(Received for publication, November 1, 1994; and in revised form, January 23, 1995)
From the
Voltage-gated potassium channel subunits are cytoplasmic
proteins that co-purify with the pore-forming
subunits. One of
these subunits, Kv
1 from rat brain, was previously demonstrated to
increase the rate of inactivation of Kv1.1 and Kv1.4 when co-expressed
in Xenopus oocytes. We have cloned and characterized a novel
voltage-gated K
channel
subunit. The cDNA,
designated Kv
3, has a 408-amino acid open reading frame. It
possesses a unique 79-amino acid N-terminal leader, but is identical
with rat Kv
1 over the 329 C-terminal amino acids. The Kv
3
transcript was found in many tissues, but was most abundant in aorta
and left ventricle of the heart. Co-expression of Kv
3 with
K
channel
subunits shows that this
subunit
can increase the rate of inactivation from 4- to 7-fold in a Kv1.4 or Shaker B channel. Kv
3 had no effect on Kv1.1, unlike
Kv
1 which can increase rate of inactivation of this
subunit
more than 100-fold. Other kinetic parameters were unaffected. This
study shows that voltage-gated K
channel
subunits are present outside the central nervous system, and that at
least one member of this family selectively modulates inactivation of
K
channel
subunits.
Voltage-gated potassium channels are present in all excitable
and most nonexcitable eukaryotic cell types. They are involved in a
diverse array of functions, including electrogenesis, secretion, and
cell motility. In mammals, a diverse group of more than 50 members of
an extended gene family homologous to the Shaker gene of Drosophila have been identified that are responsible for
generating many voltage-gated K currents(1, 2, 3, 4, 5) .
Shaker-type K channel proteins, expressed
in Xenopus oocytes or mammalian cell lines, form tetramers of
subunits that selectively conduct K
ions and
reproduce many properties of native channels. However, there are
inconsistencies between native and cloned K
channel
currents, suggesting a failure to reconstitute the native channels
fully in heterologous expression systems. These inconsistencies have
several possible explanations. Some native K
channels
are heteromultimeric associations of more than one type of
subunit. These associations can modify the current phenotypes, making
them distinct from currents produced by either
subunit
alone(6, 7, 8) . It has also been suggested
that differences between cloned and native channels are due to
differences in post-translational processing or intracellular
environment between heterologous expression systems and the
channel's native
environment(9, 10, 11) . Finally,
K
channel structure in heterologous expression systems
may be incomplete(12, 13) . Two voltage-gated
K
channels recently purified from mammalian brain (14, 15) were shown to consist of an
subunit,
identical with previously described Shaker-like K
channel proteins, and a smaller
subunit (M
= 40,000). This suggests that complete reconstitution of
K
channels in heterologous expression systems requires
the presence of appropriate
subunits.
Partial amino acid
sequencing of the bovine K channel
subunit led
to the cloning of one bovine brain (14) and two rat brain (16)
subunit cDNAs. They were divided into two classes
based on differences in sequence: Kv
1, found in rat, and Kv
2,
found in both rat and bovine. These
subunits consisted of 401 and
367 amino acids, respectively, and had 85% identity in their 329
C-terminal amino acids. The respective N termini of 72 and 38 amino
acids exhibit no conservation. Co-expression of rat Kv
1 with a
delayed rectifier type K
channel (RCK1; Kv1.1)
increased the rate of inactivation more than 100-fold. Site-directed
mutagenesis showed that the increase in inactivation rate was mediated
through the N-terminal region of the
subunit. The Kv
2
subunit did not affect the properties of RCK1 or of a fast-inactivating
Kv1.4 channel, RCK4(16) .
Kv1 mRNA was detected
exclusively in brain(16) . To determine if novel K
channel
subunits might be found in other tissues, we
attempted to clone these proteins from heart. The large array of
K
currents in heart (17, 18) made it
a logical source of unknown
subunits. A cDNA was isolated from a
ferret ventricular cDNA library. It contained a single open reading
frame highly conserved with previously described voltage-gated
K
channel
subunits(16, 19) .
Its deduced amino acid sequence contained a unique leader sequence,
followed by 329 amino acids identical with rat Kv
1. The Kv
3
transcript was most abundant in aorta and left ventricle, although it
was also found in other locations in the heart as well as brain,
skeletal muscle, and kidney. Kv
3 is conserved in humans and rats.
Kv
3 was co-expressed in Xenopus oocytes with K
channel
subunits from ferret (Kv1.4(20) ), rat
(Kv1.1(21) ), and Drosophila (Shaker B
6-46(22) ). It accelerated inactivation in
Kv1.4 in a manner analogous to that of Kv
1(16) . However,
unlike Kv
1, which increased the inactivation rate of Kv1.1
100-fold, Kv
3 had no effect on this
subunit. As Kv
3
was also capable of modulating inactivation of Shaker B, the
failure of Kv
3 to alter inactivation of Kv1.1 was not due to
strict specificity for Kv1.4
subunits.
Unless otherwise specified, standard molecular biological methods were used(23, 24) . Enzymes were used according to manufacturers' directions.
Figure 1:
Deduced amino acid sequence of ferret
Kv3. The sequence is shown aligned with the other known
K
channel
subunit sequences: rat Kv
1, rat
Kv
2, and bovine Kv
2(16, 19) . Amino acids
identical with those in Kv
3 are designated with a hyphen in the Kv
1 and -2 sequences. The nucleotide sequence of
ferret Kv
3 has been deposited in GenBank under the accession
number U17966.
Human and rat Kv3 cDNAs were
isolated by standard PCR of cDNA synthesized as above. Human heart
whole cell RNA was purchased from Clontech. The primers used were
FK
3/58 (5`-GTATAAACCTGCCTGTGC-3`; covering amino acids 4-9
in ferret Kv
3) and FK
3/37 (5`-AGCCTTTCCAGCAGCATAGACTTC-3`;
covering amino acids 129-135; Fig. 1). PCR was performed
as described(30) . After a 5-min 94 °C denaturation step,
the reactions were cycled 35 times at 94 °C for 30 s, 53 °C for
45 s, and 72 °C for 1 min. The procedure was completed with a 5-min
incubation at 72 °C. The PCR products were cloned into
pBS-SK
, and two independent clones of each were
sequenced. Partial cDNA sequences of the rat and human Kv
3 have
been deposited in GenBank
under the accession numbers
U17967 and U17968, respectively.
Figure 2:
A, strategy for detection of Kv3
transcripts by competitive PCR. Ferret transcripts were detected using
oligonucleotides Kv
3/56 and Kv
3/37, which give a 460-bp DNA
molecule (B). The internal standard, added to PCR reactions in B, was a 183-bp deletion of Kv
3 that preserved the
nucleotide sequence complementary to the oligonucleotides; its product
was 272 bp. The open boxes represent unconserved regions, and
the shaded boxes represent the conserved region of Kv
3. B, competitive PCR of cDNAs from various ferret tissues. Five
µg of whole cell RNA were reverse-transcribed and amplified by PCR
in the presence of 0.2 fmol internal standard. Lanes 1, cDNA
alone; 2, internal standard alone; 3, right
ventricle; 4, left ventricle; 5, atrium; 6,
aorta; 7, cultured endothelial cells; 8, brain; 9, skeletal muscle; 10, liver; and 11,
kidney. Competitive PCR of sham reverse transcription controls showed
no detectable signal (data not shown).
Fig. 1shows an alignment of the deduced amino acid
sequence of ferret Kv3 to K
channel
subunits from rat (Kv
1 and Kv
2) and bovine (Kv
2).
Strikingly, Kv
3 and rat Kv
1 are identical over the last 329
amino acids, a region where Kv
1 and Kv
2 share 85% identity.
However, there is no similarity among the first 79 amino acids of the
ferret
subunit and the first 72 or 38 amino acids of Kv
1 and
Kv
2, respectively. Similarity searches of both the entire
subunit and the N-terminal 79 amino acids revealed no significant
similarity between ferret Kv
3 and any other protein except the
previously described
subunits. No similarity was found between
the N terminus of ferret Kv
3 and any other protein sequence.
Despite their apparent lack of similarity, the N termini of Kv
1
and Kv
3 share one potentially important structural feature: a
cysteine residue near the N terminus. Oxidation of this amino acid,
also found near the N termini of other fast inactivating channels, can
dramatically decrease inactivation rates (11, 16) .
Hydropathy and secondary structural analyses of the Kv
1 and
Kv
3 N termini fail to show any apparent similarity in predicted
secondary
structure(35, 36, 37, 38, 39, 40) .
Hydropathy analysis also suggested Kv
3 is a cytoplasmic protein,
as has been predicted for Kv
1 and Kv
2 (16, 19) . Comparison of the deduced Kv
3 amino
acid sequence with protein motif data bases (41, 42) showed the presence of 33 consensus
phosphorylation sites.
To determine the tissue localization of
Kv3, competitive PCR (32) was performed on cDNA made from
several tissues. Oligonucleotides were chosen at the 5` end of the
clone to ensure specific amplification of Kv
3 (Fig. 2A). Ferret Kv
3 transcript was most abundant
in aorta followed by left ventricle (Fig. 2B).
Transcripts were also detected in right ventricle, atrium, brain,
skeletal muscle, and kidney. None were detectable in liver or cultured
aortic endothelial cells. Aorta is rich in smooth muscle, fibrous
tissue, and endothelium; as the transcript was not present in
endothelial cells, it is likely that this transcript is present in
smooth muscle or fibroblasts, although its presence in other cell types
cannot be ruled out.
Kv3 is not unique to ferret. Cross-species
PCR using oligonucleotides FK
3/37 and FK
3/58 (which is
complementary to the unique portion of the ferret cDNA) was performed
on rat and human heart cDNA. The reactions yielded the expected
400-nucleotide DNA species, which was cloned and sequenced. There was
extensive similarity in the nucleotide sequence among ferret, human,
and rat, showing that Kv
3 is present in all three species. The
amino acid sequence conservation in the N-terminal region was 88%
between ferret and human and 80% between ferret and rat (data not
shown).
Kv1 can increase the rate of inactivation of both a
delayed rectifier and a fast-inactivating (A-type) K
channel
subunit(16) . Conservation between Kv
3
and Kv
1 suggested that Kv
3 might have a similar influence. To
test the effect of Kv
3 on inactivation and other channel
properties, it was co-expressed in Xenopus oocytes with FK1, a
ferret Kv1.4 channel(20) , and RCK1 (rat Kv1.1(21) ).
Co-injection of Kv
3 mRNA with FK1 mRNA caused subtle changes in
inactivation kinetics (Fig. 3A). Inactivation of FK1
channels has been previously shown to be biexponential, with two
closely spaced time constants at +50 mV(20) . The time
constant of the fast component of inactivation decreased from 42
± 3 (n = 20) for FK1 alone to 6.2 ± 0.2
ms (n = 28); the slow component decreased from 313
± 94 to 101 ± 2 ms. The decrease of the fast component of
inactivation was comparable to the overall decrease induced by Kv
1
expressed with rat Kv1.4. Co-expression of Kv
3 with FK1 also
changed the relative contribution of each component of inactivation;
the ratio of the amplitude of fast to slow time constants decreased
from 5.0 ± 1.7 to 0.67 ± 0.03. Because of the greater
participation of the slow component, the net result was a minor
alteration in the overall rate of inactivation.
Figure 3:
Outward K currents
elicited by depolarizing pulses to +50 mV from a holding potential
of -90 mV using a two-electrode voltage clamp. Currents were
normalized relative to the same peak current to emphasize the kinetic
changes induced by co-expression of Kv
3. Currents resulted from
depolarizations to +50 mV from a holding potential of -90
mV, stimulation rate of 0.1 Hz, and perfused with ND-96. Oocytes were
injected with mRNA encoding various K
channel subunits
types. A, FK1 alone (Kv1.4, 8 ng of mRNA/oocyte, peak current
12.7 µA) or FK1 + Kv
3 (8 and 32 ng of mRNA/oocyte, peak
current 9.7 µA). Note that expression of FK1 alone results in a
smooth inactivation with a small but slow late component of
inactivation. Co-expression of FK1 with Kv
3 results in an initial
small rapid component followed by a much larger slow component of
inactivation. B, RCK1 alone (Kv1.1, 2 ng of mRNA/oocyte, peak
current 14.4 µA) or RCK1 + Kv
3 (2 and 32 ng of
mRNA/oocyte, respectively, peak current 10.2 µA). Co-injection of
Kv
3 with RCK1 did not increase the rate of inactivation. C, FK1
2-146 (8 ng of mRNA/oocyte, peak current 5.7
µA), an N-terminal deletion mutant (see (20) ) or
FK1
2-146 + Kv
3 (2 and 32 ng of mRNA/oocyte,
respectively, peak current 1.7 µA). Co-injection of
FK1
2-146 with Kv
3 partially, but not completely,
restored inactivation. D, Shaker B
6-46 alone (2 ng
of mRNA/oocyte, 9.1 µA) or Shaker B
6-46 +
Kv
3 (8 and 32 ng of mRNA/oocyte, respectively, peak current 11.1
µA). The increase in inactivation rate was similar to the results
with FK1
2-146. Kv
3 partially restored inactivation in
the mutant Shaker channel and induced a very small additional
fast component that was not observed with
FK1
2-146.
In contrast to the
results obtained with FK1, co-injection of RCK1 (Kv1.1) with Kv3
failed to show any effect on channel inactivation (Fig. 3B). This was true despite a 16:1 (by weight)
co-injection of Kv
3:RCK1 mRNA; parallel experiments using FK1 and
FK1
2-146 (see below) showed that the Kv
3 subunit was
active (data not shown). These results are strikingly different from
those obtained by Rettig et al.(16) with Kv
1.
They showed an approximately 100-fold increase in the rate of
inactivation of RCK1. Since Kv
3 was co-injected with the identical
RCK1
subunit, and conservation of RCK4 and FK1 is
97%(20) , it is likely that the difference in inactivation is
due to the different N termini of Kv
3 and Kv
1.
It is
possible that the change in rate of inactivation caused by Kv3 may
be dependent on the ratio of expressed
subunit to
subunit.
If the
subunits failed to saturate all available binding sites on
the
subunits, the overall rate and degree of
subunit-mediated inactivation would likely be decreased. A second
explanation is that the maximal association of
subunits to
subunits was achieved, but that Kv
3 simply induced slower
inactivation, or perhaps caused inactivation by a different mechanism.
To discriminate between these possibilities, a mutant form of FK1,
FK1
2-146, in which the first 146 N-terminal amino acids have
been deleted to remove fast N-type inactivation ((20); referred to as
FK1
Nco), was co-expressed with Kv
3. As shown in Fig. 3C, co-expression with Kv
3 greatly increased
the rate at which FK1
2-146 inactivated. The time constant of
inactivation for FK1
2-146 was 1795 ± 81 ms (at
+50 mV), decreasing to 409 ± 27 ms with co-injection of
Kv
3 mRNA. The effects of co-expression of Kv
3 mRNA on the
inactivation rate of FK1
2-146 were dependent upon the ratio
of injected Kv
3 to FK1
2-146 mRNA for ratios less than 2
to 1 but were insensitive to ratios higher than 2 to 1 (Fig. 4),
suggesting that the
subunits were saturating at the mRNA
concentrations used in these experiments.
Figure 4:
Saturation of FK12-146 with
Kv
3. The alteration of inactivation time constants was sensitive
to the ratio of
subunit to
subunit for low ratios but
saturated for high ratios of
to
subunit, indicating that
the rate of inactivation reflected the maximal association of Kv
3
to FK1
2-146 channels. Two ng/oocyte of FK1
2-146
was injected into each oocyte followed by a second injection of 0, 2,
4, 8, 16, or 32 ng/oocyte Kv
3 mRNA. Inactivation was measured with
a single exponential time constant fit to the rate of current decay.
The data for each particular level of injected
subunit mRNA is
given as the mean ± S.E. (n = 11, 4, 8, 11, 6,
10 oocytes for each point, average peak currents were 4.9 ± 0.5,
3.7 ± 0.8, 3.1 ± 0.4, 2.3 ± 0.2, 2.4 ± 0.2,
1.5 ± 0.1 µA, respectively).
These data showed that
ferret Kv3 can associate with ferret Kv1.4 (FK1), but did not rule
out the possibility that this
subunit's effects might be
specific for only a limited class of
subunits. Therefore, we
co-injected Kv
3 with the distantly related channel, Shaker B from Drosophila. Since this channel is only 50%
conserved with FK1, it appeared to be an unlikely candidate to
associate with a mammalian
subunit. We used Shaker B
6-46(22) , a mutant lacking fast N-type
inactivation, to be able to clearly detect the effects of
subunit
association. Co-expression of Shaker B
6-46 with
Kv
3 resulted in an increase in the degree and rate of inactivation
similar to that observed for FK1
2-146 (Fig. 3D). Association with Shaker B suggested
that Kv
3 is probably binding to the much more highly conserved
RCK1, but for some reason is unable to induce inactivation.
Other
gating characteristics of FK1 and FK12-146 were not strongly
altered by co-expression of Kv
3. Fig. 5shows data obtained
using the cut-open oocyte clamp (34) for FK1
2-146 in
the presence and absence of
subunit. FK1
2-146 was used
to minimize the overlap of fast inactivation with activation. There was
no obvious difference in the sigmoid onset of the current during the
first 2 ms at +50 mV; however, the late rising phase of current
was terminated prematurely by what appears to be the onset of an
inactivation-like process. Although the time to peak current appears to
have decreased with co-expression of Kv
3, this is most likely due
to the increased inactivation rate. Similarly,
subunit expression
had only modest effects on recovery from inactivation when co-expressed
either with full-length FK1 (t
= 4.88
± 0.13 s (n = 4) with
subunit; t
= 2.95 ± 0.18 s (n = 4) without
subunit) or FK1
2-146 (t
= 2.94 ± 0.12 s (n = 5) with
subunit; t
=
2.20 ± 0.11 s (n = 4) without
subunit).
Figure 5:
Early onset of current activation is
unchanged by the addition of subunit. Currents recorded from
FK1
2-146 (solid line) and FK1
2-146
co-expressed with Kv
3 (dashed line) using the cut-open
oocyte voltage clamp technique as described(20, 34) .
The currents were normalized for the purpose of comparison. The peak
current occurred much earlier due to the overlap with
subunit-mediated inactivation, but was otherwise apparently unaffected.
Current amplitudes were 15 and 2.1 µA for FK1
2-146 and
FK1
2-146 +
subunit, respectively. Both currents
were recorded in response to a depolarizing pulse to +50 mV from a
holding potential of -90 mV and were capacitance and leakage
compensated. No off-line subtraction procedures were
employed.
We have cloned, sequenced, and characterized a novel K channel
subunit, Kv
3. The deduced protein sequence is
identical with Kv
1 in its 329 C-terminal amino acids, but has a
79-amino acid N terminus with no sequence identity with Kv
1 or
Kv
2. Conservation of C termini between Kv
1 and Kv
3, and
their sharp transition to a nonconserved region, suggested that one
mechanism for generating different
subunits might be alternative
splicing of precursor transcripts.
The general structural similarity
between Kv1 and Kv
3 implied that Kv
3 might induce
inactivation in K
channel
subunits. This was
true for mutant channels that have had N-type inactivation disrupted
(FK1
2-146 and Shaker B
6-46) and in
wild-type Kv1.4. However, unlike Kv
1, it did not cause
inactivation of a Kv1.1
subunit. The failure of Kv
3 to
modify RCK1 inactivation kinetics may reflect a failure of this
and
subunit to associate, although association with Shaker argues against this. Alternatively, Kv
3 and RCK1 may
associate, but specific domains required for inactivation cannot
interact.
Acceleration of inactivation by Kv1 is mediated by a
``tethered ball''(16) . If Kv
3 operates through
a similar mechanism, the implication is that the inactivation domain of
Kv
3 is specific for certain
subunits. This seems unlikely,
given the recent demonstration that a variety of inactivation domains
can induce inactivation in fast inactivating and non-inactivating
subunits (43, 44) . Another possibility is that
Kv
3 alters inactivation through another mechanism. In either case,
it seems clear that the nonconserved N-terminal region in
subunits confer some degree of specificity of interaction.
Kv3
is the third member of a family of ancillary subunits that associate
with voltage-gated K
channels. Co-expression of
Kv
3 causes alterations in K
channel function that
are distinct from the effects Kv
1 or Kv
2. This suggests that
the diversity of in vivo K
channel function
may result from expression of different K
channel
subunits, as well as diversity of expression and assembly of
subunits. This mechanism is similar to that of Na
and Ca
channels, which are known to generate
diversity through assembly of different subunits(45) . Given
the wide variety of associated subunits for these other channels, it
seems likely that Kv
1, Kv
2, and Kv
3 are the first
members of a large group of ancillary K
channel
subunits that differ in their N termini. The novel differences in
specificity of channel interaction and electrophysiological properties
of Kv
3 from Kv
1 and Kv
2 provide an additional basis for
understanding K
channel function and diversity.