Identification of Specific Pore Residues Mediating KCNQ1
Inactivation
A NOVEL MECHANISM FOR LONG QT SYNDROME*
Guiscard
Seebohm,
Constanze R.
Scherer,
Andreas E.
Busch, and
Christian
Lerche
From the Aventis Pharma Deutschland GmbH, DG Cardiovascular
Diseases, Building H821, D-65926 Frankfurt am Main, Germany
Received for publication, September 13, 2000, and in revised form, January 11, 2001
 |
ABSTRACT |
KCNQ1 inactivation bears
electrophysiological characteristics different from classical N- and
C-type inactivation in Shaker-like potassium channels.
However, the molecular site of KCNQ1 inactivation has not yet been
determined. KCNQ2 channels do not exert a fast inactivation in contrast
to KCNQ1 channels. By expressing functional chimeras between KCNQ1 and
KCNQ2 in Xenopus oocytes, we mapped the region of this
inactivation to transmembrane domain S5 and the pore loop H5 and
finally narrowed down the site to positions Gly272 and
Val307 in KCNQ1. Exchanging these two amino acids
individually with the analogous KCNQ2 residue abolished inactivation.
Furthermore, a KCNQ1-like inactivation was introduced into KCNQ2 by
mutagenesis in the corresponding region, confirming its relevance for
the inactivation process. As KCNQ1 inactivation involves the
regions S5 and H5, it exhibits a geography distinct from N- or C-type inactivation. Native cardiac IKs channels
comprising KCNQ1 and accessory MinK subunits do not inactivate because
of the functional interaction of KCNQ1 with MinK. Mutations in
KCNQ1 can lead to long QT1 syndrome, an inherited form of
arrhythmia. The long QT1 mutant KCNQ1(L273F) displays a pronounced
KCNQ1 inactivation. Here we show that when expressing mutant
IKs channels formed from KCNQ1(L273F) and
MinK, MinK association no longer eliminates KCNQ1 inactivation. This
results in smaller repolarizing currents in the heart and therefore
represents a novel mechanism leading to long QT syndrome.
 |
INTRODUCTION |
Ion channels regulate the membrane potential of excitable cells.
These are proteins containing aqueous pores and undergo conformational changes leading to the defined gating states "open" and
"closed," where open channels are conducting and closed channels
are nonconducting. In many channels there are additional
"inactivated" states, which are also nonconducting and which follow
the open states because of an activating physiological stimulus. Among
voltage-gated potassium channels, there are two major types of
inactivation. N-type inactivation is mediated by an intracellular
"ball," located within the N terminus of either the pore-forming
protein or a modulatory
-subunit, which plugs the ion channel pore
(1-4). In C-type inactivation the outer vestibule of the pore itself
undergoes conformational changes (5).
Inactivation plays an important physiological role such as determining
the sodium spike in neurons and myocytes. Genetic defects, resulting in
impaired inactivation of sodium channels, can cause myotonia and a form
of long QT syndrome
(LQTS)1 (6, 7).
The KCNQ gene family represents a group of recently
identified voltage-gated potassium channels, and disease-causing
mutations have been identified in 4 of 5 known KCNQ genes
(8-13). KCNQ1 (formerly called KvLQT1), the founding member of this
family (8), coassembles with MinK (also called IsK or KCNE1) protein
(14) generating slowly activating potassium currents. These constitute the cardiac IKs conductance, one component of
the delayed rectifier repolarizing current IK
(15-17). Mutations in either gene cause LQTS, whereby LQT1 mutations
occur in KCNQ1 and LQT5 mutations in MinK (8, 18-21).
Homomeric KCNQ1 channels are characterized by fast activation and
delayed inactivation. Delay of inactivation was explained by two open
states separating closed and inactivated states in a linear gating
scheme. Inactivation of KCNQ1 is incomplete, supposedly resulting from
a weak voltage sensitivity of the rates of both onset of and recovery
from inactivation. Alternatively, the partial inactivation can also be
caused by subconductance states. The lack of sensitivity to high
extracellular TEA and potassium, together with delayed onset
distinguish KCNQ1 inactivation from classical C-type inactivation in
Shaker-like channels. The weak voltage sensitivity of KCNQ1
inactivation and the lack of a Shaker-like ball structure
within KCNQ1 differ from N-type inactivation (22, 23).
In contrast to the existing biophysical data on KCNQ1 inactivation, its
molecular determinants have only been discussed tentatively so far.
Franqueza et al. (24) analyzed three different LQT1 mutations located within or close to the intracellular linker between
the S4 and S5 transmembrane domains. After expression in
Xenopus oocytes, some mutants showed overall altered gating characteristics compared with KCNQ1-WT, but in none of them
inactivation was abolished. The LQT1 mutation L273F (8) exhibited a
pronounced macroscopic inactivation when expressed in
Xenopus oocytes (19). (The position number according to
Shalaby et al. (19) is L272F.) The mutation is located in
transmembrane segment S5, indicating a possible involvement of the S5
region in channel inactivation.
Inactivation of KCNQ1 is abolished by coassembly with MinK (22, 23),
suggesting that interacting domains of these two proteins might also
influence the inactivation process itself. Pusch et al. (25)
have presented evidence for MinK interaction with the outer vestibule
of KCNQ1. MinK also might directly interact with the pore region of
KCNQ1 as suggested by both a yeast two-hybrid study and a
cysteine-scanning study in MinK (26, 27).
It was our aim to gain insights into the as yet unknown molecular
mechanism underlying KCNQ1 inactivation and its possible pathophysiological relevance.
 |
EXPERIMENTAL PROCEDURES |
Molecular Biology--
For the construction of chimeras, silent
point mutations were introduced producing restriction endonuclease
sites at corresponding positions in KCNQ1 and KCNQ2, namely a
SacI site (at KCNQ1 amino acids
Glu261-Leu262), an NsiI site
(at KCNQ1 amino acids
Asp301-Ala302-Leu303), and a
BamHI-site (at KCNQ1 amino acids
Gly348-Ser349). Other chimeric joining regions
were created by recombinant polymerase chain reaction resulting
in constructs described in Fig. 1.
Site-directed mutagenesis was performed by polymerase chain reaction
using cloned Pyrococcus furiosus DNA polymerase
(Stratagene). All constructs were reconfirmed by automated DNA
sequencing. For Xenopus oocyte expression, capped cRNA was
synthesized using the SP6 and T7 mMessage mMachine kits (Ambion).
GenBankTM/EBI accession numbers for sequences used are as
follows: hKCNQ1, AJ006345; hKCNQ2, NM004518; and MinK(KCNE1),
M26685.
Two Electrode Voltage Clamp Technique--
Xenopus
laevis oocytes were obtained from tricaine-anesthetized animals.
Ovaries were collagenase-treated (1 mg/ml, Worthington, type II) in OR2
solution (NaCl 82.5 mM, KCl 2 mM,
MgCl2 1 mM, HEPES 5 mM, pH 7.4) for
120 min and subsequently stored in recording solution ND96 (NaCl 96 mM, KCl 2 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 5 mM, pH 7.4) with additional sodium pyruvate (275 mg/liter), theophylline (90 mg/liter) and Gentamycin (50 mg/liter) at 18 °C.
Oocytes were individually injected with 10 ng of cRNA encoding WT or
mutant KCNQ subunits, or coinjected with 10 ng of cRNA of WT or mutant
KCNQ1 and 5 ng of hMinK cRNA. Standard two electrode voltage clamp
recordings were performed at 22 °C with a Turbo Tec 10CX (NPI)
amplifier, an ITC-16 interface combined with Pulse software (Heka) and
Origin version 5.0 (Microcal Software) for data acquisition.
Macroscopic currents were recorded 3-4 days after injection. The
pipette solution contained 3 M KCl. All fitting procedures
were based on the simplex algorithm. Student's t test was
used to test for statistical significance, which was assumed with
p < 0.05.
To calculate the fraction of inactivated channels, a double exponential
fit to the tail currents was done according to the formula:
I(t) = As × exp(
t/
s)
Af × exp(
t/
f). The faster and the
slower component represent recovery from inactivation and deactivation
respectively, whereby As and
s are the
amplitude and time constant for the slow component, and Af,
and
f are the amplitude and time constant for the fast
component. The fraction of inactivated channels is given by
Af/As, where the amplitude As is
related to the degree of activation, and the amplitude Af indicates the degree of inactivation. The method was previously described in detail (22, 23).
 |
RESULTS |
Inactivation in KCNQ1 channels becomes apparent in a hook in the
tail current when repolarizing the membrane potential after a
depolarizing pulse (Refs. 22, 23, and Fig.
1D). This trait is also
described for HERG channels where it is even more pronounced (28, 29).
The hook is attributed to rapid recovery of channels from inactivation
at a rate much faster than deactivation (Fig. 1D). Onset of
KCNQ1 inactivation can be revealed using a double-pulse protocol (23,
29, 30). A conditioning 2-s pulse to 40 mV is applied to activate and
inactivate channels followed by a 20-ms hyperpolarizing interpulse,
which transiently removes inactivation. During a final test pulse to 40 mV, onset of inactivation becomes visible (Fig. 1C).
Throughout this study we considered both the hook and the onset of
inactivation as a marker for the presence or absence of inactivation in
mutant KCNQ1 channels.

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 1.
Chimera and point mutants identifying regions
of importance for KCNQ1 inactivation. A, protein
sequence alignment of hKCNQ1, hKCNQ2, and rKCNQ3 showing the important
regions for KCNQ1 inactivation. Postulated transmembrane segments S5
and S6 and the inner pore loop H5 are indicated above. Gray
bars indicate the KCNQ2 spanning sequence in the Q1S6Q2 and
Q1S5/H5linkerQ2 chimeras. These amino acids had a low impact on the
inactivation process as shown below in B, C, and
D. Differences in S5 and H5 of KCNQ1 and KCNQ2/KCNQ3 are
marked by arrows. Amino acids that can be exchanged in KCNQ1
without affecting inactivation are marked by gray arrowheads
and those that abolish inactivation by black arrowheads.
Point mutants V308I and V310L and a double mutant T365A/L366W
were still inactivating (data not shown). KCNQ3 forms heteromers with
KCNQ2, which are not inactivating (13). Asterisk indicates
the site of the LQT1 mutant L273F. B, model of important
chimeras and mutants showing the typical backbone of a
voltage-dependent potassium channel with the intracellular
C and N terminus and six transmembrane domains. Black and
gray lines indicate KCNQ1 and KCNQ2 sequences, respectively.
C, representative current traces recorded in an oocyte
injected with cRNA from the respective construct shown in B. A double-pulse protocol was used to show the onset or lack of
inactivation. After stepping the membrane potential for 2 s from
100 mV to 40 mV, a second 1-s pulse to 40 mV was applied following
different 20-ms interpulses (to 120 mV, 90 mV, and 60 mV)
abrogating inactivation. The insets magnify the important
regions. D, representative current traces at different
voltages recorded by stepping the membrane potential for 3 s from
100 mV to 50 mV in 10 mV increments in an oocyte injected with cRNA of the respective
construct shown in B. Tail currents show a characteristic
hook reflecting recovery from inactivation. The insets
magnify the important regions. E, IV relationship of
constructs shown in B recorded as described in D
(n = 10-16). Amplitudes at the end of 3-s test pulses
were plotted against voltage. Error bars indicate S.E. All
vertical bars represent 2 µA; horizontal bars,
0.5 s.
|
|
To define the structural determinants of KCNQ1 inactivation, we
constructed a series of functional chimeras between the closely related
channels KCNQ1 and KCNQ2. KCNQ2 is a component of the neuronal
noninactivating M-current and is mutated in neonatal epilepsy (9, 10,
31). As shown in Fig. 1, KCNQ2 did not inactivate like KCNQ1. In a
gain of function approach, we transferred the ability to inactivate
from KCNQ1 to KCNQ2. Substituting the S5/H5/S6 domain from KNCQ1 into
KCNQ2, caused a dramatic change in the biophysical characteristics of
the resulting construct (Q2S5-S6Q1) compared with KCNQ2-WT (Fig. 1).
Macroscopic inactivation is conferred at positive voltages, suggesting
that this part of the protein is necessary for the inactivation process.
By contrast it was possible to substitute large segments of KCNQ2 into
KCNQ1 without abolishing inactivation. A KCNQ1 construct including
substituted KCNQ2 amino acids from the end of H5 to the end of S6
(Q1S6Q2, Fig. 1) maintained inactivation. Interestingly the KCNQ2 S6
region somehow enhances intrinsic KCNQ1 inactivation. Another KCNQ1
chimeric construct containing the KCNQ2 S5/H5 linker (Q1S5/H5linkerQ2)
revealed that exchanging this region does not remove inactivation (Fig.
1). Thus the borders of the crucial region for inactivation could be
assigned to the S5 segment and parts of the H5 pore loop.
Fig. 1A illustrates differences in the protein sequences of
the related KCNQ channels in the described regions. With site-directed mutagenesis, we exchanged the different amino acids in KCNQ1 to the
corresponding residues in KCNQ2. Expression of these point mutants
showed that two single amino acid substitutions, G272C and V307L, are
capable of abolishing inactivation (Fig. 1). Mutation G272C, adjacent
to the site of clinical mutation L273F (as described above), caused
additionally slightly slowed activation kinetics. To exclude the
possibility that the introduced cysteine might be stabilizing the
channel via formation of novel intramolecular disulfide bonds, we
substituted glycine with threonine at this position. Now, activation
and deactivation kinetics of G272T were very similar to KCNQ1-WT (Table
I); however inactivation still was
completely abolished. The other key mutation V307L located in the pore
loop showed slightly slowed activation and deactivation kinetics
compared with KCNQ1-WT (Table I), but again abolished inactivation. The
slight changes in activation kinetics are not reflected by altered
current-voltage relationships and thus shifted voltage dependence of
activation cannot account for the lack of inactivation. A further
observation was that in the noninactivating mutants, the slow component
of deactivation was decreased or abolished (Table I). Possibly the slow
component of deactivation in KCNQ1 is somehow connected to the
inactivation process.
View this table:
[in this window]
[in a new window]
|
Table I
Activation and deactivation time constants of WT-KCNQ1 and mutant KCNQ1
channels
Oocytes were injected with 10 ng of cRNA. Currents were activated by a
3-s pulse to 40 mV and followed by a hyperpolarizing pulse to 60 mV.
Kinetics were analyzed by exponential fits to the rising phase for
activation and to the tail current for deactivation. Values are
mean ± S.E., numbers of oocytes are shown in parenthesis.
|
|
To corroborate our results, we made additional point mutations in the
inactivating KCNQ2 chimera Q2S5-S6Q1. Analogous substitution of glycine
272 in S5 (KCNQ1 numbering) and valine 307 in H5 (KCNQ1 numbering) by
cysteine and leucine, respectively, again abolished inactivation in
this chimeric construct. Effects on activation and deactivation
kinetics were only minor (Fig. 2,
A and B).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Point mutants abolishing KCNQ1 inactivation
in the inactivating chimera Q2S5-S6Q1. A and
B, model of the chimera Q2S5-S6Q1 including introduced
mutations G272C (A) and V307L (B).
Black and gray lines indicate KCNQ1 and KCNQ2
sequences, respectively. Double-pulse protocol as shown in Fig. 1
demonstrate lack of inactivation in both point mutations shown.
Right, current traces at different voltages of oocytes
injected with cRNA of each construct; protocols as described in Fig. 1.
Insets show magnified tail currents. The respective IV
relationship is shown plotted from 10 oocytes for each construct.
Error bars indicate S.E. All vertical bars
represent 2 µA, horizontal bars, 0.5 s.
|
|
Using a final approach, we attempted to introduce a KCNQ1-like
inactivation into KCNQ2 through single amino acid exchanges. We
constructed the KCNQ2 mutations C242G, L243F, and L272V. These positions correspond to the residues Gly273,
Leu273, and Val307 in KCNQ1, which we
recognized as being important for KCNQ1 inactivation. Introducing a
phenylalanine at position 243 in KCNQ2 resulted in inactivating
currents interestingly similar to the KCNQ1 mutant L273F (Fig.
3A). The mutant KCNQ2(L272V)
did not exert a KCNQ1-like inactivation as shown in Fig. 3B.
In a construct containing both mutations L243F and L272V, an
inactivation was apparent although the currents were small in amplitude
(Fig. 3C). Substituting the cysteine at KCNQ2 position 242 by glycine individually or together with the mutations described above
resulted in a mutant that did not produce measurable currents under our
conditions.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 3.
Introduction of fast inactivation into KCNQ2
by point mutation. A, B, and C,
models of the KCNQ2 channel subunit including introduced mutations
L243F (A), L272V (B), and L243F/L272V are drawn
on the left side (C). Double-pulse protocols as
performed in Fig. 1 were used demonstrating existence or lack of
inactivation in the point mutants. Current traces of oocytes injected
with cRNA of each construct were measured at different voltages with
protocols as described in Fig. 1. The insets show magnified
important regions. The respective IV relationships are shown plotted
from 5-7 oocytes for each construct. Error bars indicate
S.E. All vertical bars represent 1 µA, horizontal
bars, 0.5 s.
|
|
The only crystal structure of the pore-forming part of a potassium
channel presently available derives from the KcsA channel of
Streptomyces lividans (32). In this structure, we checked the locations of the residues equivalent to those that we identified as
being involved in KCNQ1 inactivation. Interestingly, the amino acids in
the KcsA channel, Leu40 and Val70 corresponding
to the clinically relevant residue KCNQ1(L273) and the residue
KCNQ1(V307) respectively, are in close vicinity within one subunit. The
closest distances of these amino acids are around 4 Å for all four
subunits, thereby enabling attractive interactions. Within the S5/H5/S6
region, KCNQ1 shares about 36% identity and 46% homology to KcsA.
Accordingly the same residues in KCNQ1 can also be expected to be in
close proximity. This raises the possibility that intramolecular
interaction of transmembrane segment S5 and the helical part of the
pore loop H5 is involved in the inactivation process, putatively
stabilizing the three-dimensional conformation of the inactivated
channel. Exchange of glycine at KCNQ1 position 272 by a side
chain-containing amino acid can be anticipated to change the
conformation of the encompassing S5 region and therefore possibly
disrupt interaction of amino acids Leu273 and
Val307. Introduction of a KCNQ1-like inactivation into
KCNQ2 by exchanging leucine 243 with the more extensive phenylalanine
in S5 supports the hypothesis of interaction of domains S5 and H5
facilitating KCNQ1 inactivation.
We next examined the pathophysiological role of KCNQ1 inactivation by
studying kinetics of the LQT1 mutant L273F, which is located next to
Gly272 and in the KcsA channel model in close proximity to
Val307. The mutant was analyzed previously (19); the
authors reported a pronounced macroscopic inactivation of KCNQ1(L273F)
as well as a reduced current amplitude of KCNQ1(L273F)/MinK
compared with KCNQ1-WT/MinK. We expressed homomeric mutant and
heteromeric mutant/MinK channels and compared them to KCNQ1-WT and
IKs channels (Fig. 4). The greatly enhanced inactivation of
KCNQ1(L273F) is shown in Fig. 3B. Tail current analysis
after a 3-s preconditioning pulse to 40 mV resulted in 86 ± 3%
inactivated mutant channels in contrast to only 31 ± 1% in
KCNQ1-WT (Fig. 4, A and B; Table II). Around 30% inactivated WT channels
were also calculated previously using the same analysis (22, 23). Time
courses of inactivation and recovery were significantly affected
compared with WT channels as well (Table II). After coinjecting the
same amounts of KCNQ1-WT or KCNQ1(L273F) cRNA together with MinK cRNA,
we observed a decreased current amplitude for mutant
IKs channels consistent with previous data (19)
(Fig. 3, C and D). The current-voltage
relationship of activation was not shifted in mutant
IKs channels compared with WT channels (Fig.
4E). Most interestingly, using the double-pulse protocol, we
demonstrated that MinK is no longer able to completely abolish the
pronounced inactivation in KCNQ1(L273F) (Fig. 4D). Moreover,
this inactivation in mutant IKs channels was
accelerated compared with homomeric KCNQ1(L273F), further indicating
functional assembly of the mutant KCNQ1 subunit with MinK (Table II).
Tail currents of KCNQ1(L273F)/MinK channels displayed a weak hook
confirming the persistence of inactivation in these heteromers (Fig.
4D). By analyzing this hook, we calculated that more than
20% of mutant IKs channels were in the
inactivated state after a 3-s activating pulse to 40 mV (Fig.
4F). Thus, the significant number of inactivated, i.e. nonconducting, mutant IKs
channels contributes to the decreased amplitudes of KCNQ1(L273F) and
KCNQ1(L273F)/MinK compared with those of KCNQ1-WT and KCNQ1/MinK.
In vivo this is expected to result in a decreased
repolarizing IKs conductance constituting a new
mechanism leading to LQT1 syndrome.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Characterization of the LQT1 mutant L273F in
oocytes. A, representative current traces at different
voltages recorded as described in Fig. 1 in an oocyte injected with
KCNQ1-WT cRNA. Double-pulse protocol as described in Fig. 1 was used to
show the onset of inactivation. The insets magnify the
important regions. B, current traces of a L273F-coinjected
oocyte recorded as described in A. C, current
traces at different voltages of a KCNQ1-WT/MinK coinjected oocyte
recorded as described in A. Double-pulse protocol of a
KCNQ1-WT/MinK coinjected oocyte recorded as described in A.
The insets magnify important regions. D, current
traces of a L273F/MinK-coinjected oocyte recorded as described in
A. E, IV relationship of mutant L273F/MinK
(n = 14) and of KCNQ1-WT/MinK (n = 17)
from oocytes recorded as in B and D. Amplitudes
after 3-s test pulses were plotted against voltage. F, The
fraction of inactivated channels of mutant L273F/MinK and of
KCNQ1WT/MinK was determined by tail current analysis (double
exponential fit) and calculated by the amplitudes of recovery from
inactivation (Afast) and deactivation (Aslow)
(see "Experimental Procedures"). All vertical bars
represent 2 µA, horizontal bars, 0.5 s.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Characteristics of inactivation in KCNQ1-WT and KCNQ1(L273F) in
homomeric and heteromeric expression together with MinK in Xenopus
oocytes
Oocytes were injected with 10 ng of KCNQ1 cRNA. For heteromeric
expression 5 ng of MinK cRNA were added. The double-pulse protocol with
two 40-mV pulses separated by a 20-ms pulse to 120 mV was used to
analyze the onset of inactivation. The time constants of onset were
calculated by exponential fits to the decaying phase of the currents at
the beginning of the second pulse. To determine recovery from
inactivation and the fraction of inactivated channels, currents were
activated by a 3-s pulse to 40 mV followed by a hyperpolarizing pulse
to 60 mV. Tail currents were analyzed by double exponential fits. The
faster and the slower component constitute recovery from inactivation
and deactivation respectively. Values are mean ± S.E., numbers of
oocytes are shown in parenthesis.
|
|
 |
DISCUSSION |
Inactivation is best understood and intensively studied for
classical N- and C-type mechanisms in ShakerB potassium
channels. Fast N-type inactivation is realized by a ball plug-in
mechanism, where a ball-forming domain of the channel binds to its
receptor thereby blocking the ion pathway from the intracellular side
(1-5). This occurs independently of the membrane potential. KCNQ1
inactivation exerts slower inactivation gating and a weak voltage
dependence. Further stressing the differences from the N-type
mechanism, the identified residues at KCNQ1 positions 272, 273, and 307 are not expected to be accessible for a complex ball structure
from the intracellular side and thus probably cannot account for a
potential receptor site.
In classical C-type inactivation, conformational changes in the outer
vestibule of the pore are coupled to the inactivation process. This is
reflected in the dependence of the inactivation to extracellular
potassium and TEA (33, 34). In KCNQ1, TEA sensitivity of the
inactivation rate is not determinable because of very low TEA affinity
to the channel (22, 23, 35). However, KCNQ1 inactivation is independent
of extracellular potassium concentration; a principal difference from
C-type behavior. In Shaker-like potassium channels, certain
mutations within the P-region H5 and the sixth membrane-spanning region
S6 have been shown to affect the time course of inactivation (33, 36).
Notably, also in our study KCNQ1 inactivation was enhanced by
modification of this region (Q1S6Q2), but identified residues essential
for KCNQ1 inactivation within the S5 region and the helical part of the
H5 segment have not been reported to be substantially relevant for
C-type inactivation. In conclusion, both C-type and KCNQ1 inactivation
occur within the pore region spanning S5 to S6, but the defined
critical structures appear to be different.
In summary our results define geographical peculiarities for KCNQ1
inactivation, which were not yet reported for classical N- and C-type
inactivation in Shaker-like potassium channels. KCNQ1
inactivation is characterized by an involvement of the pore region H5
and the S5 transmembrane region, putatively interplaying with each
other. It is believed that KCNQ1 inactivation is abolished by
interaction with MinK in native heteromeric IKs
channels (22, 23). As suggested previously, interaction between KCNQ1
and MinK might involve the pore region of KCNQ1 (26, 27). Our results
demonstrate an essential function of the pore region for KCNQ1
inactivation, therefore allowing the interesting possibility that MinK
interaction may occur in the same region that is important for
inactivation. MinK possibly abolishes inactivation by weakening the
interplay between H5 and S5. In the LQT1 mutant L273F this interplay
might be stronger, thereby stabilizing inactivation such that MinK no
longer can abolish inactivation. This in turn may contribute to the
deleterious phenotype of the LQT1 mutant.
 |
ACKNOWLEDGEMENTS |
We thank K. Steinmeyer, H. Lerche, B. Attali,
P. Ruppersberg, A. Wei, I. Gutcher, and E. Kostenis for fruitful
discussions and careful proofreading.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed. Tel.: 33-673-846986;
Fax: 49-69-305-16393; E-mail: christian.lerche@aventis.com.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M008373200
 |
ABBREVIATIONS |
The abbreviations used are:
LQTS, long QT
syndrome;
LQT, long QT;
TEA, tetraethylammonium;
WT, wild type.
 |
REFERENCES |
1.
|
Armstrong, C. M.,
and Bezanilla, F.
(1977)
J. Gen. Physiol.
70,
567-590[Abstract]
|
2.
|
Zagotta, W. N.,
Hoshi, T.,
and Aldrich, R. W.
(1990)
Science
250,
568-571[Medline]
[Order article via Infotrieve]
|
3.
|
Hoshi, T.,
Zagotta, W. N.,
and Aldrich, R. W.
(1990)
Science
250,
533-538[Medline]
[Order article via Infotrieve]
|
4.
|
Rettig, J.,
Heinemann, S. H.,
Wunder, F.,
Lorra, C.,
Parcej, D. N.,
Dolly, J. O.,
and Pongs, O.
(1994)
Nature
369,
289-294[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Liu, Y.,
Jurman, M. E.,
and Yellen, G.
(1996)
Neuron
16,
859-867[Medline]
[Order article via Infotrieve]
|
6.
|
Lerche, H.,
Heine, R.,
Pika, U.,
George, A. L., Jr.,
Mitrovic, N.,
Browatzki, M.,
Weiss, T.,
Rivet-Bastide, M.,
Franke, C.,
Lomonaco, M.,
Ricker, K.,
and Lehmann-Horn, F.
(1993)
J. Physiol.
470,
13-22[Abstract]
|
7.
|
Bennett, P. B.,
Yazawa, K.,
Makita, N.,
and George, A. L., Jr.
(1995)
Nature
376,
683-685[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Wang, Q.,
Curran, M. E.,
Splawski, I.,
Burn, T. C.,
Millholland, J. M.,
Van Raay, T. J.,
Shen, J.,
Timothy, K. W.,
Vincent, G. M.,
De Jager, T.,
Schwarz, P. J.,
Towbin, 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]
|
9.
|
Charlier, C.,
Singh, N. A.,
Ryan, S. G.,
Lewis, T. B.,
Reus, B. E.,
Leach, R. J.,
and Leppert, M.
(1998)
Nat. Genet.
18,
53-55[Medline]
[Order article via Infotrieve]
|
10.
|
Singh, N. A.,
Charlier, C.,
Stauffer, D.,
DuPont, B. R.,
Leach, R. J.,
Melis, R.,
Ronen, G. M.,
Bjerre, I.,
Quattlebaum, T.,
Murphy, J. V.,
McHarg, M. L.,
Gagnon, D.,
Rosales, T. O.,
Peiffer, A.,
Anderson, V. E.,
and Leppert, M.
(1998)
Nat. Genet.
18,
25-29[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Biervert, C.,
Schroeder, B. C.,
Kubisch, C.,
Berkovic, S. F.,
Propping, P.,
Jentsch, T. J.,
and Steinlein, O. K.
(1998)
Science
279,
403-406[Abstract/Free Full Text]
|
12.
|
Kubisch, C.,
Schröder, B. C.,
Friedrich, T.,
Lütjohann, B.,
El Amraoui, A.,
Marlin, S.,
Petit, C.,
and Jentsch, T. J.
(1999)
Cell
96,
437-446[Medline]
[Order article via Infotrieve]
|
13.
|
Lerche, C.,
Scherer, C. R.,
Seebohm, G.,
Derst, C.,
Wei, A. D.,
Busch, A. E.,
and Steinmeyer, K.
(2000)
J. Biol. Chem.
29,
22395-22400[CrossRef]
|
14.
|
Takumi, T.,
Ohkubo, H.,
and Nakanishi, S.
(1988)
Science
242,
1042-1045[Medline]
[Order article via Infotrieve]
|
15.
|
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]
|
16.
|
Barhanin, J.,
Lesage, F.,
Guillemare, E.,
Fink, M.,
Lazdunski, M.,
and Romey, G.
(1997)
Nature
384,
78-80
|
17.
|
Attali, B.
(1996)
Nature
384,
24-25[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Chouabe, C.,
Neyroud, N.,
Guicheney, P.,
Lazdunski, M.,
Romey, G.,
and Barhanin, J.
(1997)
EMBO J.
16,
5472-5479[Abstract/Free Full Text]
|
19.
|
Shalaby, F. Y.,
Levesque, P. C.,
Yang, W. P.,
Little, W. A.,
Conder, M. L.,
Jenkins-West, T.,
and Blanar, M. A.
(1997)
Circulation
96,
1733-1736[Abstract/Free Full Text]
|
20.
|
Wollnik, B.,
Schroeder, B. C.,
Kubisch, C.,
Esperer, H. D.,
Wieacker, P.,
and Jentsch, T. J.
(1997)
Hum. Mol. Gen.
6,
1943-1949[Abstract/Free Full Text]
|
21.
|
Schulze-Bahr, E.,
Wang, Q.,
Wedekind, H.,
Haverkamp, W.,
Chen, Q.,
and Sun, Y.
(1997)
Nat. Genet.
17,
267-268[Medline]
[Order article via Infotrieve]
|
22.
|
Pusch, M.,
Magrassi, R.,
Wollnik, B.,
and Conti, F.
(1998)
Biophys. J.
75,
785-792[Abstract/Free Full Text]
|
23.
|
Tristani-Firouzi, M.,
and Sanguinetti, M. C.
(1998)
J. Physiol. (Lond.)
510,
37-45[Abstract/Free Full Text]
|
24.
|
Franqueza, L.,
Lin, M.,
Shen, J.,
Splawski, I.,
Keating, M. T.,
and Sanguinetti, M. C.
(1999)
J. Biol. Chem.
274,
21063-21070[Abstract/Free Full Text]
|
25.
|
Pusch, M.,
Bertorello, L.,
and Conti, F.
(2000)
Biophys. J.
78,
211-226[Abstract/Free Full Text]
|
26.
|
Romey, G.,
Attali, B.,
Chouabe, C.,
Abitbol, I.,
Guillemare, E.,
Barhanin, J.,
and Lazdunski, M.
(1997)
J. Biol. Chem.
272,
16713-16716[Abstract/Free Full Text]
|
27.
|
Tai, K. K.,
and Goldstein, S. A.
(1998)
Nature
391,
605-608[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Sanguinetti, M. C.,
Jiang, C.,
Curran, M.-E.,
and Keating, M. T.
(1995)
Cell
81,
299-307[Medline]
[Order article via Infotrieve]
|
29.
|
Smith, P. L.,
Baukrowitz, T.,
and Yellen, G.
(1996)
Nature
379,
833-836[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Schonherr, R.,
and Heinemann, S. H.
(1996)
J. Physiol. (Lond.)
493,
635-642[Abstract]
|
31.
|
Wang, H. S.,
Pan, Z.,
Shi, W.,
Brown, B. S.,
Wymore, R. S.,
Cohen, I. S.,
Dixon, J. E.,
and McKinnon, D.
(1998)
Science
282,
1890-1893[Abstract/Free Full Text]
|
32.
|
Doyle, D. A.,
Morais, C.,
abral, J.,
Pfuetzner, R. A.,
Kuo, A.,
Gulbis, J. M.,
Cohen, S. L.,
Chait, B. T.,
and MacKinnon, R.
(1998)
Science
280,
69-77[Abstract/Free Full Text]
|
33.
|
LopezBarneo, J.,
Hoshi, T.,
Heinemann, S. H.,
and Aldrich, R. W.
(1993)
Recept. Channel
1,
61-71[Medline]
[Order article via Infotrieve]
|
34.
|
Choi, K. L.,
Aldrich, R. W,
and Yellen, G.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
5092-5095[Abstract]
|
35.
|
Hadley, J. K.,
Noda, M.,
Selyanko, A. A.,
Wood, I. C.,
Abogadie, F. C.,
and Brown, D. A.
(2000)
Br. J. Pharmacol.
129,
413-415[Abstract/Free Full Text]
|
36.
|
Hoshi, T.,
Zagotta, W. N.,
and Aldrich, R. W.
(1991)
Neuron
7,
547-556[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.