(Received for publication, September 13, 1995; and in revised form, October 2, 1995)
From the
Voltage-gated K channels can form multimeric
complexes with accessory
-subunits. We report here a novel
K
channel
-subunit cloned from human heart,
hKv
1.3, that has 74-83% overall identity with previously
cloned
-subunits. Comparison of hKv
1.3 with the previously
cloned hKv
3 and rKv
1 proteins indicates that the
carboxyl-terminal 328 amino acids are identical, while unique variable
length amino termini exist. Analysis of human
-subunit cDNA and
genomic nucleotide sequences confirm that these three
-subunits
are alternatively spliced from a common
-subunit gene.
Co-expression of hKv
1.3 in Xenopus oocytes with the
delayed rectifier hKv1.5 indicated that hKv
1.3 has unique
functional effects. This novel
-subunit induced a time-dependent
inactivation during membrane voltage steps to positive potentials,
induced a 13-mV hyperpolarizing shift in the activation curve, and
slowed deactivation (
= 13 ± 0.5 ms versus 35 ± 1.7 ms at -40 mV). Most notably, hKv
1.3
converted the Kv1.5 outwardly rectifying current voltage relationship
to one showing strong inward rectification. These data suggest that Kv
channel current diversity may arise from association with alternatively
spliced Kv
-subunits. A simplified nomenclature for the
K
channel
-subunit subfamilies is suggested.
Voltage-gated K channels (Kv) (
)are
important regulators of membrane action potentials as well as many
other cellular functions including maintenance of the resting membrane
potential, regulating neuron firing, and
secretion(1, 2, 3) . Most tissues contain
multiple channel types belonging to one or more Kv gene
subfamilies(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
However, assigning specific K
channel clones to a
native current often is difficult since most heterologously expressed
Kv channels display either a fast inactivating or a delayed rectifier
type current, often with similar pharmacology. While possible factors
contributing to this diversity may include Kv channel glycosylation,
phosphorylation, and heterotetrameric
-subunit formation within a
gene subfamily(16, 17, 18, 19) ,
recent studies have shown that
-subunits associate with and
functionally alter Kv channel clones in heterologous
systems(20, 21, 22, 23, 24, 25, 26) .
At present, four Kv -subunits have been reported. Three
distinct K
channel
-subunits were cloned from rat
brain(21, 27) . The rat Kv
1 subunit confers rapid
A-type inactivation on the Kv1.1 delayed rectifier channel, while the
rat Kv
2 isoform does not alter K
channel current
phenotypes in the Xenopus oocyte expression
system(21) . Heinemann and co-workers (27) have
reported a third
-subunit from rat brain originally named Kv
3
that shares 68% identity with rKv
1 and induces partial
inactivation in channels of the Kv1 family. The fourth distinct
-subunit clone, also termed Kv
3, was isolated from human and
ferret heart(23, 24, 26) . Identity of this
Kv
3 to previously cloned
-subunits is greatest in the
carboxyl-terminal region with complete identity of hKv
3 and
rKv
1 in the carboxyl 329 amino acids and 85% identity to
rKv
2. However, the first 79 amino acids of hKv
3 share only
25% identity with rKv
1 and do not align with rKv
2. Fast
inactivation of Kv1.4 was accelerated when expressed with hKv
3 (23, 24, 25) and fast inactivation and a
20-mV hyperpolarizing shift in the activation curve was conferred on
the delayed rectifier Kv1.5(23, 26) . Human Kv
3
has no functional effect on the Kv1.1, Kv1.2, and Kv2.1 channel
clones(23) , although these channels have been postulated to
associate with accessory subunits(28, 29) .
The
complete amino acid identity between rKv1 and hKv
3 in the
carboxyl terminus suggests that Kv channel
-subunit isoforms are
encoded by a single
-subunit gene. Alternative splicing was
suggested previously (21, 26, 30) since the
point of divergence between rKv
1 and hKv
3 cDNA contained a
potential splice junction and the nucleotide sequence identity in the
carboxyl terminus was >90%. Calcium channel
-subunits are
encoded by four different genes with alternative splicing of the
1
and
2 genes giving rise to multiple
-subunits within these
subfamilies(31, 32) . Cell-specific alternative
splicing of Kv
-subunits may be one mechanism responsible for the
diversity of Kv channel current among cell types.
We report here the
cloning and characterization of a cDNA from human heart that encodes a
unique K channel
-subunit designated hKv
1.3.
The hKv
1.3 subunit uniquely alters the functional properties of
hKv1.5, converting it from a delayed rectifier to a channel with rapid,
but partial, inactivation. In addition, this current activates at lower
voltages, rectifies at depolarized potentials, and has slowed
deactivation. Nucleotide sequence comparison of cDNA and genomic DNA
encoding human Kv
1.3, Kv
3, and Kv
1 indicate that these
subunits are encoded by a common
-subunit gene, here designated
the Kv
1 subfamily gene. We suggest that the nomenclature be
changed to reflect that the Kv
1 subunit family members are
generated through alternative mRNA splicing (Table 1).
Isolation
of hKv1.1 cDNA was completed by generating PCR fragments
corresponding to nucleotides 1-216 of rKv
1 and screening 2.8
10
unamplified recombinants from a newly
constructed
ZAPII (Stratagene) cDNA library made from human
cerebral cortex mRNA (Clontech). A 4-kb clone was isolated, and
plaque-purified clones were recovered by in vivo excision
yielding hKv
1.1 in pBluescript (SK-). Nucleotide sequence in
various regions was determined as described above.
Defolliculated Xenopus oocytes were prepared as described previously (19) and
injected with approximately 40 nl (4-20 ng) of in vitro transcribed cRNA. These dilutions resulted in peak currents of
1-10 µA. Electrophysiological recordings have been described
in detail previously(19, 26) . Oocytes were bathed in
an extracellular solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl, 1 mM
MgCl
, 5 mM Hepes (pH 7.5 with NaOH). Membrane
currents were recorded using a two-microelectrode voltage clamp
amplifier from Warner Instruments (New Haven, CT). Values are expressed
as mean ± S.E. unless indicated otherwise. All experiments were
performed at room temperature.
Screening an
amplified human heart cDNA library at low stringency with a
PCR-generated cDNA probe corresponding to nucleotides 435-1089 of
rKv2.1 yielded two partial cDNA clones as determined by nucleotide
sequencing. The deduced amino acid sequence of one of these clones
(h
2-1) was found to be nearly identical with rKv
2.1 and
likely represents the human homologue of this previously cloned rat
subunit(21) . The other cDNA (h
8-82) was most similar to
rKv
1.1 but exhibited little amino acid identity within the
postulated amino terminus and lacked a likely translation start site.
Additional screens of cDNA libraries did not yield a full-length clone.
Screening a human genomic library produced the 26 nucleotides missing
from the 5` end and an obvious translation start site. The initiating
methionine was assigned because it represents the first in-frame ATG
positioned 3` of termination codons in an open reading frame encoding a
419-amino acid protein (47 kDa). Hydropathy analysis did not reveal a
hydrophobic domain, suggesting that similar to other
-subunits,
hKv
1.3 is likely a cytoplasmic protein. An amino acid sequence
comparison between hKv
1.3, hKv
1.2, rKv
1.1, and
rKv
2.1 is illustrated in Fig. 1. The carboxyl-terminal 328
amino acids of hKv
1.3 are 100% identical with rKv
1.1 and
hKv
1.2 and share 85% identity with rKv
. However,
the first 91 amino acids of hKv
1.3 share <10% identity with
hKv
1.2, rKv
1.1, and rKv
2.1.
Figure 1:
Comparison of hKv1.3, hKv
1.2,
rKv
1.1, and rKv
2.1 amino acid sequences. Identical amino acid
residues are indicated by dashes. The rKv
1.1,
hKv
1.2, and rKv
2.1 sequences are from Refs. 21, 23 and
26.
To determine whether
hKv1.3 and hKv
1.2 represent splice variants of the same gene,
3`-untranslated regions were compared. Alignments of this 1800-base
pair region showed 99% nucleotide identity between clones isolated from
separate individuals, suggesting that hKv
1.3 and hKv
1.2
represent splice variants with minor allelic differences. To confirm
that Kv
1.1 is also derived from this gene, Kv
1.1 was cloned
from a human cerebral cortex cDNA library. Three different regions from
the 3`-untranslated region corresponding to
250,
750, and
1500 bp 3` of the translation stop codon were sequenced and showed
complete identity with Kv
1.2 and Kv
1.3 in this
3`-untranslated region suggesting that all three
-subunits are
encoded by this gene. To confirm the splice junction, we attempted to
clone the entire Kv
1 gene from a human genomic library by
screening with a probe corresponding to the carboxyl-terminal 328 amino
acids of Kv
1.1. Although several clones were 10-20 kb in
length, a full-length gene was not isolated based on the finding that
no single clone hybridized to either hKv
1.2 or hKv
1.3
amino-terminal specific probes. Likewise, when the unique amino termini
of hKv
1.3 and hKv
1.2 were used to isolate additional genomic
clones, these clones did not hybridize to the carboxyl-terminal probe.
The complete gene encoding the hKv
1 subfamily likely exceeds 40
kb.
Fig. 2illustrates the genomic sequences of Kv1.2
and Kv
1.3 surrounding the predicted splice site. Both Kv
1.2
and Kv
1.3 genomic sequences correspond to their respective cDNA in
the region marked exon. Both genomic clones contain a consensus
sequence for donor/acceptor splice sites as shown by the underlined
sequence(43) . These data provide further evidence that at
least three
-subunits result from alternative splicing. Therefore,
differential regulation of Kv
-subunit expression and alternative
splicing are likely to be two mechanisms regulating Kv channel
diversity. Further in situ analysis and antibody-based
immunohistochemical localization of the Kv
-subunits will further
our understanding of Kv channel
- and
-subunit association.
Figure 2:
Comparison of hKv1.3 and hKv
1.2
genomic sequences surrounding the proposed splice junction. Genomic
sequences corresponding to the variable amino termini cDNA sequences of
hKv
1.3 and hKv
1.2 are shown. Genomic and cDNA sequences match
in the region marked exon and diverge at the exon/intron border.
Consensus splice site sequences are indicated by the underlining(43) .
Figure 3:
Functional effects of hKv1.3 on
hKv1.5. Whole cell potassium current was recorded from Xenopus oocytes expressing hKv1.5 in the absence (A) and presence (B) of hKv
1.3, and each cell was normalized to peak
current at +50 mV (= 1). The cells were voltage-clamped at
a holding potential of -80 mV for 30 s prior to a variable test
potential (shown as inset voltage protocol) and then stepped to
-40 mV to record deactivating tail currents. In A, the
test step was 75 ms in duration which allowed steady state current
levels to be attained at each potential. This duration was increased to
100 ms in the presence of the hKv
1.3 subunit (B) to
permit steady state to be achieved. Normalized tail currents of hKv1.5
in the presence (closed circles) and the absence (closed
squares) of hKv
1.3 are plotted as a function of test step
potential (C). D represents the steady-state
current-voltage relationship for Kv1.5 alone (open squares)
and Kv1.5 coexpressed with hKv
1.3 (open circles).
Potassium current was measured at steady state (see open symbols in A and B) and plotted as a function of the
test potential. In order to compare different cells, the current was
normalized by dividing the current at each membrane potential by the
value measured at 0 mV (= 1). Note that hKv
1.3 causes an
apparent rectification and that the channels begin to open at more
negative membrane potentials compared to Kv1.5 alone. Symbols represent between 5 and 10 observations and are plotted as the
mean ± 2
S.E.
An additional effect observed during co-expression of Kv1.5 with
Kv1.3 was that the threshold for Kv1.5 channel activation occurred
at more negative potentials. The shift in the activation curve toward
more negative potentials is illustrated in Fig. 3C where the amplitude of the tail currents is plotted as a function
of the membrane potential. Since the driving force is constant during
this measurement, the curve reflects the fraction of channels open at
each membrane potential. The average midpoint of the activation curve
for the hKv1.5 was -7.1 ± 0.5 mV (n = 6)
whereas in the presence of the hKv
1.3 it was -20 ±
0.5 mV (n = 6). Fig. 3D shows steady
state outward current measured during depolarizing steps. Note that
hKv1.5 current is observed at more negative potentials when hKv
1.3
is present, even at potentials that do not show apparent inactivation (i.e. -20 mV). At membrane potentials greater than
approximately 0 mV, hKv1.5 current in the presence of hKv
1.3 is
suppressed relative to the Kv1.5 alone, thereby converting this
apparent outwardly rectifying current voltage relationship to one that
shows inward rectification. Future detailed analysis of these
interactions will elucidate the underlying effects of hKv
1.3.
Discovery of the novel Kv1.3 subunit and that
alternative mRNA splicing generates multiple function altering
-subunits further complicates determination of the relationship
between cardiac clones and native currents. Future cell-specific
localization and co-purification studies using Kv
-antibodies will
enable us to understand both the pattern of Kv
-subunit isoform
expression and the Kv channels with which these subunits associate. In
addition, analysis of the mechanisms by which Kv
1.3 alters the
voltage sensitivity, inactivation, and rectification of Kv1.5 will
advance our understanding of Kv channel function.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L47665[GenBank].