Kv1.4 but not with Kv2.1(8) . A detailed characterization
of purified bovine brain dendrotoxin acceptors, which were later found
to contain Kv
1 and Kv
2(3) , showed that these
K
channel complexes contain Kv1.1, Kv1.2, Kv1.4, and
Kv1.6 (22) . Our findings provide a first step toward
understanding the molecular determinants of
/
-subunit
interaction by showing that the subunit selectivity observed in rat
brain can be recapitulated in transfected cell lines, indicating that
selectivity is mainly determined by the primary structure of the
interacting subunits.
The voltage-gated K channel
-subunit genes segregate into four subfamilies based on the
similarity of primary structure of each member(23) . As
discussed above, our results show that Kv
1 and Kv
2
interaction seemed to be restricted to Shaker- and Shal-related subfamilies. Interestingly, proposed phylogenetic
trees place the Shaker (Kv1) and Shal (Kv4)
subfamilies on one major branch, while Shab (Kv2) and Shaw (Kv3) members are placed on a separate
branch(1, 24) . Thus, the ability to interact with
Kv
1 and Kv
2 appears to reside in the relatedness of their
primary sequences as evidenced by their phylogenetic grouping and
allows for the design of structure-function analyses aimed at defining
the domains of
-subunits mediating
/
-subunit
interaction. In the case of voltage-sensitive Ca
channel
/
-subunit interaction, the
-subunit binds
to a conserved cytoplasmic motif in the
-subunit(25) . Taken together with the fact
that K
channel
-subunits are also cytoplasmic
proteins, it is likely that the interaction domain on K
channel
-subunits is present on a cytoplasmic domain.
No
distinct domain of -subunits stands out as a clear candidate for
mediating interaction with
-subunits based simply on the positive
interaction of both Kv1 and Kv4 family members. However, our
experiments using SDS treatment to disrupt
/
-subunit
interaction imply that the interaction of
-subunits with Kv1.2 and
Kv4.2 are somewhat distinct. In addition, only Kv1 and not Kv2, Kv3, or
Kv4 subfamily
-subunits have been found associated with Kv
1
and Kv
2 in rat brain in situ(8) . (
)Together, these data may imply that the only
physiologically relevant subunit interactions are between Kv1 (Shaker-related)
-subunits and Kv
1 and Kv
2.
Using this assumption, a conserved N-terminal, presumably cytoplasmic
domain of about 130 amino acids is striking in that it is highly
conserved among Kv1
-subunits but not among members of the other
(Kv2, Kv3, and Kv4) subfamily members. This highly conserved region,
known as the ``T1'' (26) or ``NAB'' (27) domain, is thought to be important in mediating efficient
/
-subunit interaction(26, 27, 28) .
This may raise the interesting scenario whereby both
/
- and
/
-subunit interactions are mediated through similar domains.
Extensive mutational analysis of
-subunit proteins will lead to
the elucidation of the specific
-subunit binding region.