From the Section of Molecular Cardiology, Departments
of Medicine and Molecular Pharmacology, Albert Einstein College of
Medicine, Bronx, New York 10461 and the ¶ Down Syndrome
Research Group Medical and Molecular Genetics Center-IRO, Hospital
Duran i Reynals Gran Via Km 2,7 08907 L'Hospitalet,
Barcelona, Spain
Received for publication, November 28, 2000
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ABSTRACT |
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KvLQT1 is a Shaker-like voltage-gated potassium
channel that when complexed with minK (KCNE1) produces the
slowly activating delayed rectifier Iks. The emerging family of
KCNE1-related peptides includes KCNE1 and KCNE3, both of which complex
with KvLQT1 to produce functionally distinct currents. Namely
Iks, the slowly activating delayed rectifier current, is
produced by KvLQT1/KCNE1, whereas KvLQT1/KCNE3 yields a more rapidly
activating current with a distinct constitutively active component. We
exploited these functional differences and the general structural
similarities of KCNE1 and KCNE3 to study which physical regions are
critical for control of KvLQT1 by making chimerical constructs of KCNE1 and KCNE3. By using this approach, we have found that a three-amino acid stretch within the transmembrane domain is necessary and sufficient to confer specificity of control of activation kinetics by
KCNE1 and KCNE3. Moreover, chimera analysis showed that different regions within the transmembrane domain control deactivation rates. Our
results help to provide a basis for understanding the mechanism by
which KCNE proteins control K+ channel activity.
KvLQT1 is a voltage-activated Shaker-type potassium channel widely
expressed in heart, kidney, colon, pancreas, and inner ear (1-3).
KvLQT1 (also known as KCNQ1) complexes with minK (KCNE1) to form
a slowly activating delayed rectifier current, Iks (4, 5).
KCNE1 is a member of a gene family that includes a number of
one-spanning membrane proteins whose association with potassium
channels controls their activity (6, 7). Iks, when compared
with KvLQT1 homotetrameric channels, shows an augmentation of whole
cell current amplitude, a depolarizing shift of the voltage dependence
of activation, removal of inactivation (8), and a slowing down of both
activation and deactivation kinetics (4, 5). Mutations in either
KvLQT1 (1, 9) or KCNE1 (10, 11) genes can
lead to Romano-Ward and Jervell and Lange-Nielsen syndromes, both
characterized by a prolonged Q-T interval due to delayed cardiac
repolarization and, in the case of Jervell and Lange-Nielsen syndrome,
congenital sensorineural deafness. The nature of the interaction
between KCNE1 and KvLQT1 has been the subject of much investigation. An
extensive mutagenesis study of KCNE1 (12) originally uncovered a region
of the protein within the N-terminal portion of the cytosolic domain
that appeared critical for KCNE1 function; changes within this stretch
were poorly tolerated. Romey et al. (13) provided
biochemical evidence in support of an interaction between the pore
region of KvLQT1 and the intracellular C terminus of KCNE1.
Cysteine-scanning mutagenesis (14, 15) provided evidence that residues
in the transmembrane domain line the channel conduction pore. Tapper
and George (16), using KCNE1 deletions and chimeras with a sodium
channel Recently, a novel KCNE family member, KCNE3, was shown to interact with
KvLQT1 to produce a potassium current with a constitutive component
(2). This current resembles that seen in colonic epithelia, where both
KCNE3 and KvLQT1 are expressed. Here we have exploited the marked
differences in the regulation of KvLQT1 by these two proteins to
investigate which regions of these two proteins mediate the differences
in effects on KvLQT1. The approach of KCNE1/KCNE3 chimeras has the
advantage of placing structural portions of KCNE1 in a homologous
environment, but one that can modulate KvLQT1 function quite
differently from KCNE1. By using this approach, we find that a
three-amino acid segment within the transmembrane domain of KCNE1 is
sufficient and necessary to determine the specificity of KCNE control
of channel activation. Additionally, our results show a physical
separation of determinants of activation and deactivation.
Cloning of KCNE3--
We detected human ESTs with homology to
KCNE1 via TBLASTN search. By overlapping the ESTs we constructed the
full-length cDNA. Forward and reverse primers from the 5'- and
3'-ends of the predicted coding region (5'-ATGGAGACTACCAATGGAACGGAG and
3'-TTAGATCATAGACACACGGTTCTT) were used in 5'- and 3'-rapid
amplification of cDNA ends PCR using the Marathon Ready cDNA
kit (CLONTECH) and human stomach cDNA. Several
rapid amplifications of cDNA end clones were sequenced and
assembled to get the full-length cDNA. The sequence has been reported to GenBankTM under accession number AF302494.
Construction of KCNE1/KCNE3 Chimeras--
Chimeras were
constructed using a PCR-based approach. Briefly, using PCR with
Pfu turbo (Stratagene) and 5'-phosphorylated primers, we
produced blunt-ended PCR fragments that corresponded to the regions of
interest from KCNE1 and KCNE3. Blunt-ended fragments from KCNE1 were
gel-purified, ligated overnight to a blunt-ended PCR product
corresponding to the appropriate region of KCNE3, and re-amplified by
PCR for subcloning into PCR Blunt II TOPO (Invitrogen). Chimera
cDNAs were digested using HindIII and EcoRI and ligated into the mammalian expression plasmid pcDNA3
(Invitrogen). Nomenclature of the primers and chimeras corresponds to
the order of fragments (M is minK (KCNE1) and K is KCNE3),
followed by the splice site in human KCNE1 when the sequences are
aligned as shown in Fig. 1b.
Thus, a chimera corresponding to the N-terminal 47 residues of KCNE1
followed by KCNE3 residues for the remainder of the protein is named
M(1-47)K. All primers used are in Fig. 1a. Primers below
the shaded gray line are complementary to fragments of both
KCNE1 and KCNE3 and were used to generate swaps of short (<7) amino
acids.
Cell Culture and Transfection--
CHO cells were maintained in
Ham's F-12 media supplemented with 10% fetal calf serum and
penicillin/streptomycin at 37 °C and 5% CO2. Gene
transfer was performed using 15 µg of Qiagen Midiprep purified
plasmid DNA. Cells were electroporated at 225 V, 72 Electrophysiology--
Cells were grown on sterile glass
coverslips and placed in an acrylic/polystyrene perfusion chamber
(Warner Instruments, Inc.) mounted on an inverted microscope outfitted
with fluorescence optics and patch pipette micromanipulators.
Extracellular solution was 150 mM NaCl, 1.8 mM
CaCl2, 4 mM KCl, 1 mM
MgCl2, 5 mM glucose, and 10 mM
HEPES buffer, pH 7.4, at room temperature. Intracellular pipette
solution was 126 mM KCl, 4 mM K-ATP, 2 mM MgSO4, 5 mM EGTA, 0.5 mM CaCl2, 25 mM HEPES buffer, pH
7.2, at room temperature. The whole cell configuration of the patch
clamp technique was used to measure potassium currents (20). An
Axopatch 1D patch clamp amplifier was used, and protocols were
controlled via PC using pCLAMP8. Data were filtered using an 8-pole
Bessel filter at 1 kHz and sampled at 4 kHz. For the protocol in Fig.
3c, lower trace, sampling was at 40 kHz.
Activation curves are derived from tail currents at 0 mV following a
series of 2 s. depolarizations and fitted to a Boltzmann curve
using the equation, I = 1/(1 + exp((Vh KCNE1 and KCNE3 Control KvLQT1 Activity--
Fig.
2 shows whole cell current recordings
from CHO cells transfected with KvLQT1 cDNA alone or in combination
with either KCNE3 or KCNE1. As previously reported, coexpression with
KCNE1 results in an increase in whole cell current amplitude (2.2-fold, see Table I) and the kinetics of both
activation and deactivation are slowed (Fig. 2, a and
b, and Table I) (4, 5). Coexpression of KCNE3/KvLQT1, as
previously reported (2), resulted in a current with a constitutively
active component (Fig. 2c), as viewed on faster time scales
and hyperpolarizations to KCNE Transmembrane Regions Determine Specificity of KCNE Control of
KvLQT1 Kinetics--
To determine the regions of KCNE1 and KCNE3 that
are responsible for the different effects on KvLQT1 function, we
swapped various segments of each to produce recombinant chimera
cDNAs. We then coexpressed the chimerical cDNA with KvLQT1 for
functional analysis of the resulting currents. Fig.
3, a and b, shows
the results of swapping the N-terminal 46 residues of KCNE1 with the corresponding segment of KCNE3, a stretch encompassing the
extracellular domain as well as four residues of the outer
transmembrane domain. Kinetics of both activation and deactivation of
chimeras K(1-47)M and M(1-47)K (rise times: K(1-47)M, 442 ± 15 ms, and M(1-47)K, 27 ± 1.6 ms) resemble KCNE1 and KCNE3,
respectively (rise times: 704 ± 27.5 and 21.1 ± 1.9 ms, see
also Fig. 3, a and b, and Fig. 5). Chimeras that
exchanged the intracellular domains plus 4 amino acids of the
transmembrane domain, M(1-63)K (rise time: 591 ± 70 ms) and
K(1-63)M (rise time: 14.3 ± 0.6 ms), resembled KCNE3 and KCNE1,
respectively (Fig. 3, c and d, and Fig. 5). These
chimeras thus define the transmembrane region of KCNE1 from Val-48 to
Leu-63 (corresponding to KCNE3 residues Leu-62 to Leu-77) that
appears to mediate the specific effects of these proteins, a hypothesis that we confirmed with chimera KM(48-63)K (Table I and Figs. 3e and 5).
Specificity of Control of KvLQT1 Activity Lies in a Small Region
within the Transmembrane Segment--
To examine the transmembrane
region in finer detail, we subdivided it by constructing chimeras
KM(48-56)K that had a clear constitutive component present and
activated with a rise time of 42.5 ± 3.2 ms (Fig.
4c), and KM(48-59)K (Fig.
4a) which, like KCNE1, showed no constitutive component and
slow activation (rise time 691 ± 34.9 ms). These chimeras showed
that activation kinetics follow amino acids 57-59 in the KCNE1
transmembrane domain (KCNE3 amino acids 71-73). This result indicated
that a region of only three residues was potentially responsible for
the activation differences seen in KvLQT1-KCNE1 complexes. We confirmed
this hypothesis by constructing three amino acid-swapped chimeras
KM(57-59)K and MK(57-59)M. As seen in Fig. 4, d and
e, and Fig. 5, kinetics of
activation of KM(57-59)K (rise time: 633 ± 26.2 ms), a chimera with 3 KCNE1 amino acids inserted into KCNE3, approximate those of
KCNE1. Activation kinetics of MK(57-59)M (rise time: 65 ± 6.4 ms), in turn, resemble those of KCNE3. A constitutively active current
was detected for MK(57-59)M; however, it composed a smaller fraction
of the total than that seen with wild-type KCNE3 (Table I), indicating
that additional residues outside this region may be involved in
generating the constitutive component.
Separation of KCNE Determinants of Activation and Deactivation
Rates--
In contrast to currents seen with KCNE1 or other
Iks-like chimeras (Table I and Figs. 5 and
6b), the kinetics of deactivation of KM(57-59)K are greatly accelerated (39 ± 1.0 ms versus 146 ± 13.5 ms for KvLQT1/KCNE1 currents). The
voltage-dependent current of chimera MK(57-59)M shows
activation kinetics that are somewhat slower than the KCNE3
voltage-dependent current alone (MK(57-59)M, 65 ± 6.4 ms, KCNE3, 21.1 ± 1.9 ms, and Fig. 6a) but nonetheless are ~9-fold faster than the rise time of
KvLQT1/KCNE1(704 ± 27.5 ms). Deactivation kinetics with this
chimera (123 ± 4.8 ms) are nearly as slow as that of KCNE1
(146 ± 13.5 ms), however. Comparison of deactivation rates for
chimeras KM(48-59)K and KM(57-59)K implicates a role for KCNE1
residues 48-56, lying N-terminal to this stretch, in determining the
slow kinetics of deactivation seen with Iks. In contrast,
incorporation of KCNE3 residues either N-terminal (MK(48-59)M) or
C-terminal (MK(57-63)M) to this stretch resulted in fast kinetics of
deactivation, similar to KCNE3 (Table I). A possible explanation is
that residues 48-56 of KCNE1 confer slow deactivation, whereas in
KCNE3, C-terminal residues (60) control fast deactivation. When
both segments are present in the same protein, however, the KCNE3
residues may act in a dominant fashion, but only when residues 57-59
are present as well (compare KM(48-56)K and MK(57-63)M, Table I).
KCNE3 Induces a Constitutively Active K+ Current
through Complex Physical Interaction with KvLQT1--
We determined
the presence of a constitutively active component to KCNE3 currents by
looking for the appearance of an instantaneous current following
depolarization, manifested as sharp corner before the onset of
voltage-activated current (see Figs. 2c, 3, and 4). From the
height of this step we estimated the fraction of total current that was
attributable to the constitutive component (Table I, last column). The
presence of constitutively active current was also seen using very fast
sampling rates and membrane hyperpolarizations to eliminate the
possibility of a very fast-activating voltage-dependent current (Figs. 2c and 4e). All chimeras
containing KCNE3 amino acids 71-73 exhibited such a current. In
contrast, chimera KM(57-59)K which consists essentially of KCNE3 with
amino acids 71-73 replaced with KCNE1 residues 57-59 does not exhibit
any observable constitutive component to its current, demonstrating
that these residues are necessary for generating the constitutive
component (Fig. 4, d and e) The fraction of total
current provided by this constitutive component, however (0.18 ± 0.03), was smaller for chimeras containing these and surrounding
residues than for KCNE3 (constitutive component is 0.302 ± 0.03 of total current). Thus, although residues 71-73 are necessary for
constitutive activation, they are not sufficient for its full
expression. Our results suggest that full control of the constitutively
active component may be more complex than kinetics of voltage
activation. Interestingly, Cd2+ treatment of a T59C KCNE1
mutant (T58 in human KCNE1) leads to currents with a constitutively
active component (14). The equivalent residue of KCNE3 is a valine, a
substitution that like T59C abolishes the hydrogen bonding potential of
the threonine hydroxyl group and introduces a larger residue in its place.
Our work has determined that small regions within the
transmembrane domain of KCNE family members control the activation and deactivation rates of KvLQT1. Specifically, a three-mino acid stretch
is sufficient and necessary to confer specificity of activation kinetics to KvLQT1, but neighboring regions appear to control the rates
of deactivation. These same three amino acids also appear sufficient
and necessary for at least partial generation of the constitutive
component of current seen with KCNE3/KvLQT1 complexes.
Several groups have provided evidence that the KCNE1 C terminus is a
region critical for its function (12, 13, 21). Most recently, Tapper
and George (16) have shown that the C-terminal domain alone is not
sufficient for slow activation when placed in a chimera with a sodium
channel Our results indicate a separation of structural determinants of
activation and deactivation kinetics. Thermodynamics can help elucidate
the mechanism of KCNE control of channel activity by relating
experimentally determined rates to energy states of the channel as it
opens and closes. Changes in the voltage dependence of activation
(Vh) are indicators of changes in the relative free
energy of the closed and open states of the channel ( That we observed nearly identical kinetics of activation between KCNE1
and chimera KM(57-59)K suggests that this region is necessary and
sufficient to modulate the slow gating of activation seen with
KvLQT1-KCNE1 complexes. We observed a ~30-mV difference in the
Vh between the two, however. This result indicates that the substitution of the KCNE1 residues 57-59 in KCNE3 was able to
confer the KCNE1-like kinetics of activation of KvLQT1 (by increasing
Our data are consistent with a model in which the conformation of
KvLQT1, when altered by the change in voltage across the membrane,
presents different regions of the channel for interaction with KCNE1
and KCNE3. Following a depolarization, it appears that KCNE1 residues
57-59 (KCNE3 residues 71-73) are available to interact with a
transition state to modulate the activation kinetics. Upon repolarization, the different conformations of KvLQT1 and KCNE1/3 allow
other neighboring transmembrane regions of the proteins to interact.
This interaction then modulates the stability of the reverse transition
state ( Our study has thus elucidated structural components of KCNE1 and KCNE3
that are necessary and sufficient to modulate the gating kinetics of
KvLQT1 by interacting with several transition states. The technique of
perturbing specific structural components by moving them into a
homologous environment can serve to complement results from gating
charge (23), crystallographic (24), and structural data (25, 26).
Further work and analysis of the interactions of KCNE family of
proteins with K+ channel
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit, showed that the KCNE1 C terminus alone cannot
modify the KvLQT1 channel but rather that the presence of the KCNE1
transmembrane domain is also needed for this function.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Construction of KCNE1/KCNE3 chimeras.
a, list of primers used for chimera construction. For
details see "Materials and Methods." Primers below the
shaded gray line are complementary to splice regions of
previous chimeras to allow construction of smaller interpositions.
b, alignment of KCNE1 and KCNE3 transmembrane and proximal
C-terminal domain. Shaded gray indicates transmembrane
domain. Arrows indicate splice sites used in chimera
construction. Numbers refer to the amino acid of KCNE1 where
splicing was performed.
, 1800 microfarads with cytomix media (17). Cells were studied between 24 and
48 h after transfection. Molar ratios of 7:7:2 of
KvLQT1:chimera:green fluorescent protein plasmid were used, to allow
identification of transfected cells as previously described (18,
19).
V)/k)), where I is
the measured tail current; V is the applied membrane
voltage; Vh is the voltage at half-maximal
activation; and k is the slope factor. Whole cell current
amplitudes were compared daily to a KCNE1/KvLQT1 control and normalized
to this value to control for variations in transfection efficiency.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
150 mV (Fig. 2c, inset). We also
observed a distinct time voltage-dependent component of
activation and deactivation for the KvLQT1/KCNE3 current (Fig. 2,
c, e, and f). The voltage-dependent
component had altered activation (KvLQT1, 24.2 ± 2.3 ms, and
KCNE3, 21.1 ± 1.9 ms) and deactivation kinetics (KvLQT1, 39 ± 1.1 ms, and KCNE3, 16 ± 2.1 ms) with a shift in
Vh (KvLQT1,
15.2 ± 0.52 mV, and KCNE3,
52.2 ± 2.15 mV), and lacked obvious voltage-dependent inactivation (Fig. 2c), indicating that it too was a
consequence of the association of KvLQT1 with KCNE3. Whole cell current
amplitudes were also augmented relative to KvLQT1 alone, to a similar
extent as KCNE1 (KCNE1, 2.13 ± 0.14-fold, and KCNE3, 2.22 ± 0.09-fold, relative to KvLQT1, 1.00). Expression of KCNE3 alone did not
yield currents (Fig. 2d).
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Fig. 2.
Differential control of KvLQT1 channel
activity by KCNE1 and KCNE3. a, whole cell current recording
in response to depolarizing steps in CHO cells transfected with KvLQT1
alone. b, cotransfection with KCNE1. c,
cotransfection with KCNE3; inset, same cell recorded at
faster time scale. d, transfection with KCNE3 alone.
e, voltage-dependent activation curves.
f, normalized activation curves were fitted using the
Boltzman equation. y scale bars are a and
d, 400 pA; b and c, 10,000 pA;
e, 200 pA; c (lower trace), 11,000 pA.
x scale bars are c (lower trace), 50 ms; all others, 500 ms.
Quantitative analysis of currents from KCNE1/KCNE3 chimeras
120 mV following depolarization using a single
exponential. The ratio of constitutive component to total current was
determined by taking the height of the instantaneous rise in current
following depolarization to 100 mV to the total steady-state current
after voltage-activated current had reached a plateau.
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Fig. 3.
Specificity of KvLQT1 activation lies within
the transmembrane domain. a-e, whole cell currents from
cells cotransfected with KvLQT1 and the indicated chimera.
Schematics depict the transmembrane domain and surrounding
residues to show swapped regions. Top row shows chimeras
with N termini swapped, and bottom row shows the C termini
swapped. e, chimera that retains the transmembrane domain of
KCNE1 only still activates and deactivates with similar kinetics.
Arrows show zero current. y scale bars are as
follows: a, 10,000 pA; b, 3000 pA;
c, 300 pA; d, 7000 pA; and e, 1000 pA.
All x scale bars are 500 ms.
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Fig. 4.
A three-amino acid stretch of the
transmembrane domain controls activation but not deactivation of KvLQT1
heteromeric channels. Chimeras containing only small parts of the
KCNE1/3 transmembrane domains nonetheless retain activation kinetics of
the corresponding protein as long as they contain KCNE1 residues 57-59
(KCNE3 residues 71-73). Traces d and e show
traces from chimeras swapping only these residues. d, inset
shows response at fast time scales to both depolarizations and
hyperpolarizations. The instantaneous presence of current at
hyperpolarizations as negative as 150 mV indicates a constitutive
component to the current when only three KCNE3 amino acids (71) are
present. Note that activation kinetics resemble the appropriate KCNE
protein but that deactivation kinetics are different (see also Table
I). y scale bars are as follows: a, 5000 pA;
b, 1000 pA; c, 6000 pA; d, 4000 pA;
inset, 1000 pA; e, 400 pA. x scale
bars are 500 ms except d, inset, where scale bar
is 40 ms.
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Fig. 5.
Graphical comparison of activation and
deactivation rates for KCNE3/KCNE1 chimeras. a, comparison
of rise times to half-maximum following an 80-mV depolarizing pulse.
b, comparison of mono-exponential time constants fit to tail
currents at 120 mV. Bars in gray represent
three amino acid-swapped chimeras. Note discordance of activation and
deactivation kinetics as compared with KCNE1, KCNE3, and all other
chimeras.
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Fig. 6.
Voltage-dependent activation and
deactivation of chimeras containing a three-amino acid stretch of
either KCNE1 or KCNE3. KCNE1 amino acids 57-59 and KCNE3 residues
71-73 determine properties of activation but not deactivation
kinetics. a, variation of rise times with voltage of
depolarization for KCNE1, KCNE3, MK(57-59)M, and KM(57-59)K.
b, variation of monoexponential time constant for tail
currents at the indicated voltages.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit and that the C-terminal half of the transmembrane
domain was also necessary. Our work defines that portion of the
transmembrane domain that is sufficient and necessary to confer
functional specificity to KCNE1 and -3. Our results do not, however,
rule out a role for the C terminus in KCNE1 function. The greatest
homology between KCNE1 and KCNE3 resides in the previously determined
critical C-terminal amino acids (Fig. 1). This region may be needed for
anchoring, binding, or positioning the transmembrane domain that we now
identify as the effector. This would explain the results of earlier
mutagenesis studies that showed the C-terminal region to be relatively
intolerant to amino acid substitutions, as well as an analysis of
KvLQT1 mutations that affect KvLQT1 function only in association with KCNE1 (22).
G
indicates change in Gibbs' free energy). Rates of opening and closing
of K channels, in contrast, reflect changes in the activation energy (
G
). This is the energy required to
reach the transition state, the least stable intermediate conformation
along the reaction coordinate of a molecule undergoing a physical
change. KCNE1 and KCNE3 affect the rates of both opening and closing of
the channel and the voltage dependence of activation. Our results
provide a physical basis for each of these effects.
G
) independently of the
Vh (
G). The implication therefore is
that KCNE1 residues 57-59 interact with the transition state of the
channel during the closed
open transition but do so somewhat independently of the relative free energy of the closed and open states
(
G). In addition, our results have dissected the region of KCNE1 controlling activation rate from that controlling deactivation rate. This result is not consistent with a view of KCNE1 acting simply
as an "enzyme," modulating the activation energy of a reversible reaction. If that were true, we would expect the regions controlling activation and inactivation to be identical. Our observations imply
that KCNE1 modulates the kinetics of KvLQT1 by interacting with
different transition states during activation (the closed
open
transition) and deactivation (the open
closed transition). While
such a scenario is impossible in simple chemical equilibria, in the
case of voltage-gated ion channels the two transitions take place under
conditions of different electric field across the membrane. Channel
structure is expected to be different during activation
(depolarization) and deactivation (repolarization). Thus, the
transition states for each process are also expected to be different.
G
rev).
-subunits may help to improve
our understanding of gating mechanisms in a new way.
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FOOTNOTES |
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* This work was supported in part by NHLBI Grant R01 HL 57388 from the National Institutes of Health (to T. V. M) and the Spanish CICYT Grant SAF99-0092-CO2-01 (to S. L).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF302494.
§ Supported by a National Institutes of Health grant from the Medical Scientist Training Program.
To whom correspondence should be addressed. Tel.:
718-430-3370; Fax: 718-430-8989; E-mail: mcdonald@aecom.yu.edu.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M010713200
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ABBREVIATIONS |
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The abbreviations used are: ESTs, expressed sequence tags; CHO, Chinese hamster ovary; PCR, polymerase chain reaction.
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1. | Neyroud, N., Tesson, F., Denjoy, I., Leibovici, M., Donger, C., Barhanin, J., Faure, S., Gary, F., Coumel, P., Petit, C., Schwartz, K., and Guicheney, P. (1997) Nat. Genet. 15, 186-189[Medline] [Order article via Infotrieve] |
2. | Schroeder, B. C., Waldegger, S., Fehr, S., Bleich, M., Warth, R., Greger, R., and Jentsch, T. J. (2000) Nature 403, 196-199[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Yang, W. P.,
Levesque, P. C.,
Little, W. A.,
Conder, M. L.,
Shalaby, F. Y.,
and Blanar, M. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4017-4021 |
4. | Barhanin, J., Lesage, F., Guillemare, E., Fink, M., Lazdunski, M., and Romey, G. (1996) Nature 384, 78-80[CrossRef][Medline] [Order article via Infotrieve] |
5. | Sanguinetti, M. C., Curran, M. E., Zou, A., Shen, J., Spector, P. S., Atkinson, D. L., and Keating, M. T. (1996) Nature 384, 80-83[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Takumi, T.,
Ohkubo, H.,
and Nakanishi, S.
(1989)
FASEB J.
5,
331-337 |
7. | Abbott, G. W., and Goldstein, S. A. (1998) Q. Rev. Biophys. 31, 357-398[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Tristani-Firouzi, M.,
and Sanguinetti, M. C.
(1998)
J. Physiol. (Lond.)
510,
37-45 |
9. | Wang, Q, C. M., Splawski, I., Burn, T. C., Millholland, J. M., VanRaay, T. J., Shen, J., Timothy, K. W., Vincent, G. M., de Jager, T., Schwartz, P. J., Toubin, J. A., Moss, A. J., Atkinson, D. L., Landes, G. M., Connors, T. D., and Keating, M. T. (1996) Nat. Genet. 12, 17-23[Medline] [Order article via Infotrieve] |
10. |
Duggal, P.,
Vesely, M. R.,
Wattanasirichaigoon, D.,
Villafane, J.,
Kaushik, V.,
and Beggs, A. H.
(1998)
Circulation
97,
142-146 |
11. |
Tyson, J, T. L.,
Bellman, S.,
Wren, C.,
Taylor, J. F.,
Bathen, J.,
Aslaksen, B.,
Sorland, S. J.,
Lund, O.,
Malcolm, S.,
Pembrey, M.,
Bhattacharya, S.,
and Bitner-Glindzicz, M.
(1997)
Hum. Mol. Genet.
6,
2179-2185 |
12. |
Takumi, T.,
Moriyoshi, K.,
Aramori, I.,
Ishii, T.,
Oiki, S.,
Okada, Y.,
Ohkubo, H.,
and Nakanishi, S.
(1991)
J. Biol. Chem.
266,
22192-22198 |
13. |
Romey, G.,
Attali, B.,
Chouabe, C.,
Abitbol, I.,
Guillemare, E.,
Barhanin, J.,
and Lazdunski, M.
(1997)
J. Biol. Chem.
272,
16713-16716 |
14. | Tai, K. K., and Goldstein, S. A. (1998) Nature 391, 605-608[CrossRef][Medline] [Order article via Infotrieve] |
15. | Wang, K. W., Tai, K. K., and Goldstein, S. A. N. (1996) Neuron 16, 571-577[Medline] [Order article via Infotrieve] |
16. |
Tapper, A. R.,
and George, A. J.
(2000)
J. Gen. Physiol.
116,
379-390 |
17. | van den Hoff, M. J., Moorman, A. F., and Lamers, W. H. (1992) Nucleic Acids Res. 20, 2902[Medline] [Order article via Infotrieve] |
18. |
Kagan, A., Yu, Z.,
Fishman, G. I.,
and McDonald, T. V.
(2000)
J. Biol. Chem.
275,
11241-11248 |
19. | McDonald, T. V., Yu, Z., Ming, Z., Palma, E., Meyers, M. B., Goldstein, S. A. N., and Fishman, G. I. (1997) Nature 388, 289-292[CrossRef][Medline] [Order article via Infotrieve] |
20. | Hammill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pfluegers Arch. 391, 85-100[Medline] [Order article via Infotrieve] |
21. |
Sesti, F.,
and Goldstein, S. A.
(1998)
J. Gen. Physiol.
112,
651-663 |
22. | Chouabe, C. N. N., Richard, P., Denjoy, I., Hainque, B., Romey, G., Drici, M. D., Guicheney, P., and Barhanin, J. (2000) Cardiovasc. Res. 45, 971-980[CrossRef][Medline] [Order article via Infotrieve] |
23. | Seoh, S. A., Sigg, D., Papazian, D. M., and Bezanilla, F. (1996) Neuron 16, 1159-1167[Medline] [Order article via Infotrieve] |
24. |
Doyle, D. A.,
Cabral, J. M.,
Pfuetzner, R. A.,
Kuo, A.,
Gulbis, J. M.,
Cohen, S. L.,
Chait, B. T.,
and MacKinnon, R.
(1998)
Science
280,
69-77 |
25. | Cha, A., Snyder, G. E., Selvin, P. R., and Bezanilla, F. (1999) Nature 402, 809-813[CrossRef][Medline] [Order article via Infotrieve] |
26. | Glauner, K. S., Mannuzzu, L. M., Gandhi, C. S., and Isacoff, E. Y. (1999) Nature 402, 813-817[CrossRef][Medline] [Order article via Infotrieve] |