From the Institut für Neurale
Signalverarbeitung, Zentrum für Molekulare Neurobiologie der
Universität Hamburg, Hamburg, Germany and the
§ Insitute for Pharmacology, Department of Physiology,
University of Leeds, Leeds LS2 9JT, United Kingdom
Received for publication, January 18, 2001, and in revised form, April 6, 2001
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ABSTRACT |
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The accessory Shaker-related voltage-gated potassium
(Kv)1 channels are assembled
from membrane-integral pore-forming Kv Three Kv We constructed chimeras between Kv In Vitro Mutagenesis and cRNA Synthesis--
Kv
DNA sequences amplified by the polymerase chain reaction were verified
by sequencing using BigDye terminator cycle sequencing kit (PerkinElmer
Life Sciences). The sequencing reactions were analyzed on an ABI 377 automated sequencer (PerkinElmer Life Sciences).
Point mutations in rat Kv Recording Techniques and Data Analysis--
Xenopus
oocytes were prepared and injected with cRNA, and electrophysiological
recordings were made as previously described (18). Briefly, oocytes
were injected with 50 nl of a solution containing equal amounts (25 ng)
of cRNA for rKv1.5 Kv Failure of Kv
To identify the C-terminal domains responsible for the failure of
Kv Inactivating Activity of Kv Voltage Dependence of Kv1.5 Current Activation Affected by Kv Kv1.5/Kv Mutations of Putative Catalytic Residues Attenuate Kv It has been shown that rapid N-type inactivation of
Shaker Kv channels requires the presence of an N-terminal
inactivating domain and of a receptor close to the inner entrance of
the Shaker channel pore (4). Upon depolarization the
inactivating domain rapidly binds to the receptor and thereby occludes
the pore. Kv subunits of
voltage-dependent potassium (Kv) channels form tetramers
arranged with 4-fold rotational symmetry like the membrane-integral and
pore-forming
subunits (Gulbis, J. M., Mann, S., and MacKinnon,
R. (1999) Cell. 90, 943-952). The crystal structure
of the Kv
2 subunit shows that Kv
subunits are oxidoreductase
enzymes containing an active site composed of conserved catalytic
residues, a nicotinamide (NADPH)-cofactor, and a substrate binding
site. Also, Kv
subunits with an N-terminal inactivating domain like
Kv
1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C.,
Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature
369, 289-294) and Kv
3.1 (Heinemann, S. H., Rettig, J., Graack,
H. R., and Pongs, O. (1996) J. Physiol.
(Lond.) 493, 625-633) confer rapid N-type inactivation to
otherwise non-inactivating channels. Here we show by a combination of
structural modeling and electrophysiological characterization of
structure-based mutations that changes in Kv
oxidoreductase activity
may markedly influence the gating mode of Kv channels. Amino acid
substitutions of the putative catalytic residues in the Kv
1.1
oxidoreductase active site attenuate the inactivating activity of
Kv
1.1 in Xenopus oocytes. Conversely, mutating the
substrate binding domain and/or the cofactor binding domain rescues the
failure of Kv
3.1 to confer rapid inactivation to Kv1.5 channels in
Xenopus oocytes. We propose that Kv
oxidoreductase
activity couples Kv channel inactivation to cellular redox regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits associated with
auxiliary cytoplasmic Kv
subunits (4, 5, 6). The membrane topology
of Kv
subunits shows six membrane-spanning segments, S1-S6, a
pore-forming loop structure between S5 and S6, and cytoplasmic N and C
termini. A tetramerization domain resides in the N terminus and directs
assembly of Kv
subunits (7, 8). The tetramerization domain also
associates with the auxiliary Kv
subunits (9, 10). Crystallographic
analysis of Kv
2 tetramers showed that each subunit contains an
interface for association with Kv
subunits (11) and an
oxidoreductase active site (1), but the specific substrate is unknown.
genes have been identified: Kv
1,
Kv
2, and Kv
3 (12). Kv
1.1 and Kv
3.1
subunits possess an N-terminal inactivating domain that confers rapid
N-type inactivation to Kv channels (2, 3). Thus, the association of
Kv
1.1 and Kv
3.1 subunits with certain Kv1
subunits leads to
the expression of rapidly inactivating A-type channels (2, 3).
Interestingly, Kv
3.1 confers rapid inactivation to Kv1.5 channels
only when coexpressed in mammalian cells (12) but not in
Xenopus oocytes (13). By contrast, Kv1.5/Kv
1.1 channels mediate rapidly inactivating currents both in mammalian cells
(14, 15) and in Xenopus oocytes (2).
3.1 and Kv
1.1 to identify
possible domains correlated with the apparent lack of function of
Kv
3.1 in particular with reference to the N-terminal inactivating domain (2, 3), the interface for association with Kv
subunits (11),
and the oxidoreductase active site (Ref. 1; see Fig. 1, B
and C). The results of our structure-function analysis
demonstrate that the failure of Kv
3.1 to confer rapid inactivation
to Kv1.5 channels in the Xenopus oocyte expression system is
associated with two C-terminal domains of Kv
3.1, which contain
structural elements of the oxidoreductase active site. According to the
crystal structure of Kv
2, the two Kv
3.1 domains occur in Kv
protein regions, which are part of the NADPH cofactor binding pocket
and the substrate binding site, respectively. Chimeric replacement of
Kv
3.1 residues by Kv
1.1 residues within these domains rescued the
Kv
3.1 inactivating activity, and point mutations of Kv
1.1 active
site residues attenuated the Kv
1.1 inactivating activity. We propose
that Kv
oxidoreductase enzymatic activity and the biophysics of
Kv
inactivating activity are coupled.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.1pAKS (2)
and Kv
3.1pAKS (3) were used for Kv
1.1 and Kv
3.1 cRNA synthesis
as described previously (16). The cDNAs for the Kv
chimeras
between rat Kv
1.1 (GenBankTM accession number X70662)
and rat Kv
3.1 (GenBankTM accession number X76723) were
obtained exploiting appropriate restriction enzyme sites and/or using
an overlap polymerase chain reaction (17). The chimeric cDNAs were
cloned into the pAKS vector (16). For the construction of the different
chimeras the following DNA fragments were joined together (note that
numbers in parentheses refer to Kv
-cDNA nucleotides):
Kv
chiA, Kv
3.1-(1-1080) and Kv
1.1-(1000-1546);
Kv
chiArev Kv
1.1-(1-997) and
Kv
3.1-(1075-1599); Kv
chiB, Kv
3.1-(390-1260) and
Kv
1.1-(1180-1534); Kv
chiC, Kv
3.1-(390-1260) and
Kv
1.1-(1180-1534); Kv
chiD Kv
3.1-(390-1380) and 32 Kv
1.1-(1300-1534); Kv
chiE, Kv
3.1-(390-1534) and
Kv
1.1-(1453-1534); Kv
chiF, Kv
3.1-(390-1450), Kv
1.1-(1370-1453), and Kv
3.1-(1535-1606); Kv
chiG,
Kv
3.1-(390-1376), Kv
1.1-(1304-1385), and Kv
3.1-(1457-1606);
Kv
chiH, Kv
3.1-(390-1222), Kv
1.1-(1142-1237),
Kv
3.1-(1318-1440), Kv
1.1-(1370-1453), Kv
3.1-(1535-1606); Kv
chiI, Kv
3.1-(390-1222), Kv
1.1-(1142-1270),
Kv
3.1-(1285-1440), Kv
1.1-(1370-1453),
Kv
3.1-(1535-1606).
1.1 were introduced by site-directed
mutagenesis using appropriate mutation primers (17). Polymerase chain
reaction products were cloned into Kv
1.1pGEM using a
DraIII and a NcoI restriction site. DNA
constructs were sequenced prior to use. RNA synthesis was done using
the mMessage mMachine in vitro transcription kit according
to the manufacturer's protocol (Novagen Inc.).
and rKv
subunits (wild-type or
Kv
chi). Deviations from these cRNA concentrations are
indicated in the figure legends. Oocytes were then incubated at
20 °C for 24-48 h in multiwell tissue culture plates (one
oocyte/well) containing modified Barth's solution (88 mM
NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM
Ca(NO3)2, 0.4 mM CaCl2,
7.5 mM Tris-HCl, pH 7.6, 10,000 units/l penicillin, 100 mg/l streptomycin). To record expressed membrane currents, the
oocytes were held in a recording chamber (50 µl volume) and
continually perfused (2 ml/min) with Ringer's solution (115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 10 mM HEPES adjusted to pH 7.2 with
NaOH). Membrane currents were recorded with the two-microelectrode
voltage-clamp technique (microelectrodes filled with 3 M
KCl) using a Geneclamp 500 amplifier (Axon Instruments), and signals
were filtered at 2 kHz. Twenty 10 mV hyperpolarizing steps (1 s
duration, 0.5 Hz) were applied and used to remove leak and capacitance
currents. To construct current-voltage (I-V) relationships, the cell
was held at
80 mV, 1-s duration test depolarizations at 0.1 Hz were
applied in 10 mV increments from
70 to +120 mV, and peak and end
current amplitudes were measured. I-V curves were fitted with Boltzmann functions of the form I = (V
Vr) × Gmax/(1 + exp((V0.5
V)/k) where
Vr equals
98 mV. The inactivation time course was
fit by a sum of two exponentials by the least squares technique.
Student's t test was used to test for statistical
significance. In some experiments cDNA (vector pcDNA3) for
Kv1.5 (25 ng/µl) together with Kv
chi cDNA (500 ng/µl) were microinjected into Chinese hamster ovary (CHO) cells.
Whole-cell currents were measured with the patch clamp technique on the
following day using an EPC9 amplifier and PULSE software (HEKA,
Lambrecht, Germany). The extracellular solution contained (in
mM) NaCl 135, KCl 5, CaCl2 2, MgCl2
2, HEPES 5, sucrose 10 (pH 7.4, NaOH), and recording pipettes (2-5 M
) were filled with intracellular solution containing (in
mM) KCl 125, CaCl2 1, MgCl2 1, EGTA
11, HEPES 10, glutathione 2, K2ATP 2 (pH 7.2, KOH). All
experiments were conducted at room temperature (20-22 °C).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.1 but Not Kv
3.1 Confers Rapid Inactivation to Kv1.5
Channels in Xenopus Oocytes--
Kv
1.1 and Kv
3.1 subunits have
functional inactivating domains, but Kv
3.1 confers rapid
inactivation to Kv1.5 channels only when coexpressed in mammalian cells
(12). In agreement with previous results (13), wild-type Kv
3.1
subunits did not cause rapid inactivation when we coexpressed it with
Kv1.5 channels in Xenopus oocytes (Fig.
1A; Table
I). Upon depolarization of the oocyte
membrane, slowly inactivating outward currents were recorded, and 84%
of the initial peak amplitude remained at the end of a 1-s test pulse
to +80 mV (Fig. 1, A and D; Table I). By
contrast, Kv1.5/Kv
1.1 channels mediate rapidly inactivating currents
both in mammalian cells (14, 15) and in Xenopus oocytes (2).
In our experiments they decayed to ~13% of the initial maximum
current amplitude at the end of a 1 s test pulse to +80 mV (Fig.
1, A and D; Table I). The inactivation time
course (Fig. 1A) was fitted with two time constants,
1 (13.0 ± 0.9 ms) and
2 (75.0 ± 0.5 ms; n = 14; Table I). The fast time constant
1 accounted for 90 ± 10% of the total current
decay. We examined the structural motifs in Kv
3.1 responsible for
its inactivation failure in Xenopus oocytes by constructing
chimeras between Kv
3.1 and Kv
1.1. Possible structural
determinants correlated with the apparent lack of function of Kv
3.1
may be located in the N-terminal inactivating domain (2, 3), the
interface for association with Kv
subunits (11) and/or the
oxidoreductase active site (Ref. 1; Fig. 1, B and
C).
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Fig. 1.
Inactivating activity of
Kv 1.1, Kv
3.1, and the
chimeric Kv
subunits
Kv
chiA - I when coexpressed with
Kv1.5. A, currents were elicited in Xenopus
oocytes expressing Kv
/
combinations as indicated on
top of each trace. The current traces were recorded using
depolarizing steps from a holding potential of
80 mV to a test
potential of +80 mV of 1 s. Leak currents have been subtracted.
Bars show current and time calibrations. B, bar
diagrams show in gray relative positions of domains
and, respectively, amino acid residues of Kv
1.1 corresponding to the
N-type inactivation domain, the Kv
oxidoreductase active site, and
the interface for assembly with Kv
subunits. C, bar
diagrams of chimeric constructs Kv
chiA - I
(open bars, Kv
1.1; filled bars, Kv
3.1).
Numbers on top refer to first and last Kv
1.1 amino acid
residue; numbers at bottom refer to first and last Kv
3.1
amino acid residue in each construct. D, mean percentage
(I1s / Ipeak) of peak current
(Ipeak) remaining after 1 s of inactivation
(I1s) for Kv1.5 coexpressed with Kv
1.1, Kv
3.1, and
each of the constructs as indicated (experimental protocol as in
A; n = 9-20).
Summary of gating parameters for Kv1.5 currents in the presence of
wild-type and chimeric Kv subunits
1,
2, and the percentage of the total decay accounted for by
1. The amount of inactivation was accessed by the fractional
current remaining after 1 s (I1s / Ipeak).
V0.5 values and respective slope factors
(k) for steady-state activation were obtained as described
under "Experimental Procedures." Numbers of oocytes (n)
are given for both kinetic analysis and voltage dependence of
activation. In all cases 25 ng of Kv1.5 and Kv
cRNA was injected per
oocyte.
3.1 in N-type Inactivation Linked to C-terminal
Domains--
We connected in the first chimera the Kv
3.1 N terminus
(residues 1-229), which contains the N-terminal inactivating domain and the conserved catalytic residues (1) of the Kv
3 oxidoreductase (Fig. 1B), with the Kv
1 C terminus corresponding to
Kv
1.1 residues 223-401 (Kv
chiA; Fig. 1C).
The N-terminal Kv
3.1 inactivating domain became fully functional
when connected to the Kv
1 C terminus (Fig. 1, A and
D), in agreement with the previous observation that the
Kv
3.1 inactivating domain becomes functional when connected to
Kv
2.1 (3). The results obtained with Kv
chiA indicated that C-terminal Kv
3.1 domain(s) must be responsible for the lack of
functional interaction with Kv1.5 channels. To test this hypothesis, we
constructed a reverse chimera (Kv
chiArev), in
which the Kv
1 N-terminal half was linked to the Kv
3 C-terminal
half (Kv
1 residues 1-222 and Kv
3 residues 230-404; Fig.
1B). The Kv1.5/Kv
chiArev-mediated currents exhibited inactivation kinetics similar to the ones observed for Kv1.5 with wild-type Kv
3.1, with 80 ± 1% of the maximal
current amplitude remaining at the end of a 1-s depolarizing pulse
(Fig. 1A; Table I). These results confirmed the idea that
the ability of Kv
chiA to inactivate Kv1.5 channels was
due to the presence of the Kv
1.1 C terminus.
3.1 in N-type inactivation we constructed further Kv
chimeras.
For this purpose we expanded the Kv
3 portion in Kv
chiA in a stepwise fashion. Additional replacement of Kv
1.1 residues 223-282 by those of Kv
3.1 (Kv
chiB) included the
Kv
1 interface domain for assembly with Kv
subunits (Ref. 11; Fig.
1, B and C). The respective
Kv1.5/Kv
chiB channels mediated outward currents with a
relatively small reduction in the extent of inactivation (Fig. 1,
A and D; Table I). The small reduction would be
compatible with a subtle difference in the affinities of Kv
1.1 and
Kv
3.1 subunits for the Kv1.5
subunits (11). Nevertheless, the
results demonstrated that the Kv
3.1 domains for N-type inactivation
and the subunit interface for complex formation with Kv1.5
subunits were not responsible for the observed inactivation failure of Kv
3.1.
The exchange of Kv
1.1 residues by those of Kv
3.1 was extended to
residues 283-346 (Kv
chiC, -D), which may cover the entire substrate binding site of the Kv
oxidoreductase (Ref. 1; Fig.
1, B and C). The respective
Kv1.5/Kv
chiC and Kv1.5/Kv
chiD currents
showed a significant reduction in the extent of inactivation (Fig. 1,
A and D) because of an increase in
2 as well as an alteration in the relative weights of
the fast (
1) and slow (
2) components (Table I). We extended the exchange of Kv
1.1 by Kv
3.1 residues further to residues 347-374 (Kv
chiE), which encompassed
a part of the Kv
oxidoreductase active site that is essential for
binding the cofactor NADPH (Ref. 1; Fig. 1, B and
C). The resulting Kv1.5/Kv
chiE currents showed
an ineffective and slow inactivation (Fig. 1, A and
D) due to an increase in both
1 and
2 (Table I). The results suggested that C-terminal
Kv
3.1 domains encompassing substrate and cofactor binding sites were
responsible for the failure of Kv
3.1 to inactivate Kv1.5 channels.
3.1 Rescued by Swapping with Kv
1.1
Domains--
With chimeras Kv
chiF - I, we tried to
rescue the inactivation failure of Kv
3.1 by exchanging Kv
3.1
domains with appropriate Kv
1.1 domains (Fig. 1, B and
C). Exchanging Kv
3.1 residues 354-381 for those of
Kv
1.1 produced Kv
chiF, which conferred to the slowly inactivating Kv1.5 currents a rapid inactivation (Fig. 1, A and D; Table I). By contrast, replacement of Kv
3.1 residues
328-353 by those of Kv
1.1 (Kv
chiG) had no significant
effect on inactivation (Fig. 1, A and D; Table
I). Supplementation of Kv
chiF with an additional Kv
1.1
sequence replacing Kv
3.1 residues 278-309 (Kv
chiH) did not markedly alter the activity of Kv
chiF toward
Kv1.5 channels. However, extending the replacement in
Kv
chiH by an additional 21 amino acids
(Kv
chiI) generated Kv1.5/Kv
chiI currents
with rapid and nearly complete inactivation (Fig. 1, A and
D; Table I). At the end of the 1-s test pulse to +80 mV the
current was reduced to ~6% of the initial maximum current amplitude
similar to Kv1.5/Kv
1.1 currents (Fig. 1D). The data
demonstrated that the exchange of two C-terminal Kv
3.1 domains
(domains I and II in Fig. 1C) by those of Kv
1.1 sufficed
to rescue the inactivation failure of Kv
3.1 in Xenopus
oocytes. Kv
domains I and II provide amino acid residues to the
Kv
oxidoreductase active site. Domain I contributes to the Kv
NADPH cofactor binding site (Ser-325, Gln-329, Glu-332, and Asn-333 in
Kv
2.1; Ref. 1). Residues in domain II (Kv
1.1 amino acids
303-323; Fig. 1B and C; see also Fig.
4A) have been proposed to participate in substrate binding in Kv
2 (1).
Chimeras--
The voltage-gating properties of Kv1.5 channels were
affected by the presence of different Kv
1.1/Kv
3.1 chimeras (Table
I). This is typically observed upon assembly of Kv
with Kv
subunits (3, 14, 19-21). Voltages of half-maximal current activation (V0.5 = +17 to +22 mV) and slope factors
(k = 19-27 mV) of the conductance-voltage
relationships were similar for Kv1.5/Kv
1.1, Kv1.5/Kv
3.1, and
Kv1.5/Kv
chi currents except for
Kv1.5/Kv
chiB (V0.5 = +36 mV),
Kv1.5/Kv
chiC (V0.5 = +38 mV), and
Kv1.5/Kv
chiF currents (V0.5 = +27
mV) (Table I). Obviously, the distinct inactivation time courses of the
various Kv1.5/Kv
chi currents at +80 mV, especially the
rapid decay of Kv1.5/Kv
chiI currents, are not due to an
altered voltage-dependent steady-state activation.
chi Coexpression Mediates A-type Currents in CHO
Cells--
We considered the possibility that the structural
differences at specific sites between Kv
1.1 and Kv
3.1 lead to
improper protein folding and thereby to the observed functional
inactivity of the Kv
chimeras. Because the wild-type Kv
3.1 is
functionally active in CHO cells (12), we tested the activity of
certain Kv
chimeras in the same expression system. We chose
Kv
chiE, Kv
chiF, and Kv
chiG
for these experiments because this series exhibited both gain of
functional activity (from Kv
chiE to Kv
chiF) and loss of functional activity (from Kv
chiF to
Kv
chiG) in Xenopus oocytes. When we
coexpressed Kv1.5 channels with these chimeras in CHO cells, all the
combinations gave rise to rapidly inactivating currents (Fig.
2A). We used a high excess of
Kv
chi cDNA (20-fold; see "Experimental
Procedures") to ensure a high expression level of
subunits
expected to yield a maximal fraction of Kv
/
complexes (22).
Therefore, we also performed control experiments in Xenopus oocytes with a similar excess of Kv
3.1 cRNA over Kv1.5 channel cRNA.
The results are shown in Fig. 2, B and C. They
were not significantly different from what we obtained in the
experiments with equal amounts of
and
subunit cRNA (see Fig.
1). The results suggested that improper folding or insufficient protein
expression represented the most unlikely explanations for the
functional inactivity of Kv
chimeras in Xenopus
oocytes.
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Fig. 2.
Functionality of Kv 3
subunits depends on the expression system. A,
Kv1.5/Kv
chi currents recorded with the patch clamp
technique in CHO cells. Kv
chi cDNA was microinjected
at a 20-fold higher concentration than Kv1.5 cDNA. Note that all
tested Kv
chimeras conferred rapid inactivation to Kv1.5 channels.
B, oocyte currents mediated by Kv1.5 and Kv1.5/Kv
3.1
channels. Oocytes were injected with 1 ng of Kv1.5 and 20 ng of
Kv
3.1cRNA (1:20 ratio). Despite the high expression level, Kv
3.1
subunits failed to rapidly inactivate Kv1.5 channels in the
Xenopus oocyte expression system. Note, however, that the
relative current amplitude (in %) remaining at the end of a 1-s pulse
was smaller in the presence of Kv
3.1 as shown in C.
1.1
Inactivating Activity--
Because the differing inactivating
activities of Kv
1.1 and Kv
3.1 were correlated to sequence
differences in their oxidoreductase active sites, we mutated the
putative catalytic residues in Kv
1.1 (Asp-119, Tyr-124, and
Lys-152), which are highly conserved in the superfamily of
oxidoreductase enzymes (1). We investigated the effect of the mutations
on Kv
1.1-mediated N-type inactivation. Coexpression of the mutants
Kv
1.1D119A, Kv
1.1Y124F, and Kv
1.1K152A with Kv1.5 at cRNA
ratios of 1:1 (Fig. 3A) and
1:5 (Fig. 3C) showed that the respective outward currents
did not decay as rapidly as Kv1.5/Kv
1.1 currents (Table
II). Each mutation affected rapid inactivation behavior to a different degree. The most marked effect was
observed with Kv
1.1D119A, which did not confer rapid inactivation to
Kv1.5 channels. However, 15-s test pulses revealed that Kv
1.1D119A did accelerate the slow inactivation of Kv1.5 currents (Fig. 3, B and D), which most likely represents a C-type
inactivation (23, 24). As N-type and C-type inactivation are coupled
(25), the observed acceleration of Kv1.5 inactivation was probably due
to some residual inactivating activity of the Kv
1.1D119A
subunit.
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Fig. 3.
Examples of normalized outward currents
mediated by Kv1.5 alone or coexpressed with wild-type
Kv 1.1 (Kv
1.1wt) or
the Kv
1.1 mutants
Kv
1.1D119A,
Kv
1.1Y124F, and
Kv
1.1K152A, respectively, as indicated by the
numbers 1-5. Currents were elicited in Xenopus oocytes
using depolarizing steps from a holding potential of
80 mV to a test
potential of +80 mV in A and B and to a test
potential of +60 mV in C and D. Leak currents
have been subtracted. Bars indicate time calibrations.
Recordings in A and B are from oocytes injected
with 3 ng of Kv1.5 and 3 ng of Kv
cRNA (1:1 ratio), whereas in
C and D 3 ng of Kv1.5 and 15 ng of Kv
cRNA
were injected (1:5 ratio).
Summary of gating parameters for Kv1.5 currents in the absence or
presence of wild-type (wt) and point-mutated Kv1.1 subunits
1,
2, and
percentage of the total decay accounted for by
1). The
amount of inactivation was accessed by the fractional current remaining
after 1 s (I1s/Ipeak). The fitting parameters for
Kv1.5 alone and for Kv1.5 + Kv
1.1D119A were obtained from 15-s
pulses. V0.5 values and respective slope factors
(k) for steady-state activation were obtained as described
under "Experimental Procedures." The values in brackets are from
oocytes, which were injected with a 1:5 ratio of Kv1.5 versus Kv
1.1
cRNA and pulsed to +60 mV for kinetic analysis. In all cases 3 ng of
Kv1.5 cRNA was injected per oocyte. Numbers of oocytes (n)
are given for both kinetic analysis and voltage dependence of
activation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3.1, like Kv
1.1, contains a functional inactivating
domain (3) and thus may confer rapid N-type inactivation to
Shaker type channels. However, the inactivating activity of
Kv
3.1 depends on the in vitro expression system; in CHO
cells Kv
3.1 confers rapid inactivation to Kv1.5 channels (12) but
fails to do so in Xenopus oocytes (3, 13). Our results
demonstrate that the lack of function of Kv
3.1 in Xenopus
oocytes was correlated with two C-terminal Kv
3.1 domains
encompassing the NADPH and substrate binding sites, respectively, of
the Kv
oxidoreductase active site. Domain I in Fig. 1C
and Fig. 4 contributes to the Kv
NADPH
cofactor binding site (Ser-325, Gln-329, Glu-332, and Asn-333 in
Kv
2.1; Ref. 1). Seven of the eight domain I residues that differ
between Kv
1.1 and Kv
3.1 (Fig. 4A) are near or at the
Kv
adenosine-binding pocket (Fig. 4, B and C).
Residues in domain II (Kv
1.1 amino acids 303-323; Fig.
4A) have been proposed to participate in substrate binding,
in particular Kv
1.1 residue Trp-306 that corresponds to Trp-272 in
Kv
2 (1). When domains I and II in Kv
3.1 were replaced by those in
Kv
1.1, the resulting Kv
3.1/Kv
1.1 chimeras were able to confer
rapid inactivation to Kv1.5 channels in the Xenopus oocyte
expression system. The results indicated that the functional activity
of Kv
3.1 in Xenopus oocyte could be rescued by replacing
Kv
3.1 amino acid residues in the oxidoreductase site by the ones of
Kv
1.1. In agreement with the assumption that C-terminal Kv
3
domain(s) are responsible for the observed lack of function, Kv
1.1
was rendered non-functional when the C-terminal half of the protein was
replaced by Kv
3.1 sequences.
View larger version (34K):
[in a new window]
Fig. 4.
Localization of domains I and II in
the Kv 2 crystal structure. A,
alignment of domain I and II sequences of Kv
1.1, Kv
2.1, and
Kv
3.1. Numbers at right and left refer to
first and last amino acid residue shown. Asterisks indicate
amino acid residues that are in close contact with the adenosine moiety
of the NADPH cofactor bound in the Kv
2 active site (1). A
Black dot marks the Kv
2 amino acid residue proposed to
contribute to the substrate binding site (1). Identical amino acid
residues are in red. Amino acid residues are given in the
single letter code. B, ribbon representation of the
three-dimensional structure of Kv
2 tetramer according to Ref. 1.
Domains I and II as defined in Fig. 1C are colored
red. NADPH cofactor (green) is shown as
Corey-Pauling-Koltun model. Domain I residues 325, 329, 332, and
333 and domain II residue 272 are labeled. Side-chains are in
stick representation and have been colored
magenta. C, Kv
monomer to illustrate domain I
side-chains in NADPH binding pocket and domain II side-chain in
substrate binding site. Model was prepared with the program INSIGHT
(Molecular Simulations Inc., San Diego, CA) and based on the Kv
2
structure as available in the Protein Database accession number
1 QRQ.
Three main alternatives may be considered to understand the results: i)
Kv3.1 is not active because the oxidoreductase site is not properly
folded; ii) Kv
3.1 activity is inhibited in Xenopus oocytes by an as yet unknown factor; and iii) Kv
3.1 is not active because the Xenopus oocytes do not provide a substrate for
the Kv
3.1 oxidoreductase. The results showed that Kv
3.1 and Kv
chimeras are functionally active in CHO cells. This demonstrates that
active and properly folded Kv
3.1 protein can be expressed in
in vitro expression systems. Most likely, translation and
folding of Kv
3.1 protein in Xenopus oocytes is not
different from that in CHO cells. Therefore, it is unlikely that
Kv
3.1 (and the Kv
chimeras) is not properly folded when expressed
in Xenopus oocytes. The existence of a putative inhibitory
factor in Xenopus oocytes, which is specific for Kv
3.1,
cannot be rigorously excluded but seems to be also unlikely. Thus, we
assume that Kv
3.1 fails to confer rapid inactivation to Kv1.5
channels because its oxidoreductase activity is not functioning in
Xenopus oocytes. In agreement with this assumption we find
that a replacement of the NADPH and the putative substrate binding
domains by those of Kv
1.1 reconstitutes the inactivating activity of
Kv
3.1 in Xenopus oocytes.
Because Kv3.1 oxidoreductase activity is apparently important for
conferring rapid inactivation to Kv1.5 channels, we explored the
possibility that mutations of catalytic residues in Kv
1.1 (Asp-119,
Tyr-124, Lys-152) may attenuate the Kv
1.1 inactivating activity. The catalytic residues are highly conserved among the active
sites of oxidoreductases (1), and comparable mutations in established
oxidoreductase enzymes have been shown to impair catalytic activity
(26, 27). The results showed that the mutations severely affected the
ability of Kv
1.1 to confer rapid inactivation to Kv1.5 channels.
Although the putative Kv
enzymatic activity could not be tested
directly, it is likely that the mutations of catalytic residues in
Kv
1.1 affected its putative oxidoreductase activity. We propose that
Kv
oxidoreductase catalytic activity is required for the
inactivating activity of Kv
1.1. Thus, manipulations of the putative
Kv
1.1 and Kv
3.1 oxidoreductase active sites were correlated with
a loss and, respectively, gain of inactivating activity in the
Xenopus oocyte expression system.
Although we have not carried out biochemical experiments to test
directly the binding of Kv3.1 to Kv1.5, the effects of Kv
3.1 and
the different Kv
1.1/Kv
3.1 chimeras on the voltage-gating properties of Kv1.5 are a clear indication that Kv
3.1 binds to Kv1.5. Changes in the voltage-gating properties of Kv1 channels are
typically observed upon assembly of Kv
with Kv
subunits (3, 14,
19-21). In conclusion, Kv
3.1 assembles with Kv1.5 channels, but the
activity of the N-terminal Kv
3.1 inactivating domain is impaired.
Apparently, the effects of Kv
3.1 on the
voltage-dependent activation of Kv1.5 channels are distinct
from those leading to rapid inactivation. This is in agreement with the
previous observation that removal of 10 amino acids from the Kv
1.3 N
terminus eliminated the inactivation activity but not the voltage shift
of activation of Kv1.5 channels (28).
Kv2 subunits do not have an N-terminal inactivating domain. When
coexpressed with Kv
subunits, Kv
2 may also alter the
voltage-gating properties of Kv channels and, in addition, enhance
trafficking of Kv channels to the plasma membrane. In agreement with
our results, it has been shown in a recent report (29) that mutating
active site residues in Kv
2 did not interfere with its binding to
Kv1.4 channels. These Kv
2 mutants still affected the voltage-gating properties of Kv1.4 channels like wild-type Kv
2, but the enhancing effects on Kv1.4 channel surface expression were attenuated.
Apparently, the putative Kv
oxidoreductase activity is important for
distinct aspects of Kv
function.
Previously, we have shown that N-type inactivation can be prevented by
a NIP-domain in Kv1.6 subunits (15). Now, we show that N-type
inactivation of Kv channels may be coupled to the putative Kv1.1 and
Kv
3.1 oxidoreductase activity. This observation indicates that the
gating mode of Kv channels linked to N-type inactivation may be
regulated by a variety of cellular mechanisms. We propose that the
presence of an oxidoreductase activity in Kv channels may couple
cellular redox regulation to the gating mode of Kv channels allowing
the channels to switch between a rapidly inactivating and a
non-inactivating mode. Identifying the Kv
oxidoreductase
substrate(s) will bring us closer to understanding the cellular
function of such potential energetic coupling.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Maudsley for performing initial
experiments, M. Berger and S. Sewing for help with the construction of
KvchiA - E chimeras, D. Clausen for technical help with
the figures, and C. Legros for modeling Kv
2.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft (O. P.) and the Wellcome Trust (D. W.).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: Zentrum für Molekulare Neurobiologie der Universität Hamburg, Martinistr. 52, 20246 Hamburg Germany. Tel.: 049-40-2803-5081/5082; Fax: 049-40-42803-5102; E-mail: Pointuri@uke.uni-hamburg.de.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M100483200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Kv, voltage-dependent potassium; CHO, Chinese hamster ovary.
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