(Received for publication, May 11, 1995; and in revised form, July 21, 1995)
From the
Voltage-gated potassium (K) channels are
assembled by four identical or homologous
-subunits to form a
tetrameric complex with a central conduction pore for potassium ions.
Most of the cloned genes for the
-subunits are classified into
four subfamilies: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), and Kv4 (Shal).
Subfamily-specific assembly of heteromeric K
channel
complexes has been observed in vitro and in vivo,
which contributes to the diversity of K
currents.
However, the molecular codes that mediate the subfamily-specific
association remain unknown. To understand the molecular basis of the
subfamily-specific assembly, we tested the protein-protein interactions
of different regions of
-subunits. We report here that the
cytoplasmic NH
-terminal domains of Kv1, Kv2, Kv3, and Kv4
subfamilies each associate to form homomultimers. Using the yeast
two-hybrid system and eight K
channel genes, two genes
(one isolated from rat and one from Drosophila) from each
subfamily, we demonstrated that the associations to form
heteromultimers by the NH
-terminal domains are strictly
subfamily-specific. These subfamily-specific associations suggest a
molecular basis for the selective formation of heteromultimeric
channels in vivo.
Potassium (K) channels comprise a large family
of homologous membrane proteins. They regulate cardiac pacemaking,
action potentials, and neurotransmitter release in excitable
tissues(1, 2, 3, 4) . In
non-excitable cells, they play important roles in hormone secretion,
cell proliferation, cell volume regulation, and lymphocyte
differentiation(5) . Four membrane bound
-subunits can
form a channel, either as a homotetramer or
heterotetramer(6, 7, 8) . The heterogeneity
of the K
channel function is reflected in part by a
large number of K
channel genes and their splice
variants(9) . The K
channel diversity may also
arise from the formation of heteromultimeric channels with novel
properties when compared with their parental
homomultimers(10, 11, 12) . A combination of
differential gene expression and selective formation of
heteromultimeric channels by different membrane-bound
-subunits
and hydrophilic
-subunits (13, 14, 15, 16) may allow
individual cells to acquire their own characteristic properties of
K
currents.
The known -subunits of
voltage-gated K
channels share a common design
composed of a hydrophobic core region with six putative transmembrane
segments flanked by hydrophilic cytoplasmic amino (NH
)- and
carboxyl (COOH)-terminal domains(17, 18) . There is
about 40% amino acid identity within the transmembrane core regions
between
-subunits of different subfamilies, compared with
70%
identity within each of the Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), and
Kv4 (Shal) subfamily(8, 9, 19) . Studies of
coexpressing different subunits in Xenopus oocytes have
indicated that the formation of heteromultimeric channels is
subfamily-specific(8, 10, 11, 12, 20, 21) .
In addition, coimmunoprecipitation of K
channel
protein in culture cells and in rat brain has detected the physical
association of different subunits in the Kv1
subfamily(22, 23, 24) . The
subfamily-specific association of
-subunits provides one mechanism
to prevent different subunits expressed in a cell from random mixing,
thereby permitting the cell to maintain several distinct K
current systems(8) . However, the molecular
determinant(s) for such selectivity of subunit assembly is unknown.
Analysis of homomultimer formation has resulted in the
identification of regions in Kv1 -subunits that play a role in
subunit assembly (21, 25, 26, 27) .
Possible regions of K
channel protein involved in
subfamily-specific association were inferred by electrophysiological
studies on cells that heterologously coexpress two different subunits.
1) Substitution of the NH
-terminal domain of a distantly
related mammalian K
channel polypeptide Kv2.1 (DRK1, (34) ) with that of ShB in Kv1 subfamily permits the chimeric
polypeptide to coassemble with ShB(21) . 2) Deletion of the
conserved regions within the NH
-terminal hydrophilic
domains of Kv2.1 and hKv1.4 channels resulted in
100- and
20-fold decreases in channel expression in Xenopus oocytes(27, 28) and the loss of
subfamily-specific assembly(27) . Consistent with this
hypothesis, Shen and Pfaffinger (29) recently showed that the
NH
-terminal domains of AKv1.1 of the Kv1 subfamily and that
of AKv3.1 (Kv3 subfamily) form only homomultimers, as determined by
coimmunoprecipitation.
To systematically determine the regions that
mediate the specific interaction between different subunits, we have
studied eight K channel genes from the four different
subfamilies, two genes (one isolated from rat and one from Drosophila) from each subfamily. We report here that the
NH
-terminal domains of Kv1, Kv2, Kv3, and Kv4 subfamilies
each associate to form homomultimers. Using the yeast two-hybrid
system, designed to analyze heteromultimeric interactions (30, 31, 32) , and homomeric interactions (32, 33) of proteins in a physiological environment,
we demonstrated that the associations to form heteromultimers by the
NH
-terminal domains are strictly subfamily-specific.
Sequence analysis of the regions required for the associations suggests
that the subfamily-specific associations are mediated by a motif of a
common structural design. The subfamily-specific associations of the
NH
-terminal domains provide a molecular basis for the
selective formation of heteromultimeric channels in vivo.
Figure 1:
Physical association of
ShB(6-46) with NShB
(6-46) detected by
coimmunoprecipitation. CHO cells that express ShB
(6-46) were
metabolically labeled after transfecting the cells with constructs that
express either GST-NShB
(6-46) or GST-NKv4.2. The associated
products were brought down by monoclonal antibody 12CA5 specific to the
tag present in GST-fusion proteins (see ``Experimental
Procedures''). The immunoprecipitated polypeptides were visualized
by either autoradiography (lanes 1-3) or by immunoblot
using rabbit antiserum against COOH-terminal domain of ShB (see
``Experimental Procedures'') (lanes 4-6). Lanes 1 and 4, ShB
(6-46) expressing cells
with no transfection; lanes 2 and 5, cells
transfected with pCMV.GST-NShB
(6-46); lanes 3 and 6, cells transfected with
pCMV.GST-NKv4.2.
Figure 2: Association of NShB detected by the yeast two-hybrid system. YGH1 yeast cells were transformed by different pairwise combinations of the two-hybrid constructs that express either fusion proteins of DNA binding domain of GAL4 (DB, pPC86) or transcription activation domain of GAL4 (TA, pPC97). The transformed cells expressing the two different fusion proteins were first selected by dextrose synthetic medium with supplement of histidine but no supplements of leucine and tryptophan (SDH). Identical numbers of cells in each combination were dotted on both SDH and SD (synthetic medium with no supplements of histidine) medium and allowed to grow at 30 °C for 65 h. Box 1, GAL4(DB)bz-fos and GAL4(TA)bz-jun; box 2, GAL4(DB)bz-fos and GAL4(TA)NShB; box 3, GAL4(DB)NShB and GAL4(TA)bz-jun; box 4, GAL4(DB)NShB and GAL4(TA)NShB; box 5, GAL4(DB) and GAL4(TA)NShB; box 6, GAL4(DB)NShB and GAL4 (TA).
For a quantitative estimate of the extent of
NShB-NShB interaction, a standard enzymatic assay for determining
-galactosidase activity in cell lysate was performed (Table 2). The specific association of human c-fos and
murine c-jun through the leucine zipper domains was used as an
internal standard. The Leu
Trp
transformants expressing GAL4DB-NShB and GAL4TA-NShB exhibited
80% of the
-galactosidase activity conferred by c-jun and c-fos interactions, which is at least 100-fold higher
than the background as determined using transformants of GAL4-DB and
GAL4-TA (Table 2, number 6). Transformants expressing either one
of the NShB fusion proteins along with the complementary GAL4 domain
showed only a background level of
-galactosidase activity
consistent with the results from the tests of growth selection (Fig. 2). Coexpression of GAL4DB-NShB with GAL4TA-c-jun in either YGH1 or PCY2 cells led to no detectable growth on triple
selection plates and only a background level of
-galactosidase
activity ( Table 2and Fig. 2), revealing no nonspecific
interaction between NShB to the GAL-mediated transcription machinery.
Thus, it is most likely that association of NShB-GAL4 fusion proteins
is responsible for the production of
-galactosidase activity and
the activation of the HIS3.
If the
involvement of NH-terminal domain in K
channel
-subunit assembly is a general mechanism, one would
expect the interaction of NH
-terminal domains of other
subfamilies. To test this hypothesis, we have subcloned cDNA fragments
of the NH
-terminal domains of three rat K
channels into GAL4DB and GAL4TA vectors, which include NKv4.2
(amino acids 1-183 of Kv4.2, a member of the Kv4 or Shal
subfamily), NKv2.1 (amino acids 1-182 of Kv2.1, Kv2, or Shab
subfamily), and Kv3.1 (amino acids 1-180 of Kv3.1, Kv3, or Shaw
subfamily). To test the possible homophilic association of these
NH
-terminal domains, pairwise combinations of plasmids were
cotransformed into PCY2 and the
-galactosidase activity was
measured. Table 2(numbers 12-20) summarizes the results
from these experiments. Activity of
-galactosidase was detected
when GAL4TA-NKv4.2 and GAL4DB-NKv4.2 were coexpressed in yeast cells.
In contrast, coexpression of GAL4DB-NKv4.2 and GAL4TA produced only
background
-galactosidase activity, indicating that the
NH
-terminal domain of Kv4.2 does not have transcription
activation activity nor does it show nonspecific association with
GAL4-TA domain. Results for the NH
-terminal domains of
Kv2.1 and Kv3.1 were also similar (Table 2, numbers 15, 16, 18,
and 19). Thus, the homophilic interactions of the hydrophilic
NH
-terminal domains were detected for each of the four
subfamilies.
To test the ability and specificity of
NH-terminal domains to form heteromultimers, eight
K
channel genes were selected as representatives of
the four subfamilies: Kv1, Kv2, Kv3, and Kv4. Among them, four genes
are from Drosophila (ShB, fShabll, fShaw2,
and fShal2) and four from rat (Kv1.4, Kv2.1, Kv3.1, and
Kv4.2). The cDNA fragments that represent the coding sequences of the
putative NH
-terminal domains were cloned and fused in frame
to GAL4TA and GAL4DB coding sequences. The ability of different fusion
proteins to interact was determined by testing pairwise combinations of
different NH
-terminal domains. For example, GAL4DB-NShB was
coexpressed with GAL4TA fusion proteins of NH
-terminal
domains of all eight genes, two genes (one isolated from rat and one
from Drosophila) from each subfamily. Fig. 3shows that
these yeast transformants all grew in double selection of SDH medium
with supplement of histidine, indicating the cells containing both
plasmids. But the triple selection medium (SD) only allowed the growth
of cells that have fusion proteins containing the
NH
-terminal domains of the same subfamily.
Consistent results were also obtained using
-galactosidase assay (Table 3). In summary, the heteromultimer formation of
NH
-terminal domains is strictly subfamily-specific. These
results further strengthen the notion that the association of
hydrophilic NH
-terminal domains plays an important role in
determining the specificity of
-subunit association to form
heteromultimeric K
channels.
Figure 3:
Subfamily-specific association of
NH-terminal domains of the Kv1 subfamily. Associations of
NShB with different NH
-terminal domains were tested by the
two-hybrid system using growth selection as described in the legend to Fig. 2. In these experiments, GAL4(DB)NShB fusion protein was
coexpressed with one of the following GAL4(TA) fusion proteins: NShB
(from Drosophila, Kv1 subfamily) (box 1), Nfshal2 (Drosophila, Kv4) (box 2), Nfshabl1 (Drosophila, Kv2) (box 3), Nfshaw2 (Drosophila, Kv3) (box 4), NKv1.4 (rat, Kv1) (box
5), NKv4.2 (rat, Kv4) (box 6), NKv2.1 (rat, Kv2) (box
7), NKv3.1 (rat, Kv3) (box 8). The corresponding
-galactosidase activity was shown in Table 2and Table 3.
Figure 4:
Determination of four conserved motifs
that mediate subfamily-specific association of NH-terminal
domains from Kv1, Kv2, Kv3, and Kv4 subfamilies. A, the
schematic diagram of Kv4.2 coding region was shown. Black boxes represent the six putative transmembrane segments. The minimal
region sufficient for NH
-terminal domain association of the
Kv4.2 was determined by deletion analysis. The fragments representing
different coding regions of Kv4.2 were obtained, expressed, and tested
by the two-hybrid system. The ability to associate with NKv4.2 (amino
acids 1-183) was determined by either growth selection or
-galactosidase activity. The + indicates that the growth;
- indicates no growth. The results of
-galactosidase assay
were shown as percent of the activity conferred by jun and fos interaction (Table 2). A region of amino acids
40-159 (indicated by the gray box) is sufficient to
mediate the association. B, minimal regions responsible for
the associations of the other three subfamilies were mapped by deletion
analysis as described above. Alignment of amino acid sequences of the
four mapped motifs (NAB
NAB
NAB
, and NAB
corresponding to the four
subfamilies of Kv1, Kv2, Kv3, and Kv4) was shown. The conserved
positions were shaded. Single letter amino acid codes are: A, Ala; R, Arg; N, Asn; D, Asp; C, Cys; Q, Gln; E, Glu; G, Gly; H, His; I, Ile; L, Leu; K, Lys; M, Met; F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr; V,
Val.
Using a similar approach, regions responsible for associations of
NH-terminal domains of other subfamilies were identified:
Kv1.4 (Kv 1 subfamily), amino acids 177-279; Kv2.1 (Kv2
subfamily), amino acids 26-136; and fshaw2 (Kv3 subfamily), amino
acids 9-110. Within the mapped regions, 20 amino acid residues
were found to be conserved in these four
-subunits (Fig. 4B). These residues are present in the majority
of the 56 members of the four subfamilies (Fig. 5).
Figure 5:
Amino
acid sequence comparison of NAB domains of the cloned genes. The NAB
regions of 56 genes encoding the -subunit were aligned. The amino
acid residues were indicated by single letter codes (see Fig. 4). A and B boxes were assigned according to Drewe et
al.(42) . The four clones that have no access number were
obtained from: AKO1(47) , Raw3(48) , Kv4(49) .
The numbers from 0 to 6 at the top of Kv1 subfamily
sequences indicate the extent of the conservation at the given
position. 0, the residue is conserved in all four subfamilies; 1, four subfamilies share residues with conserved changes; 2, no specific conservation; 3, two different types
of amino acid residues are present in that position, one is present in
three subfamilies and the other is present in one subfamily (1/3
+1/1); 4, 2/2+2/2; 5,
1/1+1/1+2/2; 6, 1/1+1/1+1/1+1/1. Thus
the residues with high scores are
subfamily-specific.
Previous
sequence comparison of K channels in different
subfamilies has revealed significant conservation within the
amino-terminal domains. Drewe et al.(42) have
identified two such regions and named them A box and B box. We note
that the A and B boxes are contained in our mapped regions for
subfamily-specific association. Thus we name these four classes of
motifs as NAB
(N, NH
-terminal domain; AB, the
AB boxes; Kv1, the subfamily), NAB
, NAB
,
and NAB
(Fig. 5).
Using coimmunoprecipitation and the yeast two-hybrid system,
we demonstrated that different NH-terminal domains interact
to form heteromultimers only if they are from the same subfamily. These
reported subfamily-specific associations of NH
-terminal
domains support the hypothesis that the specific interactions of
NH
-terminal domains determine the compatibility of
different
-subunits in forming heterotetrameric channels.
Previous studies using pulse-chase metabolic labeling have suggested
that the heteromultimeric assembly of Kv1 subfamily K channels is cotranslational(24) . The extracellular
NH
-terminal domains of muscle nicotinic acetylcholine
receptor (AChR), a ligand-gated ion channel, are necessary for channel
assembly, and their interactions may precede the association of
hydrophobic domains to form the functional channel (43) .
Perhaps the association of NH
-terminal domains prior to the
completion of translation helps to determine specificity and efficiency
of K
channel assembly. Consistent with this
hypothesis, we have found that the coexpression of
-subunit of ShB
together with its NH
-terminal domain as a fusion protein
leads to their physical association.
Subunit assembly of functional
K channels involves two aspects: efficiency and
specificity. Both are critical for cells to establish their
characteristic current systems in vivo. Thus, a region
directly involved in subunit assembly should meet at least two
criteria. 1) It is a site for protein-protein interaction(s). 2)
Changes in this region alter the efficiency and/or specificity of
assembly. The identified NAB domains meet this definition. First, the
association of the NAB region has been demonstrated in Kv1 channels (21, 25) and AKv3.1(29) . In this report we
have shown that the homomultimeric association of
NH
-terminal domains can be generalized to all tested
K
channels in all four subfamilies. Second, deletions
of regions, including NABs, abolish the homomeric expression of ShB (9) and mKv1.1 (26) or reduce the expression of
Kv2.1(Kv2.1) by more than 100-fold (28) and hKv1.4 by
20-fold(27) . Finally, Kv2.1 of the Kv2 subfamily does not
coassemble with ShB subunits of Kv1 subfamily unless its
NH
-terminal domain is replaced with that of
ShB(21) . This observation is now supported at the molecular
level by evidence in this report, namely, the NH
-terminal
domains of the four subfamilies associate to form heteromultimers in a
subfamily-specific manner. Hence, compatible association of NAB regions
is important for both the efficiency and specificity of the
K
channel assembly.
The yeast two-hybrid system
detects the associated fusion protein complexes inside yeast nuclei.
This system works only if the proteins to be tested do not possess a
dominant signal that dictates their subcellular localization outside
the nucleus. We found no detectable difference in the ability of
different NAB domains to reunite the two domains of GAL4 for the
transcription activation regardless of whether they were attached to
nuclear localization signal (NLS) of ``MPKKKRKV'' from simian
virus 40 large tumor antigen. In contrast, if the first putative
transmembrane region (S1) was included as a part of fusion protein, the
association was no longer detectable (Fig. 4A). These
results suggest that the NAB regions do not contain any dominant
signals for membrane binding or cell surface localization in yeast.
Thus, the ability and specificity of the NH-terminal domain
to associate do not appear to involve membrane binding but rather the
specific interactions between these hydrophilic portions of
-subunits.
To directly compare the amino acid sequences of the
mapped regions of the known K channels, we have
searched protein and/or DNA data bases. A total of 56 genes that encode
K
channels of the four subfamilies have been retrieved
and aligned using FFDR sequence that is conserved in all cloned genes
listed. Guided by the computer-based neural network alignment
procedures(38) , several gaps were introduced to allow higher
scores in alignment between different subfamilies. Three major features
were seen from this analysis. First, the percentage of residue identity
of the alignment (IDE) scores for the different genes encoding NAB of a
given subfamily indicates remarkably high sequence conservation: IDE
scores are 80-86% for the genes within Kv1 subfamily, 67% in the
Kv2 subfamily, 68% in the Kv3 subfamily, and 68-76% in the Kv4
subfamily. The weighted similarity of the alignment (WSIM) scores are
even higher as some residues are conserved changes in amino acid side
chains. Table 4summarizes the comparison of NAB
,
NAB
, NAB
, and NAB
using the
sequences of ShB, fShab11, fShaw2, and fShal2 genes of Drosophila. NAB
is most closely
related to NAB
(43%) and most distantly related to
NAB
(33%) in agreement with results from comparing
full-length polypeptides(44) . Similar scores have also been
obtained using NABs from other species (data not shown). Second, a
major gap was introduced between box A and box B regions to permit
significantly higher IDE scores between subfamilies. The length and the
sequence of insertions within this gap as seen in NAB
do
not appear to be critical for the binding specificity, since the
NH
-terminal domains of Kv3.1 and fshaw2, which
have different sequences of different lengths between the A and B
boxes, nevertheless associate in a subfamily-specific manner (Table 3). Third, positions with subfamily-specific conservation
were identified and assigned with higher scores ( Fig. 5and
legend). Although it is not known whether all subfamily-specific
conserved positions are required for the association of
-subunits,
it is likely that multiple residues of one subunit, perhaps including
some of those positions with high assigned scores in the alignment, are
involved in the recognition of the two adjacent subunits to form the
predicted tetrameric ring structure.
The involvement of the
NH-terminal domain of
-subunits in the formation of a
tetrameric channel with a central pore would juxtapose two parts of the
NAB surface to their respective complementary surfaces of the adjacent
subunits, similar to what was seen in the three-dimensional structure
of neuraminidase(45) . We speculate that the ability to form
homo- or heterotetramers then requires one of these two surfaces of NAB
to interact with the complementary surface of an adjacent NAB. Since
the NABs from each subfamily have to possess these structural
requirements, one possibility would be that they are folded in a
similar way to present certain amino acid residues that dictate
subfamily-specific interactions. This notion is supported by the
following evidence. First, despite the fact that NAB regions of
different subfamilies do not interact, pairwise comparison reveals that
amino acid conservation between subfamilies is
30% (Table 4), suggesting that they are structurally related and are
likely to share a common scaffold. Second, although it is difficult to
assess the secondary structural prediction in the absence of any
structural information of NABs, analysis of a large number of NAB
regions of different subfamilies using a neural network prediction
program (38) suggests that NABs from different subfamilies
exhibit similar secondary structure or pattern of side chain
properties. This secondary structural pattern is also shared by the
newly cloned Kv5.1 K
channel that represents yet the
fifth subfamily (46) (data not shown). Hence, a
three-dimensional structure obtained from any one NAB would be likely
to help our understanding on the NAB motifs of different subfamilies of
K
channels.
Since mutations within the regions
involved in subunit assembly are likely to result in the loss of
channel expression, analysis of protein interactions using the yeast
two-hybrid system provides an alternative method to study the
interactions of NH-terminal domains in the subunit assembly
of K
channels. This approach would allow one to screen
a large number of mutants that have altered properties in K
channel subunit interaction without the requirements for such
mutants to form functional channels. Combining this mutational analysis
with electrophysiological and cell biological methods may make it
possible to analyze the molecular basis and physiological functions of
subunit interactions of K
channels.