(Received for publication, September 18, 1995; and in revised form, November 8, 1995)
From the
The voltage-sensitive currents observed following hKv1.5
subunit expression in HEK 293 and mouse L-cells differ in the kinetics
and voltage dependence of activation and slow inactivation. Molecular
cloning, immunopurification, and Western blot analysis demonstrated
that an endogenous L-cell Kv
2.1 subunit assembled with transfected
hKv1.5 protein. In contrast, both mRNA and protein analysis failed to
detect a
subunit in the HEK 293 cells, suggesting that functional
differences observed between these two systems are due to endogenous
L-cell Kv
2.1 expression. In the absence of Kv
2.1, midpoints
for activation and inactivation of hKv1.5 in HEK 293 cells were
-0.2 ± 2.0 and -9.6 ± 1.8 mV, respectively.
In the presence of Kv
2.1 these values were -14.1 ±
1.8 and -22.1 ± 3.7 mV, respectively. The
subunit
also caused a 1.5-fold increase in the extent of slow inactivation at
50 mV, thus completely reconstituting the L-cell current phenotype in
the HEK 293 cells. These results indicate that 1) the Kv
2.1
subunit can alter Kv1.5
subunit function, 2)
subunits are
not required for
subunit expression, and 3) endogenous
subunits are expressed in heterologous expression systems used to study
K
channel function.
Voltage-gated K channels represent a diverse
group of membrane proteins in terms of both function and structure.
These channels establish the resting membrane potential and modulate
the frequency and duration of action potentials in nerve and
muscle(1, 2, 3) . In addition, K
channels are involved in processes not usually associated with
electrically excitable membranes such as T-lymphocyte activation, cell
volume regulation, and pancreatic beta cell
function(4, 5, 6, 7) . Multiple Shaker-like K
channel
subunit genes
have been cloned from mammalian brain, heart, skeletal muscle,
pancreas, and smooth muscle(5, 8, 9) . In
order to assess channel function, these proteins have been expressed in
heterologous expression systems as diverse as Xenopus oocytes,
mouse L-cells, CHO (
)cells, and HEK 293
cells(10, 11, 12, 13, 14, 15) .
Although there are some functional differences between these expression
systems, it has been generally assumed that these systems faithfully
reproduce native channel activity.
The differences in structure and
function between the cloned voltage-gated K channel
subunits, combined with the finding that they can form functional
heteromeric structures, indicate that potassium channel contribution to
excitable membrane function is
complex(13, 16, 17, 18) . In
addition, the recent discovery of function-altering
subunits,
some of which can convert a delayed rectifier into a rapidly
inactivating channel, has added yet another layer of
complexity(19) . Five different
subunits have been
described in detail, one from Drosophila(20) and the
others from
mammals(19, 21, 22, 23, 24, 25) .
Three of these subunits, Kv
1.1, 1.2, and 1.3, induce a variable
degree of rapid inactivation to delayed
rectifiers(19, 22, 23, 24) . The
fourth protein, Kv
2.1, was reported to have no effect on Kv1.1 or
1.4 currents in the Xenopus oocyte system(19) .
However, most recently Kv
2.1 has been shown to increase the N-type
inactivation of Kv1.4 2.3-fold(26) . The Kv
1.1, 1.2, and
2.1 subunits were originally designated
1, 3, and 2, respectively.
The Kv
1.1-1.3 proteins arise by alternative splicing from
the same gene, whereas Kv
2.1 is derived from a distinct gene (24, 26) . The nomenclature used here reflects a
subfamily classification based on genomic structure as previously
proposed(24) . An issue that must be resolved with all
heterologous expression systems is whether they contain endogenous,
function-altering
subunits.
The Kv1.5 delayed rectifier has
been cloned from heart, insulinoma tissue, gastrointestinal smooth
muscle, and skeletal muscle from rat, mouse, canine, and human
species(10, 11, 27, 28, 29, 30) .
The present study was undertaken to determine why the human Kv1.5
channel has different properties when expressed in the HEK 293 system
compared with L-cells. Specifically, in L-cells, the voltage
sensitivity is shifted 10 mV in the negative direction and slow
inactivation is increased. The data presented here indicate that the
L-cells express a Kv2.1 subunit isoform that assembles with the
transfected Kv1.5
subunit. The HEK 293 cells lack a
subunit
based on both mRNA and protein analysis. Coexpression of the mouse
L-cell
subunit with the Kv1.5 channel in the HEK 293 cells
reconstitutes the L-cell current phenotype. These results indicate that
the functional differences observed between the L-cell and HEK 293
expression systems are due to the presence of an endogenous L-cell
subunit and represent the first description of functional effects
of Kv
2.1 on delayed rectifier function.
For functional analysis, transient expression of
hKv1.5 with or without mKv2.1 in HEK 293 cells was obtained using
the lipofectamine method according to suppliers directions. GFP was
coexpressed with the channel subunits to assess transfection efficiency
(10-30%) and identify cells for voltage clamp
analysis(36) . The transient transfection used 0.5 µg of
hKv1.5/pBK
, 2 µg of Kv
2.1/pBK
, and
4 µg of GFP/pRC
mixed with 25 µl of lipofectamine
reagent. The lipofection mixture was applied for 2-3 h, after
which the standard culture medium was restored. The duration of the
lipofection was reduced because the standard 6-h exposure routinely
resulted in expression levels exceeding 5 nA (at 50 mV). The cells were
removed from the dish using brief trypsinization, washed twice with
maintenance medium, and stored at room temperature for recordings
within the next 12 h. Voltage clamp recordings revealed typical hKv1.5
currents in 100% of the cells expressing GFP. Control cells
(transfected without channel subunits or nonfluorescing cells) did not
display these currents, although an endogenous current of variable but
small size (50-150 pA) was observed in a subset of these cells.
This problem was minimized by using cell lines of low passage number.
The voltage dependence of channel opening and
inactivation (activation and inactivation curves) were fitted with a
Boltzmann equation y = 1/{1 +
exp[-(E - E)/k]} in which k represents the slope factor and E
represents
the voltage at which 50% of the channels are open or inactivated,
respectively. Because inactivation was incomplete, data were normalized
after subtraction of the noninactivating fraction at the test
potential. The time course of tail currents and slow inactivation were
fitted with a sum of exponentials. Activation kinetics were fitted with
a single exponential to the latter 50% of activation to obtain the
dominant time constant of activation(37, 38) . The
curve fitting procedure used a nonlinear least squares (Gauss-Newton)
algorithm; the results were displayed in linear and semilogarithmic
format together with the difference plot. Goodness of the fit and
required number of exponential components was judged by statistically
comparing
values (F-test) and by inspection for
systematic nonrandom trends in the difference plot.
The results are expressed as the means ± S.E. Analysis of variance with appropriate post hoc comparisons were used to compare the differences in mean values; p < 0.05 was considered significant. Specific n values are presented in the text or figure legends. All pooled data were collected from at least three separate transfections.
Figure 1:
Northern analysis of K channel
subunit expression in heterologous expression
systems. Total RNA (10 µg/lane) from L-cells, Madin-Darby canine
kidney (MDCK), LLC-PK, CHO, and HEK 293 cell lines as well as
from Xenopus oocytes and rat brain was analyzed for
subunit expression as described under ``Experimental
Procedures.'' The blot was exposed for 1 h. Exposure for 24 h
failed to detect
subunit expression in the Madin-Darby canine
kidney, LLC-PK, HEK 293, or oocyte lanes. The positions of the
ribosomal subunits are indicated.
In order to identify
the subunit isoform present in L-cells, a cDNA library was
constructed and screened as described under ``Experimental
Procedures.'' Two clones (1.5 and 1.0 kilobases) were purified to
homogeneity and excised into pBluescript. The longer clone was
sequenced in both directions, yielding an open reading frame of 1101
nucleotides. An in-frame 5` stop codon was found at -30, thus
ensuring that the full coding region was obtained. Translation of the
coding region predicted a 367-amino acid 41-kDa protein identical to
rat Kv
2.1 with 96.4% nucleotide identity in the coding
region(19) . The nucleotide and amino acid sequence of the
mouse Kv
2.1 (mKv
2.1) is depicted in Fig. 2. The
sequence between the arrows represents the region used to
generate the Kv
2.1-specific antisera. The shaded residues represent amino acids within this region that differ between the
Kv
2.1 isoform and the Kv
1.1, 1.2, and 1.3 proteins. Despite
81% sequence identity in this region between all known
subunits,
the antiserum raised was specific for the Kv
2.1 subunit as
determined by Western analysis as described under ``Experimental
Procedures.''
Figure 2:
Nucleotide and amino acid sequence of the
Kv2.1 subunit cloned from mouse L-cells. The COOH-terminal region
used for antibody production is located between the arrows. Shaded residues represent amino acids not shared in this
region with the Kv
1 subfamily. Upstream start codons at -97
and -136 are indicated by boxes. The upstream stop
codon, in-frame with mKv
2.1, is indicated by a bar above
the sequence at -43.
L-cells transfected with
either hKv1.5 or a sham vector were labeled with
[S]methionine, solubilized, and immunopurified
with anti-hKv1.5 antisera as described under ``Experimental
Procedures'' (Fig. 3, lanes 1 and 2,
respectively). Comparison of the two lanes indicates that two prominent
bands with electrophoretic mobilities corresponding to molecular
weights of approximately 40,000 and 75,000 are present exclusively in
the Kv1.5 transfected cells. Similar results were obtained when the
channel proteins were immunopurified with either anti-Kv1.5 (S1-S2 or
NH
-terminal) antisera. The larger protein represents the
Kv1.5
subunit, whereas the smaller band of 40 kDa has the
predicted mass of the cloned mKv
2.1 subunit protein. Additionally, in vitro translated
[
S]methionine-labeled mKv
2.1 comigrated
with the immunopurified lower mass protein (data not shown). The
Western blot analysis illustrated in lane 3 of Fig. 3demonstrates that the 40-kDa protein copurifying with
Kv1.5 is Kv
2.1. The Kv1.5 channel was immunopurified from
nonlabeled hKv1.5- or sham-transfected L-cell membranes (Fig. 3, lanes 3 and 4, respectively), fractionated by SDS gel
electrophoresis, transferred to nitrocellulose, and incubated with the
anti-Kv
2.1 antiserum. The 40-kDa band was specifically detected by
anti-Kv
2.1 antibodies in the material purified from the
hKv1.5-expressing cells, demonstrating that this band represents the
mKv
2.1 protein, as opposed to a proteolytic fragment of the
subunit.
Figure 3:
Assembly of Kv1.5 and Kv2.1.
Immunopurification of hKv1.5 associated proteins from hKv1.5- and
sham-transfected L-cells (lanes 1 and 2) and hKv1.5
and sham-transfected HEK 293 cells (lanes 5 and 6) is
shown. Subunit assembly was detected by antibody based copurification
from [
S]methionine/cysteine-labeled cultures as
described under ``Experimental Procedures.'' Western analysis
of immunopurified hKv1.5 from hKv1.5- and sham-transfected L-cells (lanes 3 and 4) was performed with Kv
2.1
specific antiserum. The immunopurification shown was performed with
anti-Kv1.5 (N-term) antiserum. The position of the primary IgG antibody
from the immune purification as detected with the goat anti-rabbit
secondary antibody is indicated by the arrow. The bands representing the
and
subunits are labeled accordingly.
The mobility of the molecular weight standards is
indicated.
The absence of detectable subunit mRNA in HEK 293
cells does not guarantee a complete absence of
subunits. Another
family of subunits could exist that does not cross-hybridize, even at
low stringency, with the cDNA probe used in the Northern analysis.
Therefore, the hKv1.5 protein was immunopurified from transfected and
radiolabeled HEK 293 cells, looking specifically for a copurifying
protein of 30-50 kDa. As shown in Fig. 3(lanes 5 and 6), when HEK 293 cells transfected with hKv1.5 were
immunopurified (lane 5), a unique doublet, probably
representing the immature and glycosylated forms of hKv1.5, appeared at
66 and 75 kDa. These
bands were absent when nontransfected cells
were used as the starting material (lane 6). Low molecular
weight bands representing putative
subunits are noticeably absent
from lane 5, demonstrating that
subunits capable of
associating with hKv1.5 are absent from the HEK 293 cells. Because
functional channels are obtained after transfection of the
subunit alone,
subunits are not required for the synthesis of
functional channels. It is also unlikely that the
subunit plays a
role in regulating cell surface channel density because the current
densities are similar between the L-cell and HEK 293 expression systems
(10, 14).
Fig. 4shows
typical recordings from HEK 293 cells transiently transfected with
hKv1.5 alone or with mKv2.1, (A and B,
respectively). Tracings for depolarization from -80 mV to
potentials between -30 mV and +50 mV were superimposed for
comparison. No voltage-activated current was observed between
-100 and -40 mV, but a progressively larger outward current
was recorded with depolarization above -30 mV. In both A and B of Fig. 4, the activation of the current
proceeded with a sigmoidal time course, and the rate of activation
increased with depolarization. Upon repolarization to -30 mV,
outward tail currents were recorded. These hKv1.5 currents were in
qualitative agreement with observations in various expression
systems(10, 11, 12, 13, 14) .
However, when hKv1.5 was coexpressed with Kv
2.1, substantially
more current was activated at the lowest depolarizations as indicated
by the arrows in A and B. Although slow
inactivation was limited in both cases, the current declined more when
hKv1.5 was coexpressed with mKv
2.1, especially at the strongest
depolarizations.
Figure 4:
Functional analysis of Kv1.5 and
Kv2.1 coexpression in HEK 293 cells. A and B show the expression of hKv1.5 in HEK 293 cells without and with
the mKv
2.1 subunit, respectively. Outward currents were elicited
by a series of 250-ms step depolarizations from a holding potential of
-80 mV to potentials between -30 and +50 mV in 10-mV
increments. Note that the 0-mV stimulus is indicated in both panels. C shows the voltage activation curve, in the presence and the
absence of the mKv
2.1 subunit. D illustrates the voltage
dependence of the activation time constant plus and minus mKv
2.1. E illustrates the voltage dependence of inactivation with and
without the mKv
2.1 subunit. Pulse protocols are as described
previously(14) . F summarizes effects of the
mKv
2.1 subunit on hKv1.5 slow inactivation. The data are plotted
as the percentage of inactivation (relative to peak current) at the
indicated pulse duration. The asterisks represent significant
differences (p < 0.01). In A, B, C, and E, representative data from a single paired
experiment are shown, i.e. the curves in C and E were obtained from the same cells represented in A and B. For D and F, data were derived from five
or six separate cells from three independent transfections. The dashed lines in C and D represent data
obtained from hKv1.5 expression in mouse
L-cells(14) .
To directly quantitate the effects of mKv2.1
coexpression, we analyzed the voltage-dependence of activation from the
amplitude of the decaying tail currents. Fig. 4C shows
that coexpression with mKv
2.1 results in a negative displacement
of the activation curve. The sigmoidal voltage-dependence was fitted
with a single Boltzmann equation resulting in half-activation voltages
of -0.2 ± 2.0 mV (n = 8) and -14.1
± 1.8 mV (n = 9; p < 0.01) without
and with mKv
2.1, respectively. The slope factors were not
significantly different (6.2 ± 0.6 mV without and 5.6 ±
0.4 mV with mKv
2.1, p > 0.1). Because the steady-state
voltage dependence and the kinetics of activation are determined by the
same underlying rate constants, we determined whether a similar shift
existed in the activation kinetics. For comparison with previous
results, the dominant time constant of activation was determined (see
``Experimental Procedures''). Fig. 4D shows
that coexpression of mKv
2.1 resulted in a faster activation rate
between -10 and +60 mV, resulting in a parallel shift of the
same magnitude as for the activation curve of C. The results
obtained in the absence of mKv
2.1 correspond closely to those of
Fedida et al. who expressed hKv1.5 in HEK 293
cells(10) . The dashed lines in C and D of Fig. 4illustrate the corresponding results previously
obtained in L-cells under identical conditions(14) . In both
cases, the results for coexpression of hKv1.5 with mKv
2.1
correspond closely to hKv1.5 expressed in L-cells.
Like many delayed
rectifiers, hKv1.5 displays partial and slow inactivation, presumably
of the C-type(41) . Often, hKv1.5 expressed in oocytes displays
less extensive inactivation than in the L-cells. Therefore we compared
both the voltage dependence of slow inactivation and the degree of slow
inactivation at 250 ms, 1 s, and 5 s during depolarizations to 50 mV. Fig. 4E shows the results from the cells shown in A and B. Coexpression with mKv2.1 resulted in a
hyperpolarizing displacement of the normalized inactivation curve with
average values E
= -9.6 ± 1.8
mV (n = 6) and -22.1 ± 3.7 mV (n = 5; p < 0.01) without and with mKv
2.1,
respectively. Slope factors were not significantly different (5.2
± 0.4 and 5.1 ± 0.2, p > 0.1). Because the
voltage dependence of C-type inactivation appears to be linked to that
of activation(41) , these results are consistent with the
effect of Kv
2.1 on activation. Indeed, when the difference in the
midpoints of activation and inactivation were determined for each cell
individually, we observed a similar displacement between both curves of
9.8 ± 1.3 mV (n = 6) and 8.9 ± 2.8 mV (n = 5) for hKv1.5 alone or with mKv
2.1,
respectively (p > 0.1). This displacement is consistent
with the 8.6-mV displacement observed in L-cells(14) . The
presence of the mKv
2.1 subunit also appeared to enhance the degree
of slow inactivation as illustrated in Fig. 4(A and B). Fig. 4F shows that the average amount of
inactivation (with respect to the peak current) at 250 ms, 1 s, and 5 s
was greater when hKv1.5 was coexpressed with mKv
2.1. The
percentage of inactivation in the HEK 293 cells expressing both hKv1.5
and mKv
2.1 compares well with the results for hKv1.5 in L-cells
(69% at 60 mV)(14) . It should be noted that the comparison is
somewhat complicated by the high temperature sensitivity of this
process(14) . Nevertheless, the results from the HEK 293 cells
reported here were obtained at the same temperature. The mKv
2.1
subunit had no effect on ion selectivity based on the finding that the
reversal potential was unchanged in the presence of mKv
2.1:
-82.3 ± 1.4 mV (n = 4) with
and
-83.7 ± 0.9 (n = 8) without
. No
effect of mKv
2.1 on channel surface density was detected, making
it unlikely this subunit plays a regulatory role in channel
biosynthesis, although such a role may be apparent only in the native
cell.
The data presented here are the first to indicate that
Kv2.1 modifies delayed rectifier potassium channel function.
Immunopurification of hKv1.5 from HEK 293 cells indicated that these
cells do not contain a
subunit that assembles with hKv1.5.
Because these cells are capable of expressing hKv1.5 current when
transfected with only the
subunit, the
subunit is not
required for expression. Coexpression of Kv
2.1 with Kv1.5 in HEK
293 cells resulted in a 10-mV leftward shift in the activation curve
and an almost 2-fold increase in the degree of slow inactivation. The
resulting HEK 293 cell current closely mimics that recorded from hKv1.5
transfected CHO and L-cell lines (which contain endogenous
subunits). In addition to demonstrating a functional effect of
Kv
2.1 on Kv1.5, these data illustrate the need to test for and
identify the
subunits present in heterologous expression systems.
The Kv1.5 K channel is expressed most abundantly in
cardiac myocytes and vascular smooth muscle(32) . Because the
Kv
2.1 subunit has also been cloned from cardiac
tissue(23) , the potential exists for Kv1.5 and Kv
2.1
coassembly in heart. Such in vivo assembly would result in an
earlier activation of the rectifying potassium current, perhaps serving
as a regulatory mechanism controlling action potential duration. The
Kv
2.1 subunit has several potential phosphorylation sites that
could regulate assembly and/or interaction with the
subunit,
allowing for a rapid response system controlling heart rate. A search
for potential pathophysiologic conditions resulting from altered
Kv
2.1 expression must wait until the cell-specific expression and in vivo
subunit association is confirmed.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L48983[GenBank].