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INTRODUCTION |
Voltage-gated potassium channels, the largest and the most diverse
group of ion channels, play a central role in the propagation of
signals and the determination of cellular excitability (1). At least
four subfamilies of voltage-gated K+ channels have been
identified. These subfamilies encode the Shaker (Kv1),
Shab (Kv2), Shaw (Kv3), and Shal (Kv4)
channel polypeptides and their mammalian homologues, which are highly
conserved across species. Each channel is synthesized as a monomeric
subunit, which assembles into a pore-forming tetrameric channel.
These channels share several common architectural designs, such as a conserved core domain which is comprised of six transmembrane segments,
and a teteramerization domain (T1 or NAB) at the amino terminus
(2).
During the past several years, we have learned a great deal about the
rules governing the assembly and multimerization of voltage-gated
potassium channels. It is now established that the co-assembly of
monomeric subunits occurs primarily within the same subfamily of Kv
channels, resulting in the formation of either homo- or
heterotetrameric complexes (3-7). The domain that determines the
specificity of subunit interactions consists of about 114 amino acids
and is located at the NH2 terminus. This domain is referred
to as the tetramerization (T1) or NAB domain (3-7). Other regions in
the central core may also be involved in channel co-assembly (8).
The role of the first transmembrane segment (S1) in the co-assembly of
the Shaker-related and Aplysia K+
channels was first characterized in our report and the reports of
others (9-11). These reports were based on deletion analyses of these
channels studied by several different approaches, including dominant
negative expression assays, co-immunoprecipitation assays, and
hydrodynamic analysis of co-assembled complexes (9-11). It was
hypothesized that the S1 segment played a direct allosteric role in
stabilizing the assembly of the functional channel complexes (9, 10).
The S1 segments of all four subfamilies have two completely conserved
amino acids, which in Kv1.1 correspond to Ile-177 and
Ser-180. Here we examine the role of these residues in Kv1.1. Our
results provide evidence that rather than playing a role in the
assembly and targeting to the plasma membrane, these residues are
directly involved in interactions that are critical to the proper
functioning of Kv1.1 channels.
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MATERIALS AND METHODS |
Preparation and Quantitation of cRNA--
Capped T7 run-off
transcripts of gel-purified ApaI linearized templates were
prepared using mMessage mMachine kit (Ambion). Accurate quantitation of
the cRNAs and their integrity were examined using spectrophotometry,
formaldehyde-agarose gel electrophoresis, and autoradiography for which
[
-32P]UTP (NEN) was added as a tracer in the reactions.
Two-electrode Voltage Clamp of Xenopus Oocytes--
The
isolation and preparation of the oocytes was carried out as described
by Stühmer and Parekh (12). For co-injections, a fixed amount of
the wild type cRNA (0.69 ng) was mixed with the mutant cRNA at 1:1 and
1:3 ratios. Whole oocyte currents were recorded 2-5 days
post-injection by a two-microelectrode voltage clamp using a GeneClamp
500 amplifier (Axon Instruments, CA). The bath solution contained (in
mM): NaCl 96, KCl 2, CaCl2 0.5, MgCl2 0.5, and HEPES 10, pH 7.5. The oocytes were held at
70 mV (in some experiments,
80 mV) and the currents were elicited by a series of 200-ms depolarization pulses from the holding potential to +50 mV in 10 mV increments followed by a repolarization to 50 mV (in
some experiments,
60 mV). A P/4 subtraction protocol was used to
minimize the leakage and the capacitative currents (13). Data were
expressed as mean ± S.E. Student's t test was used to
evaluate the statistical significance.
Binomial Distribution and Its Use to Determine the Potency of
Dominant Negative Suppression--
The fraction of the current carried
by the homo- and heterotetrameric channel complexes (n = 4) containing mutant subunits (i = 0, 1, 2, 3, or 4)
can be calculated using the binomial equation (14),
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(Eq. 1)
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The total amount of cRNA injected at 1:1 and 1:3 ratios of wild
type to mutants is increased 2- and 4-fold, respectively; therefore,
more channel complexes are expected to form. The amplitudes of the
resultant currents would be expected to be greater than that of the
wild type alone, if the subunits do not exert dominant negative
suppression. The percentage of the current will be the sum of the
conducting complexes and can be predicted if one, two, or three
subunits are required to block the channels (15, 16).
Construction of FL-Kv1.1, FL-Kv1.1Ile177, FL-Kv1.1Ser180 Mutants,
and TR-Kv1.1HA--
Kv1.1 cDNA cloned from rat soleus muscle (17)
was the starting template for creating the constructs. It was amplified
by polymerase chain reaction using the T7 primer and a redundant reverse primer carrying Arg, Leu, and Pro codons at either
Ile-177 or Ser-180 in the S1 segment and a
silent mutation to introduce an XhoI site. The polymerase
chain reaction product was subcloned and sequenced. An
XhoI/KpnI fragment from Kv1.1 cDNA was added to each mutant to generate the full-length channel construct in Bluescript II vector (Stratagene) (FL-Kv1.1). Each of these Kv1.1Ile177 and Kv1.1Ser180 mutants was digested with KpnI enzyme,
blunt-ended with T4 DNA polymerase, and then digested with
BstEII enzyme. These fragments were then used to generate
the FLAG epitope-tagged Kv1.1 (FL-Kv1.1) and the FLAG epitope-tagged
mutants (FL-Kv1.1Ile177 or FL-Kv1.1Ser180) in pCDNA3
(11). We used the gene SOEing method to construct the truncated Kv1.1
containing the NH2-terminal 205 amino acid residues tagged
with three copies of the HA epitope (TR-Kv1.1HA) (18).
Transient Transfections in COS-7 Cells--
COS-7 cells were
grown to about 70% confluency in Petri dishes (100 mm). LipofectAMINE
reagent (30 µl/10 µg of DNA) was used in Opti-MEM medium (Life
Technologies, Inc.) for 24 h and then changed to the complete
medium for a total period of 66-72 h (11). The cells were washed with
2 × 5 ml of PBS1 and
scrapped in a total 1.5 ml of chilled PBS on ice. For co-translation and co-assembly analysis, one-half of the cells transfected with either
the FL-Kv1.1 or the TR-Kv1.1HA were mixed at this stage. The cells were
pelleted and stored at
70 °C until used.
Cell Lysis, Immunoprecipitation, and Immunoblotting--
The
cells were lysed for 5 min on ice in 1 ml of lysis buffer (mM final
concentration): NaCl 150; Tris/HCl, pH 8.0, 10; EDTA 0.5; iodoacetamide
1; phenylmethylsulfonyl fluoride 0.5; 1% Triton X-100 and 4 µg each
of pepstatin A, chymostatin, leupeptin, antipain, and bestatin (Sigma).
Equal amounts of protein (19) were used for co-immunoprecipitation in
three sequential steps. In the first step, 4 µg of anti-HA antibody
(12CA5, Boehringher) was added (overnight at 4 °C). Protein
G-agarose slurry was added, mixed (4 °C/4 h), and centrifuged. The
pellets were then washed with lysis buffer. Supernatants from the first
step were used for the second round of immunoprecipitation with 12CA5
antibody exactly as described above. In the final step, supernatants
from the second step were used for immunoprecipitation with M2
anti-FLAG antibody (19 µg) (Eastman). The precipitates were
electrophoresed in 10% polyacrylamide-SDS gel, blotted onto
polyvinylidene difluoride membranes, and probed with the anti-FLAG
antibody. After washing with PBS, the blots were incubated with
horseradish peroxidase-conjugated goat anti-mouse IgG (Zymed
Laboratories Inc.) for 1 h at room temperature. The protein
bands were detected by using chemiluminescence. After stripping, the
blots were re-probed with anti-HA antibody (12CA5). The luminograms
were scanned using a Hewlett-Packard Scan Jet 4C/T scanner at 600 disintegrations/min (sharp black and white photo). The images were
analyzed and the bands of interest were quantitated as integrated
densities by Scion Image program. The values obtained were linear at
the exposure intervals we used.
Immunocytochemistry and Laser-scanning Confocal
Microscopy--
The cells were grown on coverslips in 6-well tissue
culture dishes. For co-transfections, 1 µg of each cDNA was used.
The cells were washed (3 × 1.5 ml of PBS with Ca2+
and Mg2+), fixed with 2% paraformaldehyde, permeabilized
with 0.2% Triton X-100 in PBS and reacted with the anti-FLAG antibody
(M2) (1:1000) (20). The secondary antibody used for staining was the
fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:100)
(Jackson ImmunoResearch). The cells were visualized by laser-scanning
confocal microscopy (MRC1024, Bio-Rad).
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RESULTS |
Ile-177 and Ser-180 Mutants Did Not Express Outward Potassium
Currents in Oocytes--
Fig.
1A shows the primary structure
of the first transmembrane segment (S1) of Kv1.1 and the mammalian
homologues of four voltage-gated K+ channels, Shaker,
Shaw, Shab, and Shal. The wild type Kv1.1 polypeptide used in this study was tagged with a FLAG epitope at the
NH2 terminus (FL-Kv1.1) (Fig. 1B). Ile-177 and
Ser-180, the two completely conserved amino acids in the S1
segment, were mutated to arginine, leucine, or proline (FL-Kv1.1I177R,
FL-Kv1.1I177S, FL-Kv1.1I177P, or FL-Kv1.1S180R etc.) (Fig.
1B). Oocytes injected with FL-Kv1.1 cRNA showed a robust
expression of a rapidly activating, non-inactivating outward potassium
current (Fig. 2A). The
inclusion of a FLAG epitope at the amino-terminal end did not produce
any noticeable change in the properties of this current (10, 17). In
contrast, oocytes injected with each of the mutant cRNA showed no
detectable outward potassium currents even after injection of higher
doses of cRNAs or recording after longer post-injection periods (Fig.
2, B and C). Thus, the mutation of either
Ile-177 or Ser-180 to Arg, Leu, or
Pro abolished the Kv1.1-encoded outward potassium currents.

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Fig. 1.
The primary structure of the S1 segments of
voltage-gated Kv channels. A, the sequence of amino
acids 168-186 in rat Kv1.1 (above) and the corresponding
Shaker (below) K+ channels are shown. The amino
acid sequence is shown in a single-letter code. Dashes
indicate identity. The consensus sequence at the bottom contains all
possible primary structures of the S1 segment of mammalian Kv1 through
Kv4 and Drosophila channels. Mutations introduced at Ile-177
and Ser-180 are boxed. B, schematic
representation of FL-Kv1.1 or the FL-Kv1.1Ile177 and FL-Kv1.1Ser180
constructs (above) and an HA-tagged truncated TR-Kv1.1HA (below) in
pCDNA3 vector.
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Fig. 2.
Two-electrode voltage clamp analysis of
macroscopic Kv1.1 currents expressed in Xenopus
oocytes. A-C show representative current traces
recorded from oocytes injected with 0.92 ng of cRNA encoding FL-Kv1.1,
FL-Kv1.1S180P, and FL-Kv1.1I177L, respectively. D, pulse
protocol used. E, steady-state current-voltage (I-V)
relationships of the FL-Kv1.1 current. F, current amplitudes
at 0 mV were plotted as a function of the amount of the FL-Kv1.1 cRNA
injected. Data were fitted by a linear equation: f (x) = 42.4x 1.5 (r = 0.994). Data are
shown as mean ± S.E. (n = 3-4).
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Dominant Negative Suppression of Kv1.1-encoded Currents by Ile-177
and Ser-180 Mutants--
To address the question of whether the
mutations affected the ability of these subunits to co-assemble with
the Kv1.1 polypeptide, we carried out cRNA co-injection experiments. In
these experiments, the wild type and mutant subunits were expected to
co-assemble into both homo- and heterotetrameric complexes, since they
all have an intact T1 domain. The probability of each type of homo- and
heterotetrameric complexes that would form is given by a binomial distribution (see "Materials and Methods"). The total current observed would be the sum of the currents carried by each of the conducting complexes. An accurate prediction by this equation rests
upon several critical parameters. 1) The amplitudes of the expressed
currents must be linear as a function of the amount of cRNA injected
and the protein it yielded. 2) Both the wild type and the mutant
subunits must be able to co-assemble with an equal probability. 3)
Co-assembly with one or more mutant subunits may or may not completely
suppress the assembled channel's conductance, but should not alter it.
In order to ensure the validity of the first consideration, we first
measured the amplitude of outward potassium currents expressed in
oocytes injected with increasing amounts of FL-Kv1.1 cRNA, using the
same pulse protocol as above (Fig. 2D). As clearly shown in
Fig. 2, E and F, the linearity of the current
amplitudes was maintained fairly well at different voltages with the
amounts of cRNA tested. Based on these results, we chose to use 0.69 ng of FL-Kv1.1 cRNA (expressing 10 to 30 µA current at 0 mV) in all our
co-injection experiments. Co-injection of FL-Kv1.1 with mutant cRNAs at
a 1:1 ratio resulted in a significant suppression of Kv1.1-encoded
currents (Fig. 3, A and
C-H, p < 0.01). The extent of current
suppression was greatly enhanced at a 1:3 ratio of wild type to mutant
cRNA (Fig. 3, C-H), except for the FL-Kv1.1I177P mutation,
which had no noticeable effect on the amplitude of FL-Kv1.1 encoded
currents (Fig. 3G). By contrast, we did not observe any suppression of these currents by co-injection of a 3-fold excess of
control cRNA (pTRI-Xef1) (Fig. 3B). Co-injection of the S1 mutant cRNAs did not alter the slope conductance, voltage-dependence or
kinetics of activation of the Kv1.1-encoded currents (data not shown).
Thus, suppression of the Kv1.1-encoded currents was specifically
related to the presence of the mutant cRNAs. Using the binomial
equation, the percentages of the predicted currents at both 1:1 and 1:3
ratios of co-injections are given if one or more mutant subunits are
sufficient to block the formation of functional channels (Table
I). The observed values were very close
to the predicted values at the 1:3 ratio, assuming that co-assembly
with one mutant subunit was sufficient to block the formation of
functional channels. At a lower ratio of 1:1, a somewhat weaker
suppression than predicted was observed. This phenomenon may have
resulted from the lesser amounts of protein expressed by the mutant
cRNAs as compared with the amount expressed by the wild type, thereby
shifting the actual ratios of the subunits available for co-assembly.
Indeed, we have consistently observed variations in the level of
expression of mutant proteins in transfected COS-7 cells (see protein
results below). The protein analysis also revealed that the level of
the expressed FL-Kv1.1I177P protein was dramatically reduced, thus
explaining its inability to suppress the currents in oocytes.

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Fig. 3.
Dominant-negative suppression of
FL-Kv1.1-encoded currents by the FL-Kv1.1Ile177 and FL-Kv1.1Ser180
mutants. A, typical Kv1.1 currents recorded from an
oocyte injected with 0.69 ng of FL-Kv1.1 (top), co-injected
with FL-Kv1.1 and FL-Kv1.1S180R (1:1) (middle), or
co-injected with FL-Kv1.1 and FL-Kv1.1I177L (1:1) (bottom).
B-H, I-V curves after co-injecting the FL-Kv1.1 cRNA ( )
with a control cRNA (B), FL-Kv1.1S180L (C),
FL-Kv1.1S180P (D), FL-Kv1.1S180R (E),
FL-Kv1.1I177L (F), FL-Kv1.1I177P (G), and
FL-Kv1.1I177R (H) at 1:1 ( ) and 1:3 ( ) ratios. Data
are shown as mean ± S.E. (n = 4-9).
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Table I
Effects of co-injection of the Ile-177 and Ser-180
mutant cRNA with the wild type FL-Kv1.1 cRNA on current amplitude
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FL-Kv1.1Ile177 and KL-Kv1.1Ser180 Mutants Form Both Homo- and
Heteromultimeric Complexes--
To obtain direct evidence for
co-assembly at the protein level, we carried out co-transfection
experiments in COS-7 cells, followed by co-immunoprecipitation
analyses. The FL-Kv1.1, FL-Kv1.1Ile177, FL-Kv1.1Ser180 mutants, and an
HA epitope-tagged truncated Kv1.1 construct (TR-Kv1.1HA) were
transfected (Fig. 1B). As previously established by us, an
HA-tagged deletion fragment of similar size was necessary and
sufficient to retain its ability to co-assemble with Kv1.1, Kv1.4, and
Kv1.5 channels in vitro, in GH3 cell lines and mouse heart
(10, 11, 21).
The principle of this method is based on a quantitative depletion of
the pool of one of the interacting proteins (the full-length FL-Kv1.1,
the FL-Kv1.1Ile177, or FL-Kv1.1Ser180 mutants), which is in direct
co-assembly with TR-Kv1.1HA. The heteromultimeric complexes
were precipitated by the addition of non-limiting amounts of anti-HA
antibody directed against TR-Kv1.1HA. Subsequently, anti-FLAG antibody
was used to precipitate the remaining pool of homomultimeric complexes
and unassembled FL-Kv1.1 subunits (or S1 mutants). The typical results
obtained are presented in Fig. 4,
A and B. When FL-Kv1.1 was transfected alone,
anti-FLAG antibody could immunoprecipitate multiple polypeptides with
apparent molecular masses of 56 to 59 kDa (Fig. 4A, lane 3).
These bands most likely correspond to the differentially glycosylated
and phosphorylated forms of Kv1.1
subunit (17, 22, 23). In contrast, two successive rounds of immunoprecipitation with anti-HA antibody failed to precipitate any Kv1.1 polypeptide (Fig. 4A, lanes 1 and 2). The multiple, closely moving bands of
FL-Kv1.1 and FL-Kv1.1Ile or FL-Kv1.1Ser mutants that we have observed
here have similar molecular masses and therefore reflect different maturation stages. The transfection of TR-Kv1.1HA alone followed by its
precipitation by either anti-HA antibody or anti-FLAG antibody showed
that it could only be brought down by anti-HA antibody (Fig. 4B,
lanes 4 and 5), not by anti-FLAG antibody (Fig.
4B, lane 6). TR-Kv1.1HA coded for two polypeptides with an
apparent molecular mass of ~32 kDa. Their mobility corresponded to
that of the in vitro translated TR-Kv1.1HA (not shown). When
FL-Kv1.1 and Tr-Kv1.1HA were co-transfected, most of the multiple bands that correspond to FL-Kv1.1 polypeptides were immunoprecipitated by
anti-HA antibody (Fig. 4A, lanes 7 and 8),
whereas only a small fraction immunoprecipitated with the anti-FLAG
antibody (Fig. 4A, lane 9). A corresponding depletion of the
interacting TR-Kv1.1HA protein in two sequential rounds with anti-HA
antibody can be seen in Fig. 4B (lanes 7 and 8),
confirming their co-assembly with FL-Kv1.1. A third round of
immunoprecipitation with anti-FLAG antibody did not precipitate any
TR-Kv1.1HA polypeptides (lane 9), validating the usefulness
of the scheme. In contrast, in the mixed lysates of separately
transfected COS-7 cells, the FL-Kv1.1 protein could be brought down
only by anti-FLAG antibody and not by anti-HA antibody (Fig. 4A,
lanes 10-12). This observation confirms that the co-assembly of
FL-Kv1.1 and TR-Kv1.1HA proteins depended upon their co-translation.
Mock transfection using the vector alone did not yield any specific
protein bands (Fig. 4, A and B, lanes
13-15).

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Fig. 4.
Quantitative co-immunoprecipitation of
TR-Kv1.1HA with FL-Kv1.1, FL-Kv1.1Ile177, and
FL-Kv1.1Ser180 mutants. A, immunoblot analysis,
using anti-FLAG antibody of FL-Kv1.1 (WT), FL-Kv1.1Ile177
(I177R, I177S, I177L), and FL-Kv1.1Ser180 mutant (S180R, S180L, S180P)
polypeptides immunoprecipitated with either anti-HA or anti-FLAG
antibody. The bracket denotes the identical positions of the
multiple bands of either FL-Kv1.1 or FL-Kv1.1 mutants. The constructs
transfected are indicated above by a + or by the mutant name.
B, immunoblot analysis, using anti-HA antibody of the
truncated TR-Kv1.1HA polypeptides immunoprecipitated by either anti-HA
or anti-FLAG antibody. The position of TR-Kv1.1HA polypeptides is
marked with an arrow. Lane numbers are indicated below.
C, a histogram showing the % of the FL-Kv1.1 polypeptides
(WT) or the mutants immunoprecipitated with anti-HA antibody
(12CA5) and anti-FLAG antibody. Mean ± S.D. from three
independent experiments shown.
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All the FL-Kv1.1Ile177 and FL-Kv1.1Ser180 mutants were then tested for
their ability to co-assemble by this sequential method. Fig.
4A shows the positions and the intensities of the
polypeptides encoded by FL-Kv1.1Ile177 and FL-Kv1.1Ser180 mutants.
Co-transfection of each of the FL-Kv1.1Ile177 or FL-Kv1.1Ser180 mutants
with TR-Kv1.1HA resulted in the co-precipitation of multiple protein
bands with apparent molecular masses similar to those of the FL-Kv1.1
polypeptides. However, we consistently observed variations in their
expression levels (Fig. 4A, lanes 16-33). Co-transfection
of the FL-Kv1.1I177L followed by co-precipitation yielded somewhat
elevated levels of this protein. In contrast, co-transfection of
FL-Kv1.1I177P resulted in the co-precipitation of dramatically reduced
levels of protein (Fig. 4A, lanes 31-33). Other mutants
displayed lesser variations, and their co-expression levels were
comparable to those achieved with co-transfected wild-type constructs
(Fig. 4A, lanes 7-9). Despite these variations, the mutants
were efficiently co-immunoprecipitated with the anti-HA antibody (Fig.
4A, lanes 16-33). A corresponding depletion of the
TR-Kv1.1HA with anti-HA antibody is not shown for the mutants, since it
was exactly as obtained in the controls (Fig. 4B). Fig.
4C shows the ratios of the FL-Kv1.1 and mutant polypeptides
that were immunoprecipitated with either the anti-HA or the anti-FLAG
antibody. It is evident that larger fractions of the wild type
(~83%) and the mutant proteins (70-75% for FL-Kv1.1Ile177 mutants
and 93-96% for FL-Kv1.1Ser180 mutants) were co-immunoprecipitated
with anti-HA antibody (heteromultimeric complexes), whereas much
smaller fractions precipitated with the anti-FLAG antibody
(homomultimeric complexes and unassembled subunits). The FL-Kv1.1I177P
mutant was excluded from this analysis because of the barely detectable
level of its protein.
FL-Kv1.1Ile177 and FL-Kv1.1Ser180 Mutatnts Are Targeted to the
Plasma Membrane of Sol8 Cells--
The failure of all of our mutants
to form functional channels in oocytes led us to speculate that they
were "folding mutants" which were trapped in the endoplasmic
reticulum compartment and were therefore unable to reach the plasma
membranes (11, 21, 24). Hence, we decided to study their subcellular
localization using immunofluorescence imaging of transfected cells.
This study could also shed some light on the hitherto unknown
subcellular distribution of defective K+ channels in
episodic ataxia (25) and in several types of long QT syndrome (26).
Since the FL-Kv1.1 cDNA was cloned from a rat soleus cDNA
library (17), we decided to examine its subcellular expression in Sol8,
a myogenic cell line derived from the mouse slow-twitch soleus muscle.
Indeed, transiently transfected FL-Kv1.1 could be detected on the
membranes of Sol8 cells as well as in the cytoplasm (Fig.
5B). Interestingly, we
consistently observed that plasma membrane staining was more commonly
found in regions of high confluency where some of the cells appeared to
fuse, reminiscent of myotube formation. Untransfected Sol8 cells
displayed some background fluorescence, but did not reveal any membrane
staining (Fig. 5A). Thus, these results demonstrated that
FL-Kv1.1 could be targeted to the plasma membrane of Sol8 cells. When
we tested for membrane expression of FL-Kv1.1I177R, FL-Kv1.1I177L, and
FL-Kv1.1Ser180 mutants, they could all be detected on the plasma
membranes of transfected Sol8 cells. Two representative examples,
FL-Kv1.1I177L and FL-Kv1.1S180P, are shown in Fig. 5, C and
D. Thus, the mutation of either Ile-177 or Ser-180 to
leucine, arginine, or proline did not prevent the targeting of the
Kv1.1 polypeptides to the plasma membrane. Transient transfection
experiments of FL-Kv1.1 and FL-Kv1.1 mutants into other cell lines
(including COS-7, HEK293, Chinese hamster ovary, and Madin-Darby canine
kidney) did not yield sufficient plasma membrane expression detectable
by anti-FLAG antibodies. In contrast, the transfection of a control
membrane protein, the cationic amino acid transporter (27), revealed clear membrane staining in these cells (data not shown). Taken together, these results indicate that Sol8 cells can express detectable levels of FL-Kv1.1 polypetides on the plasma membrane, with or without
mutations in the S1 region.

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Fig. 5.
Membrane immunolocalization of transiently
transfected FL-Kv1.1, Kv1.1Ile177, and Kv1.1Ser180 mutants.
A, confocal immunofluorescence imaging of untransfected Sol8
cells using anti-FLAG antibody. B-D, confocal
immunofluorescence imaging of Sol8 cells transfected with FL-Kv1.1,
FL-Kv1.1I177R, and FL-Kv1.1S180P, respectively. Laser-scanning
confocal microscopy (MRC 1024, Bio-Rad) was used to examine the cells
(10-30% laser power). The captured images were saved in an Adobe
PhotoShop format and adjusted equally by using the
"brightness/contrast" from the menu.
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DISCUSSION |
Significance of the Results of Ile-177 and Ser-180 Mutations in the
S1 Segment of Kv1.1--
A significantly high level of conservation of
amino acids in the core region of voltage-gated channels is indicative
of functionally important sites and forms an important basis for their
three-dimensional structural and functional modeling (28). The
mutations of either Ile-177 or Ser-180, the two completely conserved
amino acid residues in the S1 segments of the Kv1-4 voltage-gated
K+ channels, abolished the expression of outward potassium
currents in oocytes (Fig. 2, B and C). In
transfected COS-7 cells, all mutant cDNA constructs (except
FL-Kv1.1I177P) directed the synthesis of proteins, which were matured
normally and formed products of the expected molecular mass, albeit
with some variation in their steady-state levels (Fig. 4). It is
conceivable that the presence of the potentially helix-disrupting
proline residue at position 177 resulted in decreased protein stability.
Our analysis indicated that a block in the biosynthesis or maturation
of Kv1.1 polypeptides could not explain the generation of
non-functional channels. Indeed, the mutations did not alter the
consensus sites for the processing and maturation of Kv1.1 protein in
transfected cells (22, 23, 29, 30). Moreover, most of the wild type or
mutated Kv1.1 polypeptides formed heteromultimeric complexes with
TR-Kv1.1HA (Fig. 4C). The smaller fraction, which was
immunoprecipitated with anti-FLAG antibody in the last step, most
likely contains both homomultimeric channel complexes and unassembled
subunits. These results were in agreement with those from the
electrophysiological measurements, in which co-injection of the mutants
with the wild type cRNA in Xenopus oocytes suppressed most
of the Kv1.1 encoded currents (Fig. 3). Collectively, we conclude that
none of the Ile-177 or Ser-180 mutations obliterated the co-assembly of
homo- or heteromultimeric complexes with either the wild type or the
mutated subunits. Furthermore, the incorporation of a single mutant
subunit was most likely sufficient to suppress Kv1.1-encoded currents.
The first transmembrane segment plays a critical role in initiating the
insertion of newly translated polypeptides into the endoplasmic
reticulum membrane and in promoting the stability and clustering of the
other transmembrane segments of membrane proteins (31). Indeed, several
studies (9, 32), including ours (10, 11), have shown that the deletion
of this segment abolished the assembly of Kv1 subunits. The distinct
staining of each of the mutant FL-Kv1.1 channel protein on the plasma
membranes of the Sol8 cells is an intriguing phenomenon (Fig. 5). We
speculate that differentiated Sol8 myocytes express high levels of
membrane-associated proteins that are important for the trafficking and
membrane expression of Kv1.1 polypeptides. Assuming a co-translational
co-assembly for all subunits (shown for the wild type in Fig. 4A,
lanes 10-12), the staining probably represents homomultimeric
complexes and not a single subunit. These complexes could reach the
plasma membrane and form non-functional channel complexes. In this
context, it is worth noting that the W434F, a mutation in the pore
region of the Shaker B K+ channel which rendered
the channel non-conducting, apparently reached the membranes and
"expressed" gating currents (33).
A Proposed Role for Ile-177 and Ser-180 in Critical Subunit
Interactions--
The S1 segment contains both the hydrophobic and
hydrophilic residues (Fig. 1A). Current three-dimensional
structural models of voltage-gated K+ channels predict that
the S1 segment forms a transmembrane amphipathic
helix (34). This
model depicts the S1 segment closely packed with the S2 and S4 segments
in a cylindrical bundle of
helices in the outer half of the
channel's "open" conformation. Direct experimental evidence for
functionally critical interactions among the charged residues in the
S2, S3, and S4 segments has emerged from recent elegant biochemical
studies on the native Shaker K+ channel (35) and
from studies investigating the interactions of synthetic transmembrane
segments in phospholipid membranes (36). Our evidence for the existence
of non-functional channels in the membranes suggests that both the
Ile-177 and Ser-180 residues in the S1 segment of Kv1.1 might be
critically important in helix-helix interactions, rather than in the
assembly process per se, as we had previously hypothesized
(10). It is likely that inclusion of one "bad" subunit in the
tetrameric channel here could hinder critically important intra- or
intersubunit interactions, which could in turn impede the sequential
steps as the channel proceeds from the closed to the final open state.
This is perhaps the best interpretation of all our results, which
showed that all of the mutant proteins (except the I177P) were well
expressed, could co-assemble to form multimeric complexes, exerted a
dominant negative effect on the wild-type K+ currents, and
could also reach the plasma membranes. In summary, our results
highlight the important role that the Ile-177 and Ser-180 residues play
in the function of Kv1.1 channels. These observations will help
increase our understanding of the cellular mechanisms in long
QT-syndrome and ataxia/myokymia, which have been envisaged to arise by
dominant negative mechanisms of defective voltage-gated K+
channels, including Kv1.1 (37).