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The Journal of Neuroscience, April 15, 1998, 18(8):2842-2848
Episodic Ataxia Mutations in Kv1.1 Alter Potassium Channel
Function by Dominant Negative Effects or Haploinsufficiency
Patricia
Zerr1,
John P.
Adelman1, and
James
Maylie2
1 Vollum Institute and 2 Department of
Obstetrics and Gynecology, Oregon Health Sciences University, Portland,
Oregon 97201
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ABSTRACT |
Subunits of the voltage-gated potassium channel Kv1.1 containing
mutations responsible for episodic ataxia (EA), a human inherited neurological disease, were expressed in Xenopus oocytes.
Five EA subunits formed functional homomeric channels with lower
current amplitudes and altered gating properties compared with wild
type. Two EA mutations located in the first cytoplasmic loop, R239S and
F249I, yielded minimal or no detectable current, and Western blot
analysis showed reduced protein levels. Coinjection of equal amounts of
EA and wild-type mRNAs, mimicking the heterozygous condition, resulted
in current amplitudes and gating properties that were intermediate
between wild-type and EA homomeric channels, suggesting that
heteromeric channels are formed with a mixed stoichiometry of EA and
wild-type subunits. To examine the relative contribution of EA subunits
in forming heteromeric EA and wild-type channels, each EA subunit was
made insensitive to TEA, TEA-tagged, and coexpressed with wild-type
subunits. TEA-tagged R239S and F249I induced the smallest shift in TEA
sensitivity compared with homomeric wild-type channels, whereas the
other TEA-tagged EA subunits yielded TEA sensitivities similar to
coexpression of wild-type and TEA-tagged wild-type subunits. Taken
together, these results show that the different mutations in Kv1.1
affect channel function and indicate that both dominant negative
effects and haplotype insufficiency may result in the symptoms of
EA.
Key words:
episodic ataxia; neurological disease; K channel; oocyte
expression; haploinsufficiency; dominant negative effects
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INTRODUCTION |
The genetic lesions responsible for
many inherited neuromuscular and ataxic syndromes have been identified
and in many cases occur in the coding sequence of an ion channel gene
(Kraus and McNamara, 1995 ; Ackerman and Clapham, 1997; Greenberg,
1997). In some instances, the biophysical properties of the mutant
channel have been examined, but for many the underlying molecular
mechanism has not been established.
Episodic ataxia (EA) is an autosomal dominant neurological disorder,
affecting both central and peripheral nerve functions, with symptomatic
attacks of imbalance and uncontrolled movements (Ashizawa et al.,
1983). The attacks of ataxia may be induced by physical or emotional
stress; they usually last several minutes and may occur several times a
day. Although the symptoms are varied between and within families,
presumably reflecting the outbred nature of the human population, two
symptoms are always observed: an ataxic gait during attacks and
myokymia, characterized by a continuous muscle activity, which is
presented as a rhythmic electromyography activity with a pattern of
repeated duplets and multiplets (Gancher and Nutt, 1986; Brunt and
Weeden, 1990).
Genetic linkage studies have localized the EA syndrome locus to
chromosome 12p13 (Litt et al., 1994) and, subsequently KCNA1, the gene
encoding the voltage-gated delayed rectifier K+
channel Kv1.1, was identified as underlying EA (Browne et al., 1994).
All of the mutations occur in positions highly conserved among the
voltage-dependent K+ channel superfamily. In each
affected family, a different missense point mutation has been
identified in the coding sequence of Kv1.1, and all affected
individuals are heterozygous (Browne et al., 1994, 1995).
K+ channels are formed by the assembly of four
subunits (MacKinnon, 1991), and if wild-type and EA alleles are
expressed, both homomeric and heteromeric channels may be formed.
Our previous study showed that some of the EA alleles encode functional
channels and indicated that the autosomal dominant phenotype may be
attributed to heteromeric channel assembly of wild-type and EA subunits
(Adelman et al., 1995). In this report we show that three alleles
previously thought to encode nonfunctional subunits form functional
channels when higher levels of expression are achieved. Five EA
subunits that form functional but aberrant channels coassembled with
wild-type subunits and showed different dominant negative potencies
when expressed at a 1:1 ratio. Two EA subunits with the least dominant
negative impact on heteromeric channels also have markedly reduced
protein levels. These results show that different mechanisms may
underlie altered K+ channel function in EA-affected
individuals.
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MATERIALS AND METHODS |
Xenopus care and handling were in accordance with the
highest standards of institutional guidelines. Frogs underwent no more than two surgeries, separated by at least 3 weeks. To isolate oocytes,
frogs were anesthetized with an aerated solution of 3-aminobenzoic acid
ethyl ester. Standard recording solution contained (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.4. All chemicals were from
Sigma (St. Louis, MO). Two-electrode voltage-clamp recordings were
performed at room temperature with a Geneclamp 500 amplifier (Axon
Instruments, Burlingame, CA) interfaced to a Macintosh Quadra 800 computer. Linear leak and capacitance currents were corrected with a
P/4 leak subtraction procedure. Data collection and analysis were
performed using Pulse, PulseFit (Heka), IGOR (Wavemetrics), and
KaleidaGraph (Synergy Software). Statistical significance was
determined by an unpaired Student's t test, and
p < 0.01 was considered significant.
Human Kv1.1 cDNA was cloned into the vector pS
(Promega, Madison, WI). Site-directed mutagenesis, nucleotide
sequencing, and in vitro mRNA synthesis were performed as
described previously (Adelman et al., 1995). Equal amounts of DNA were
linearized and transcribed using common pools of reagents; mRNAs were
initially evaluated by denaturing gel electrophoresis and ethidium
bromide stain as well as by spectrophotometer. For rigorous
quantification, mRNAs were diluted in triplicate (1:20 dilution) and
dot-blotted in duplicate onto a GeneScreen filter (DuPont NEN, Boston,
MA), which was then hybridized to a Kv1.1-specific radiolabeled
oligonucleotide. To assure saturating hybridization, the radiolabeled
oligonucleotide was present in vast excess to the target mRNA
(>200-fold), and hybridization was allowed to proceed overnight in a
small volume. After washes, hybridization signals were quantified with
a scintillation counter or imaged with a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA), and the density was determined with NIH-Image
1.59 software. Based on this quantification, mRNA amounts were adjusted
to yield the ratios indicated in the text. Oligonucleotides were
purchased from Genosys.
Western blots were performed as described (Tucker et al., 1996). Only
paired groups of oocytes with wild-type or EA mRNAs in which wild-type
currents were >30 µA were used. Total oocyte membranes were prepared
using a standard method (Geering et al., 1989). Briefly, 25 oocytes
were washed twice, suspended in 1 ml of PBS containing 0.1 mM phenylmethylsulfonyl fluoride and 5 µg/ml leupeptin,
aprotinin, and pepstatin A, and homogenized, first by five passages
through a 28 gauge needle and then two passages through a 27 gauge
needle. The homogenates were centrifuged repeatedly at 2000 × g for 10 min at 4°C until all yolk granules and
melanosomes were pelleted, typically three or four times. The final
supernatant was pelleted at 60,000 × g for 30 min at
4°C to generate a total membrane fraction devoid of yolk granules.
This membrane pellet was resuspended in 25 µl of 50 mM
Tris, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, pH
8.0, and 25 µl of the loading buffer and stored at  20°C. Samples
representing five oocytes were subjected to SDS-PAGE using a 10%
resolving gel and 3% stacking gel in a Bio-Rad (Hercules, CA)
Miniprotean II apparatus. Proteins were transferred to nitrocellulose
filters, and Western blot analysis was performed using an anti-mouse
Kv1.1 polyclonal antibody (a generous gift from Dr. Bruce Tempel,
Department of Otolaryngology, University of Washington, Seattle, WA).
Antibodies were detected using the ECL detection system (Amersham,
Arlington Heights, IL) according to the manufacturer's instructions,
and signals were quantified using IP labgel software (Molecular
Dynamics).
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RESULTS |
Expression of EA subunits in Xenopus oocytes
The positions of the six EA mutations introduced into the human
Kv1.1 cDNA are shown in Figure
1A. To determine the
functional consequences of the EA mutations, equal amounts of wild-type
and EA mRNAs were separately injected into Xenopus oocytes,
and currents were examined in the two-electrode voltage-clamp
configuration. As described previously, oocytes expressing F184C and
V408A subunits had reduced current amplitudes measured at 40 mV
compared with oocytes expressing wild-type subunits (Fig.
1B) (Adelman et al., 1995). V174F, R239S, F249I, and
E325D had been reported previously as not forming functional channels.
However, when higher levels of expression were achieved, either by
injecting more mRNA or by waiting longer after injection, currents were
detected with V174F, F249I, and E325D, although in all three cases the
current amplitudes were 10- to 100-fold lower than that of wild type
(Fig. 1B). In contrast, oocytes expressing
R239S never yielded currents different from noninjected oocytes. The
current amplitude at 40 mV for each EA mutant relative to wild type is
summarized in Table 1.
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Figure 1.
A, Schematic representation of the
membrane topology of Kv1.1 subunits. The positions of the six EA point
mutations studied are indicated. B, Histogram showing
current amplitudes recorded from oocytes injected with wild-type and EA
mRNAs equilibrated as described in Materials and Methods. Currents were
measured at the end of a 200 msec pulse to 40 mV. Error bars indicate
SD; the number of cells recorded is indicated in each case.
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As described previously (Adelman et al., 1995), F184C currents showed a
shift in the voltage dependence of ~25 mV with slowed activation
kinetics, whereas V408A currents showed accelerated activation and
deactivation kinetics and increased C-type inactivation (Table 1). To
compare the effects of V174F, F249I, and E325D on gating,
representative current traces recorded at 40 mV were overlaid and
scaled to a representative wild-type current trace (Fig.
2). The comparison showed differences
between these EA and wild-type channels that may reflect shifts in
voltage dependence of activation and/or effects on channel kinetics.
Therefore, the biophysical properties for these EA mutants were
individually investigated.
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Figure 2.
Characterization of V174F, F249I, and E325D.
A, V174F; B, F249I; C,
E325D. Top panels, Traces representing currents evoked
from 60 to 50 mV depolarizing pulses (increments of 10 mV) from a
holding potential of 80 mV. Tail currents were recorded at 50 mV.
Second panels, Current traces for each EA mutation
(thick lines) were scaled and superimposed to a
representative wild-type current trace (thin lines).
Currents were evoked at 40 mV from a holding potential of 80 mV, and
tail currents were recorded at 50 mV. Third panels,
Voltage dependence of activation from a single representative
experiment. The normalized tail currents recorded at 50 mV (V174F,
F249I) or at 30 mV (E325D) were plotted as a function of the
preceding depolarizing potential (60 to 65 mV). The tail currents
were fitted with a single exponential, and the amplitude of the
exponential was used to describe the tail currents. Data points were
fitted according to the Boltzmann equation I = 1/(1 + exp-(V V1/2)/k, where
V1/2 is the potential of
half-activation, and k is a slope factor. Bottom
panels, Time constants of activation (filled
symbols) and deactivation (open symbols) from a
single representative experiment. Activation of currents was best
described with a sum of two exponentials, and the time constant of the
fast exponential was plotted here, whereas a single exponential was
sufficient for the deactivation. Data points were fitted according to
the equation  = V1/2 · e(V V1/2)/k, where
V1/2 is the
time constant at V1/2, and
k is the slope factor for the voltage dependence of the
time constants.
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V174F shifts positively the voltage dependence
of activation
The voltage dependence of activation for V174F channels was
examined by measuring tail currents at 50 mV after voltage commands to potentials between 60 and 65 mV. The normalized tail currents plotted against command potential showed that the voltage dependence of
activation was shifted to positive potentials (Fig. 2A,
third panel). The voltage dependence was quantified by
fitting the data to a Boltzmann equation, which showed a shift of the
voltage for half-maximal activation,
V1/2, of 35 mV compared
with wild-type channels (Table 1). The overlaid traces in Figure
2A show that V174F activation at 40 mV was slower and
deactivation at 50 mV was faster than in wild type. The kinetics of
activation were measured by fitting the rising phase of the current
traces evoked at different voltages with a double exponential; the fast
time constant, which accounted for >75% of the current, was plotted versus test potential (Fig. 2A, bottom panel).
Deactivation rates were determined from tail currents evoked from 10
to 70 mV after a 100 msec test pulse to 40 mV; deactivation was
fitted with a single exponential, and the time constant was plotted
versus tail potential (Fig. 2A, bottom panel).
Throughout the voltage range, V174F activation was slower and
deactivation was faster than in wild type. Because the voltage
dependence of current activation was affected, the kinetics of V174F
were compared with those of wild type by determining the time constants
of activation and deactivation at their respective
V1/2 values (Fig. 2).
Interestingly, when compared at
V1/2, the kinetics of
activation and deactivation for V174F were similar to those for wild
type (Table 1). These results showed that the effect of V174F on
channel gating may be explained solely by a shift in voltage
dependence. In addition, C-type inactivation, evaluated using a 10 sec
command pulse to 20 mV, was slightly larger than for wild type (Table
1).
F249I slows the deactivation
Analyses of F249I currents showed that the voltage dependence and
kinetics of activation were similar to those of wild type (Fig.
2B, Table 1). However, Figure 2B
shows that deactivation was slower and the corresponding time constant
at V1/2 was increased approximately twofold without a significant change in voltage steepness
(Table 1). C-type inactivation may be less pronounced for F249I (Table
1); however, the relatively small current amplitudes did not permit a
clear analysis because of contamination with endogenous currents.
E325D shifts positively the voltage dependence of activation and
increases the rate of activation, deactivation, and C-type
inactivation
The voltage dependence of activation was examined by measuring
tail currents at 30 mV. Figure 2C revealed a dramatic
shift in the voltage dependence of E325D; the
V1/2 was shifted ~60 mV, and the steepness factor was increased approximately twofold, indicating a reduced voltage dependence (Table 1). As shown in Figure
2C and summarized in Table 1, time constants of activation and deactivation at V1/2
were faster compared with wild type by a factor of ~6. However, in
contrast to the other EA mutants, the voltage dependence of the
kinetics was considerably reduced compared with wild type. Similar to
V408A, C-type inactivation of E325D was faster than for wild type,
yielding an increased amount of inactivation (Table 1).
Coexpression of wild-type and EA subunits
EA is an autosomal dominant disease, and all known individuals are
heterozygous. If both EA and wild-type Kv1.1 alleles are expressed to a
similar extent in vivo, the EA and wild-type subunits may
assemble according to a binomial distribution (MacKinnon, 1991;
Kavanaugh et al., 1992), or they may only form homomeric EA and
wild-type channels. Therefore, to examine the effects of expressing
each EA subunit with wild-type subunit, equal amounts of EA and
wild-type mRNAs were coinjected. Compared with currents recorded from
oocytes injected with the same total amount of mRNA for wild type only
(homozygous wild-type condition), the current amplitudes measured at 40 mV were reduced in oocytes coinjected with half wild-type mRNA and half
mRNA for each EA mutant (heterozygous condition) (Fig.
3). Comparison of current records
obtained at a test pulse potential of 20 mV and a tail potential of
50 mV showed that when some of the EA mutations were coexpressed with wild-type subunits, the currents demonstrated kinetics that were intermediate between those of homomeric EA and homomeric wild-type channels (Fig. 4). To examine these
differences in more detail, the voltage dependence of activation,
kinetics of activation at 20 mV and deactivation at 50 mV, and C-type
inactivation at 20 mV were assessed. Coinjection of R239S mRNA with
wild-type mRNA yielded currents with voltage dependence and kinetics
indistinguishable from those of homomeric wild type (Table
2). When coexpressed with wild type,
V174F and F184C yielded activation kinetics intermediate between
homomeric wild-type and the corresponding homomeric EA currents.
Similarly, currents from oocytes coinjected with wild-type and E325D or
V408A mRNAs yielded intermediate deactivation and C-type inactivation
kinetics, and coexpression of wild-type and V174F, F184C, or E325D
subunits showed an intermediate shift in activation voltage (Table 2).
These results suggest that V174F, F184C, E325D, or V408A subunits
coassemble with wild-type subunits, forming heteromeric channels with
altered gating properties.
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Figure 3.
Coexpression of EA and wild-type subunits.
Compared with oocytes injected with wild-type mRNA
(filled column), current amplitudes recorded from
oocytes coinjected with wild-type and EA mRNAs (open
columns) were reduced; relative amounts of mRNAs injected are
indicated below each column. Currents were measured at the end of a 200 msec pulse to 40 mV. Error bars indicate SD; the number of cells
recorded is indicated in each case.
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Figure 4.
Currents recorded from oocytes coexpressing EA and
wild-type subunits. Oocytes were injected with either EA or wild-type
mRNA alone (thin traces; * indicates wild type) or
coinjected with EA and wild-type mRNA at a 1:1 ratio (thick
traces). Currents were evoked at 40 mV (20 mV for R239S) from a
holding potential of 80 mV, and tail currents were recorded at 50
mV. For comparison of kinetics, the traces in each panel were scaled to
the same peak. Inset, Enlargement of tail currents
recorded at 50 mV.
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Heteromeric channels containing EA and wild-type subunits
To confirm this hypothesis, EA subunits were altered in
their sensitivity to external TEA (TEA-tagged EA subunits). The
Shaker K+ channel contains a tyrosine
residue within the P loop that endows homomeric channels with high
sensitivity to TEA (Ki = 0.2-0.4 mM);
substitution of a valine at this position reduces sensitivity to TEA
>100-fold (MacKinnon and Yellen, 1990). Substitution of a valine at
the equivalent position in Kv1.1 (Y379V) similarly reduced the
sensitivity to TEA ~100-fold (Fig. 5, filled squares). In
oocytes coexpressing TEA-insensitive (TEA-tagged) and TEA-sensitive subunits, the dose response to TEA reflects the number of
tyrosine-containing subunits within the channel (Kavanaugh et al.,
1992). TEA-tagged EA or wild-type mRNA was coinjected with wild-type
mRNA at a 1:1 ratio. The dose response to TEA was determined and
compared with that from oocytes injected with wild type alone (Fig.
5). The data points for wild type (Fig.
5, open circles) were fitted with a single binding isotherm
(Ki = 0.4 mM), and the data points for
wild type and TEA tagged wild type were fitted with a binomial equation
for a tetrameric channel, assuming subunit assembly without preference
for wild-type or TEA-tagged wild-type subunits (Fig. 5, filled
circles). The individual Ki values used in the
binomial equation for heteromeric channels were determined as described
previously and are given in Figure 5 (Kavanaugh et al., 1992). The
coinjection of equal amounts of wild-type and TEA-tagged V174F, F184C,
E325D, or V408A mRNAs yielded TEA dose responses similar to that for
wild-type mRNA coinjected with TEA-tagged wild-type mRNA. These results
suggest that heteromeric channels are formed from an equal number of
wild-type and V174F, F184C, E325D, or V408A subunits. However, the TEA
dose response was partially shifted for oocytes coexpressing wild-type
and TEA-tagged F249I subunits and was not significantly shifted for
wild type plus TEA-tagged R239S compared with wild type alone (Fig. 5). Application of the binomial equation to the TEA dose-response curve
showed that F249I contributed 0.3 of the total number of subunits
available for channel formation. These results are consistent with the
possibility that fewer subunits of R239S and F249I are available for
coassembly with wild-type subunits.
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Figure 5.
EA subunits coassemble with wild-type subunits.
TEA dose-response curves of oocytes injected with wild-type mRNA alone
( , n = 5), TEA-tagged wild-type mRNA alone ( ,
n = 7), or wild-type mRNA coinjected with either
TEA-tagged wild-type ( , n = 7) or TEA-tagged EA
(R239S,  , n = 4; F249I,  ,
n = 8; V174F,  , n = 6;
F184C,  , n = 2; E325D,  ,
n = 7; V408A,  , n = 5) mRNAs
at a 1:1 ratio. Currents were measured at the end of a 200 msec pulse
to 40 mV from a holding potential of 80 mV. Currents in the indicated
[TEA] were normalized by the control current. Data points for wild
type were fitted according to the equation: I = Ki/([TEA] + Ki), giving
a Ki for TEA of 0.4 mM. Data points
from the coinjection of wild type with TEA-tagged wild type, TEA-tagged
R239S, or TEA-tagged F249I were fitted according to the binomial
equation:
where f equals the fraction of wild-type subunits,
Cn4 = 4!/n!(4 n)!, and the individual Ki,n values were
0.4, 1.7, 9.8, 25.3, and 123.8 mM for n = 0-4, respectively. The values of f for wild type
coexpressed with TEA-tagged subunits of R239S, F249I, and wild type
were 0.94, 0.70, and 0.45, respectively. The TEA dose response of
homomeric TEA-tagged EA subunits was similar to that of homomeric
TEA-tagged wild-type subunits; the EA mutations do not alter
TEA sensitivity. Error bars indicate SD.
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Reduced amounts of protein for R239S and F249I
To examine the levels of EA and wild-type subunits in oocytes
individually injected with equal amounts of mRNA, total cellular membranes were isolated, and the proteins were prepared as a Western blot and probed with a Kv1.1 polyclonal antibody (Fig.
6). For the representative experiment
shown, similar amounts of protein were detected for wild type (1.0),
V174F (1.1), F184C (1.0), and V408A (1.0), whereas E325D had slightly
reduced levels (0.8). However, oocytes injected with mRNAs for R239S
and F249I contained markedly reduced protein levels, 0.4 and 0.2, respectively, compared with wild type.
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Figure 6.
Western blot of membranes from oocytes expressing
EA or wild-type subunits. Oocytes were injected with equal amounts of
wild-type or EA mRNAs. Total membranes were prepared from equal numbers
of oocytes for each sample, and a protein assay based on a Bradford
procedure was used to quantify the protein concentration. Equal amounts
of protein, representing approximately five oocytes, were loaded on
each lane and probed with the Kv1.1 polyclonal antibody. The
ladder on the left is in kilodaltons.
Doublets were observed at ~60 kDa, near the calculated molecular
weight for Kv1.1, which probably reflects glycosylated and
unglycosylated forms. The additional band detected at ~110 kDa for
V408A and wild type may be a result of to subunit dimerization. Among
the six EA mutants studied, R239S, F249I, and E325D showed lower
amounts of protein compared with wild type.
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DISCUSSION |
EA mutations in Kv1.1 have been examined for functional
differences compared with wild-type channels. Four of the functional EA
subunits that produce homomeric channels with biophysical properties different from wild-type channels are able to coassemble with wild-type
subunits, endowing the resulting heteromeric channels with partial EA
phenotypes. These alleles probably affect the symptoms of the disorder
through dominant negative interactions with wild-type subunits as well
as the intrinsic differences between homomeric EA and wild-type
channels. F249I, which also coassembles with wild-type subunits, shows
reduced levels of protein and may affect EA symptoms by both dominant
negative and haplotype insufficiency mechanisms. Finally, R239S
produces reduced but significant levels of protein but does not form
functional homomeric channels and does not coassemble with wild-type
subunits, suggesting that the R239S allele results in EA because of too
few Kv1.1 subunits, haplotype insufficiency.
Several EA subunits yielded homomeric channels with gating properties
distinct from wild-type channels. Interestingly, EA mutations in the
same domain of the subunit gave similar phenotypes. V174F and F184C
reside in the first transmembrane domain, and both showed a shift in
voltage dependence of activation of ~30 mV; F184C also had slower
activation kinetics. E325D and V408A, residing on opposite sides of the
deep pore in S5 and S6, respectively, both showed accelerated
activation and deactivation kinetics and faster C-type inactivation.
Additionally, for E325D the voltage dependence was shifted by ~60 mV,
and both activation and deactivation kinetics were less steeply
voltage-dependent. The absence or marked reduction of currents for
R239S and F249I, respectively, together with reduced amounts of
protein, suggest that the intracellular loop between transmembrane
domains 2 and 3 is critical for channel biosynthesis and subunit
stability.
To examine whether EA subunits coassembled with wild-type subunits,
mRNAs were coinjected in a 1:1 ratio, mimicking the in vivo
heterozygous condition. Current amplitudes from coinjected oocytes were
reduced compared with oocytes injected with the same total amount of
wild-type mRNA. In general, the altered functions seen from homomeric
EA channels were also observed in coinjected oocytes, although the
magnitude of the effects was less than in homomeric EA channels,
suggesting that heteromeric channels were formed. Indeed, current
measurements from oocytes coexpressing wild-type and EA subunits made
less sensitive to TEA (TEA-tagged) clearly demonstrated that the EA
mutants V174F, F184C, E325D, and V408A coassemble with wild type. The
TEA dose-response curves for these TEA-tagged EA subunits coexpressed
with wild type were similar to that seen for coexpression of wild-type
and TEA-tagged wild-type subunits. Accordingly, this suggests that
equal numbers of these EA and wild-type subunits are available for
channel assembly. However, the diminished current amplitudes of
homomeric V174F, F184C, and E325D channels, which are partially rescued
when these subunits are coexpressed with wild-type subunits, are
consistent with either a reduction in single-channel current and/or the
total number of channels. A reduction in the single-channel current caused by EA subunits may be conferred on heteromeric channels with
different potency depending on the stoichiometry of EA and wild-type
subunits. This would have been realized as a reduced shift in the TEA
dose-response experiments, which was not seen for V174F, F184C, E325D,
or V408A. In contrast, protein levels for V174F, F184C, and V408A
subunits, and to a lesser extent E325D subunits, were similar to those
of wild type, suggesting that heteromeric channels may not move to the
cell surface as readily as wild-type channels. Therefore, these EA
mutations may affect the symptoms of the disorder through both dominant
negative (altered channel function) and haplotype insufficiency effects
(reduced currents).
R239S and F249I may affect channel function in different ways.
Expression of TEA-tagged F249I subunits shifted the TEA sensitivity approximately two-thirds of that seen for coexpression for wild-type and TEA-tagged wild-type subunits, although R239S subunits had no
effect. Western blot analyses, using total cellular membranes, showed
reduced levels of protein compared with wild type for R239S (0.4) and
F249I (0.2). There is more protein for R239S than for F249I, yet R239S
has no obvious effects either when expressed alone (no current) or
together with wild type (no altered functions and no shift in TEA
sensitivity). In contrast, F249I channels are functional and only
modestly distinguished from wild-type channels, and coexpression with
wild-type subunits shifts TEA sensitivity. These results suggest that
F249I subunits are relatively unstable, but intact subunits coassemble
with wild-type subunits. Therefore, F249I subunits may demonstrate
reduced current amplitudes when expressed alone or with wild-type
subunits because of a reduction in the number of channels in the
membrane as well as possible effects on the single-channel currents. In
contrast, R239S subunits do not appear to form functional channels when
expressed alone and do not seem to coassemble with wild-type subunits,
even though there is more total cellular protein than seen for F249I.
These results suggest that R239S subunits are trapped inside the cell, unavailable for coassembly with wild-type subunits, and that the R239S
mutation gives rise to EA symptoms through haplotype insufficiency.
The EA E325D mutation is remarkable in several ways. This position,
which affects gating, is in a domain implicated as part the internal
pore region of Shaker-like potassium channels (Slesinger et
al., 1993), suggesting that E325 may participate in both gating and
conduction processes. The E325D mutation resides at the intracellular border of S5 and differs from wild type by a single side chain carbonyl. Yet E325D induces a 60 mV shift in activation voltage and a
reduction in slope; more energy is required for channel activation
compared with wild-type channels. It is possible that a D in this
position enables the side chain to form a salt bridge otherwise not
available when E occupies this position, and the strong gating effects
suggest that the counter charge may be one of the positively charged
residues in S4. The E325D phenotype is strikingly similar to those seen
after neutralization of E283 or D316 in Shaker. For D316,
there is evidence that this residue interacts with at least one of the
positively charged residues in S4 (Papazian et al., 1995; Seoh et al.,
1996). These and other results suggest that residues in S4 form salt
bridges with residues in S2 and S3 (Papazian and Bezanilla, 1997).
However, the E325D phenotype implicates additional domains beyond S2
and S3 that may couple with S4.
These studies indicate that, depending on the location within the Kv1.1
subunit, different parameters of channel function are affected, and
that each EA allele may result in different symptomatic severity.
Clinically, however, EA patients within a family and among families do
not show a pattern of severity that correlates with the underlying
alleles. This is likely caused by the heterogeneous genetic background
in humans. In contrast, introduction of different EA alleles into an
isogenic mouse background may reveal symptomatic differences obviated
by the heterologous expression studies presented here.
| |
FOOTNOTES |
Received Nov. 24, 1997; revised Jan. 20, 1998; accepted Jan 23, 1998.
This work was supported by National Institutes of Health grants to
J.P.A. and J.M. and a Medical Research Foundation grant to J.M.
P.Z. was supported in part by the Ministère des Affaires Etrangères de France and the Philippe Foundation. We thank
Zhaoping Liu for helpful discussions and Chris T. Bond for the EA
constructs.
Correspondence should be addressed to Dr. James Maylie, Department of
Obstetrics and Gynecology, Oregon Health Sciences University, L-458,
3181 SW Sam Jackson Park Road, Portland, OR 97201.
| |
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Top
Abstract
Introduction
![I=<LIM><OP>∑</OP><LL>n<UP>=</UP>0</LL><UL>4</UL></LIM> C<SUP>4</SUP><SUB>n</SUB> · f<SUP>4<UP>−</UP>n</SUP> · (1−f)<SUP>n</SUP> · K<SUB>i,n</SUB>/([<UP>TEA</UP>]+K<SUB>i,n</SUB>)](/content/vol18/issue8/fulltext/2842/img001.gif)
