From the Department of Physiology II, University of
Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany,
§ Forschungsinstitut für Molekulare Pharmakologie,
Campus Berlin-Buch, Robert-Rössle-Strasse 10, 13125 Berlin,
Germany, and ¶ Department of Biophysics and Physical Biochemistry,
University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
Received for publication, October 4, 2002, and in revised form, February 14, 2003
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ABSTRACT |
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Cumulative inactivation of
voltage-gated (Kv) K+ channels shapes the presynaptic
action potential and determines timing and strength of synaptic
transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type
inactivation gating. Different from all other Kv Fast inactivation of voltage-gated K+
(Kv)1 channels is a major
determinant for signal propagation and synaptic transmission in central
nervous system neurons (1-4). Thus, A-type K+ channels
were shown to regulate orthograde and retrograde propagation of
dendritic action potentials (APs) in CA1 pyramidal neurons and to
dynamically control presynaptic Ca2+ inflow into
hippocampal mossy fiber boutons (2, 3). In the latter, repetitive
stimulation (frequencies of The molecular determinant of the rapidly inactivating K+
current in mossy fiber boutons most probably is Kv1.4, a Kv Different from other Here we analyze the solution structure and the function of this tandem
inactivation domain of Kv1.4 channels (amino acids 1-75,
Kv1.4N-(1-75)) using NMR spectroscopy together with patch clamp
recordings in whole cell configuration and simulations of neuronal
spiking behavior.
Peptide Synthesis and Sample Preparation--
A polypeptide
covering the 75 N-terminal amino acids of Kv1.4 was expressed as a
histidine-tagged GST fusion protein in the Escherichia coli
strain BL21(DE3) and labeled uniformly with 15N using
15N-ammonium chloride (98%) as a nitrogen source. The
protein was purified to homogeneity via nickel-nitrilotriacetic
acid-agarose (Qiagen) and glutathione-agarose chromatography. The GST
fusion domain was chopped off with thrombin leaving three residues
(Gly-Ser-Thr) at the N terminus of Kv1.4N-(1-75).
For NMR Kv1.4N-(1-75) protein was dissolved in a solution containing
50 mM sodium phosphate (90% H2O, 10%
D2O (v/v), pH 4.4), 10 mM dithiothreitol, and 1 mM protease inhibitors. The final protein concentration of
Kv1.4N-(1-75) was 1 mM. For 1H chemical shift
referencing, 2,2-dimethyl-2-silapentane-5-sulfonate was added as an
internal standard.
NMR Spectroscopy--
All of the NMR spectra of Kv1.4N-(1-75)
were recorded at 283 K on Bruker Avance spectrometers. At 600 MHz proton frequency, the following spectra were recorded according to
standard procedures: 1H-15N HSQC,
1H-15N total correlation
spectroscopy-HSQC (spin-lock mixing time of 70 ms),
1H-15N NOESY-HSQC (mixing time 120 ms),
HNHA, and a two-dimensional homonuclear NOESY (mixing time 150 ms) in D2O solution. Spectral windows of 9 and 24 ppm were
employed in the 1H and 15N dimensions,
respectively. 3JHNH
Hydrogen/deuterium exchange of the backbone amide groups of
Kv1.4N-(1-75) was evaluated by 1H-15N HSQC
spectra on a uniformly 15N-labeled sample after solvent
exchange to 99.9% D2O using a NAP5 column (Amersham
Biosciences). NMR data were processed with the XWIN NMR software
(Bruker) and analyzed with the programs AURELIA (Bruker) and XEASY (ETH
Zürich) (25).
Structure Calculation--
The solution structure of
Kv1.4N-(1-75) was calculated with the DYANA program package (version
1.5) that uses a simulated annealing algorithm in the torsion angle
space (26). Distance restraints were derived from a
15N-edited three-dimensional NOESY spectrum in
H2O solution and a 1H-1H
two-dimensional NOESY spectrum in D2O solution. Volumes of
unambiguously assigned NOESY cross-peaks were converted into
proton-proton upper distance limits by the caliba routine within DYANA.
The dihedral angle
The final family of structures was generated in a calculation with 300 random starting structures and 12,000 annealing steps. The 25 best
structures had values for the target function <0.55 Å2,
no NOE violations >0.3 Å, and no violations of dihedral angle constraints. These structures were selected for further analysis and
evaluation of structural quality with the programs DYANA and MOLMOL
(27). The latter was also used to prepare the figures showing
three-dimensional structures.
Electrophysiology and Molecular Biology--
Site-directed
mutagenesis was performed with a standard cassette technique (28), and
the mutations were verified by sequencing. For functional expression,
Kv1.4 WT and mutant subunits were subcloned into pBK-CMV (29) and the
cDNA (0.5 µg/µl) was injected into Chinese hamster ovary
dhFr
Cells showing GFP fluorescence were chosen for electrophysiological
experiments 20-40 h after injection. Whole cell voltage-clamp recordings were performed with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at room temperature (21-24 °C) using quartz pipettes with resistances of 1.5-2.5 megohms when filled with
intracellular solution of the following composition (in
mM): 135 KCl, 3.5 MgCl2, 0.1 CaCl2,
5 K2EGTA, 5 HEPES, 2.5 Na2ATP, and 0.1 dithiothreitol (pH adjusted to 7.3 with NaOH). Dithiothreitol was used
to establish a stable redox milieu in the cytoplasm (9). After
establishing whole cell configuration and series resistance compensation, residual series resistance was <0.8 megohms.
Extracellular solution was (in mM) 144 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 10 HEPES, 0.7 Na2HPO4, and 5.6 glucose (pH adjusted to 7.3 with KOH). For competition experiments, TEA-Cl (1 mM) was
added to the intracellular solution and exchange of the intracellular
solution was achieved by successively repatching the same cell with
pipettes containing different intracellular solutions as described
previously (30). The absence of channel run-down during successive
experiments was verified by concluding the experiment with the control
solution used initially.
Activation and inactivation kinetics of Kv1.4 channels (Figs. 4-6)
were measured by stepping the membrane potential from
The time course of recovery from inactivation was obtained with a
three-step voltage protocol. 1) Voltage was first stepped to 60 mV for
1 s to allow for complete inactivation. 2) Voltage was then
stepped to
Currents were low pass filtered at 2 kHz (4-pole Bessel) and sampled at
10 kHz. Processing and fitting of the data were performed with IgorPro
(Wavemetrics, Lake Oswego, OR). All of the data are given as the
mean ± S.D. of n experiments.
Simulation of Neuronal Signaling--
Simulations of neuronal
input-output relations were performed with Neuron (version 4.3.1) (31,
32). This simulation environment calculates the voltage response of a
cell (of given geometry) to current stimuli based on
Hodgkin-Huxley-type voltage-gated conductances for Na+ and
K+ (non-inactivating K+ currents) and standard
values for leakage conductance and passive electrical properties
including membrane capacitance and intracellular resistance. To
implement the kinetics of Kv1.4 WT and ( Assignment and Backbone Dynamics of Kv1.4N-(1-75)--
A
polypeptide containing the first 75 amino acids of Kv1.4
(Kv1.4N-(1-75)) was overexpressed in bacteria, purified, and used for
structural analysis. A series of multidimensional (two- and three-dimensional) homonuclear and heteronuclear NMR experiments (see
"Materials and Methods") was performed in aqueous solution under
physiological salt conditions. The 1H and 15N
resonances of the polypeptide were completely assigned by standard procedures based on 15N-edited three-dimensional total
correlation spectroscopy and NOESY spectra.
As shown in Fig. 1B, the
observed NOE connectivities displayed a distinct pattern along the
Kv1.4N-(1-75) sequence with NOEs between non-adjacent amino acids
(i,i+x) only occurring on the sequence stretch extending
from Gly-20 to Ala-50. In this region, NOEs between the
This view of structurally distinct parts in Kv1.4N-(1-75) was further
supported by the chemical shift index of the
It should be noted at this point that the NOE pattern, the H
Together, NOE pattern, scalar couplings, chemical shift data, and
relaxation measurements suggest that Kv1.4N-(1-75) consists of a
highly ordered core region with mostly helical conformation flanked by
flexible N- and C-terminal ends. The absence of any stable tertiary
folding is indicated by the small chemical shift dispersion of the
backbone amide protons (~1 ppm), the fast H/D exchange of all
amide protons in D2O solution, and the missing of long
range NOEs in the NOESY spectra of Kv1.4N-(1-75).
Solution Structure of
Kv1.4N-(1-75)--
A total of 391 experimentally determined distance
restraints together with the restraints for dihedral angles and
stereospecific assignments of protons (Table I) were used to
calculate the solution structure of Kv1.4N-(1-75). After structure
calculations with the simulated annealing protocol of DYANA (26) in the
torsion angle space, a family of 25 structures with lowest values of
the target function and without NOE violations >0.3 Å was selected as
the final family of Kv1.4N-(1-75) structures (see Table I for
structural statistics).
Fig. 3 shows 22 representatives of this
family of best structures superimposed either between residues 21 and
37 (Fig. 3A) or residues 41 and 49 (Fig. 3B).
Both superpositions converged well with similar root mean square
deviation values to the mean structure (0.50 ± 0.30 Å and
0.76 ± 0.34 Å for the backbone atoms in Figs. 3A and
B, respectively), whereas superposition of the entire core
region was not meaningful because of the divergent orientation of
Ala-38 and Ala-39 that link the two convergent stretches (see Table I).
Thus, the double Ala linker divides Kv1.4N-(1-75) into an N- and
C-terminal domain. The N-terminal domain, which coincides with ID1 (9),
comprises a flexible N terminus formed by the first 20 amino acids and
a 5-turn Structure-Function Analysis Delineates Distinct Properties and
Contribution to Fast Inactivation of ID1 and ID2--
The structure
presented above (Fig. 4) was used as a guide to delineate the
functional properties of the tandem inactivation domain of Kv1.4
channels. For this purpose, the distinct structural domains were
deleted and the respective mutants were expressed in Chinese hamster
ovary cells and probed for their inactivation characteristics. Fig.
4B depicts representative current responses to a 250-ms
voltage step from
In addition to being susceptible to N-terminal deletions, rapid
inactivation in WT as well as in the
These results indicated that both ID1 and ID2 are able to inactivate
Kv1.4 channels via occlusion of the open channel pore and thus raised
the question of which domain may work as the actual ball domain.
To answer this question, recovery from inactivation, i.e.
unbinding of ID1 and ID2 from the receptor site, was investigated with
a standard three-step protocol (see "Material and Methods"). As
depicted in Fig. 5B, WT channels could be recovered
completely by a hyperpolarizing pulse to
Together, these results strongly suggest that ID1 rather than ID2 is
the pore-occluding domain of Kv1.4 channels. But as so, what is the
function of ID2 within the structural context of the Kv1.4 protein?
This question was assessed by site-directed mutagenesis that either
deleted the ID2
Different from the Structure-Function Correlation and Model for Fast Inactivation of
Kv1.4--
Here we show that the solution structure of Kv1.4N-(1-75),
a polypeptide covering the tandem inactivation domain of Kv1.4 channels
(20), consists of two domains connected by a short flexible linker. The
N-terminal half termed ID1 comprises a flexible region anchored at a
5-turn
These conclusions are based on several observations. First, although
both ID1 and ID2 block the open channel when present at the N terminus
(competition with TEA, Fig. 5A), they largely differ in
their recovery from inactivation. Thus, recovery was identical and fast
in all of the channels bearing ID1 at their N terminus (Figs.
5B and 6D). In contrast, recovery from
ID2-mediated inactivation as observed in the
Together, structural and functional data would be consistent with a
sequential model for fast inactivation in Kv1.4 channels. The
hydrophobic ID2 helix may attach ID1 to the cytoplasmic surface of the
channel protein, recently called preinactivation step (17), and thus
promotes rapid insertion of ID1 into its "receptor site" that
becomes accessible after opening (activation) of the channel pore. In
Kv1.4, this ball- or N-type inactivation may subsequently trigger a
collapse of the selectivity filter, a phenomenon known as C-type
inactivation (41, 42). Recovery from inactivation, the reverse process
of channel occlusion, should be initiated by dissociation of ID1 from
its receptor, which finally reopens the pore. At present, the
interactions between ID1 and the pore walls must remain speculative as
well as the folding adopted by ID1 inside the channel (see Supplemental
Fig. 1). Moreover, the binding site of ID2 on the cytoplasmic face of
the channel protein remains to be elucidated.
Although most probably not relevant for inactivation, pore-occlusion by
the short ID2 helix provides some new twists to where and how an ID
might interact with the surface of the inner channel pore. We probed
the insertion of this compactly folded piece into the recently proposed
"open pore structure" of MthK/KcsA (43) using the docking procedure
of the program GRAMM (44, 45). As illustrated in Supplemental Fig. 1, the ID2 helix is well able to enter the pore in helical configuration
and to proceed until right underneath the selectivity filter, very
similar to what was suggested for TEA derivatives (17).
Potential Significance of ID2 for Neuronal Signaling and for
Kv1.4-mediated Short Term Plasticity--
Because rapid N-type
inactivation is a major determinant for efficiency and short term
plasticity of synaptic transmission, we probed the significance of the
docking domain by simulating the input-output relation of a neuron
using Neuron, an environment for solving nerve equations (31, 32). For
this purpose, a spherical cell was equipped with a leakage
K+ conductance, a standard voltage-gated Na+
conductance, and a voltage-gated K+ conductance, which
either reflected non-inactivating Kv1.4 WT or Kv1.4-(
Fig. 7A illustrates spike
responses of such a neuron elicited by a depolarizing current stimulus.
Thus, with non-inactivating Kv channels, the cell responded with
regular AP of constant amplitude throughout the stimulation period. In
contrast, in the cell equipped with rapidly inactivating Kv1.4 WT, the
stimulus elicited APs that successively decreased in amplitude and
finally disappeared because of cumulative inactivation of the
voltage-gated K+ channels. This means that Kv1.4 channels
inactivated during the depolarizing phase of the AP cannot recover from
inactivation during the repolarization period that is short with
respect to the slow recovery time course (Figs. 5B and
6D). As a consequence, the cell remained depolarized once
all of the Kv1.4 channels were inactivated.
The phenomenon of cumulative inactivation appeared to be abolished in
the absence of the docking domain, and the cell equipped with
Kv1.4-(
In central nervous system neurons, cumulative inactivation implements a
"molecular memory" that operates on the time scale of seconds (2,
46, 47). This memory effect is shown in Fig. 7C where a
large amplitude excitation is preceded by a low amplitude stimulus. The
AP frequency that is determined by the set of conductances in the
Neuron platform (31, 32) was slightly above 100 Hz, in close agreement
with what is known regarding the typical spiking of granule cells in
the hippocampus (48, 49).
During this low amplitude stimulus, cells equipped with Kv1.4 WT or
subunits, Kv1.4
harbors two inactivation domains at its N terminus. Here we report the
solution structure and function of this "tandem inactivation
domain" using NMR spectroscopy and patch clamp recordings.
Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N
terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a
2.5-turn helix made up of small hydrophobic amino acids. Functional
analysis suggests that only ID1 may work as a pore-occluding ball
domain, whereas ID2 most likely acts as a "docking domain" that
attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2
slows inactivation considerably and largely impairs cumulative
inactivation. Together, the concerted action of ID1 and ID2 may promote
rapid inactivation of Kv1.4 that is crucial for the channel function in
short term plasticity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
10 Hz) led to a broadening of the
APs because of cumulative inactivation of A-type K+
channels. The prolonged AP resulted in an increased Ca2+
inflow, which in turn promoted an increased transmitter release and a
consecutive potentiation of the evoked excitatory postsynaptic currents
(2).
-subunit with prototypic ball-and-chain-type inactivation (2, 4, 5). This
classical gating process is brought about by a "plug-in" mechanism
in which a protein domain termed "inactivation ball" occludes the
ion pathway through interaction with a receptor site in the pore of the
channel (6). Structure-function analysis showed: (i) that inactivation
domains (ID) are formed by the first 20-40 amino acids at the N
terminus of certain Kv
(7-10) and Kv
subunits (11, 12); (ii)
that IDs inactivate channels independent of whether they are tethered
to the protein or offered to the channel as a synthetic peptide (8, 9,
13, 14); (iii) that IDs enter the channel pore and compete with open
pore blockers such as tetraethylammonium (TEA) (11, 15-17); and
finally (iv) that NMR-derived solution structures of the known IDs vary
from a well ordered and compact folding as in Kv3.4 ID (18) to the disordered and highly flexible ID from ShakerB (19).
/
subunits that harbor one ID in their N
terminus, Kv1.4 was found to be equipped with two IDs with the ability
to endow the channel with rapid inactivation (Fig. 1A). ID1
is made up by residues 1-37 (9), and ID2 resides between amino acids
40 and 68 (20). More explicitly, the existence of the second ID became
evident after the first 39 residues were deleted, and the resulting
mutant channels did not lack inactivation but rather displayed
rapid inactivation very similar to that observed with ID1 present at
the N terminus (20, 21). However, it remained obscure what the
functional role of the second ID might be in intact channels and,
therefore, how inactivation gating is realized by this tandem
inactivation domain.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
coupling
constants were determined from the three-dimensional HNHA spectrum as
described previously (22). The 15N relaxation measurements
(T1, T2, 1H-15N NOE)
were carried out at 500-MHz proton frequency as described previously
(23) with overall delays between scans of 2 s for the
T1 and T2 measurements and 3 s for the
heteronuclear NOE experiment, respectively. The delays used for the
T1 experiments were 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 ms, whereas delays of 8, 16, 24, 40, 64, 96, 128, 160, 256, 320, 480, and 800 ms were used for the T2 experiments. The
15N relaxation data were analyzed according to the reduced
spectral density mapping approach (24).
of residues with a
3JHNH
coupling constant of
6 Hz was
restrained to values between
90 and
30°. Structures from
preliminary DYANA runs were used to recalibrate the distance restraints
in an iterative manner. Stereospecific assignments were provided by the
glomsa algorithm implemented in DYANA.
cells with a Microinjector (Transjector 5246, Eppendorf, Germany) (30). For identification of successfully injected
cells, 10 ng/µl pBK-CMV-GFP was coinjected as a reporter. Cells were
grown in minimum essential medium-
(Invitrogen) supplemented
with 10% fetal bovine serum (Biochrom, Berlin, Germany) and
penicillin/streptomycin (100 units/ml, Invitrogen) at 37 °C and 5%
CO2.
120 mV to the
values indicated for variable periods (between 250 and 500 ms).
Steady-state inactivation curves as in Fig. 6B were obtained with a standard two-step protocol, which used a prepulse of 0.8 s
to potentials between
120 and 20 mV (10-mV increments) and a test
pulse to 60 mV that determined the fraction of channels inactivated
during the prepulse. Between individual cycles, the voltage was held at
120 mV to allow for complete recovery from inactivation.
The inactivation curve was fitted with a Boltzmann function (I = [1 + exp{(V
V1/2)/k}]
1), where
V is the membrane (prepulse) potential,
V1/2 is the potential at half-maximal inactivation,
and k is the slope factor. Activation curves (used for the
simulations in Fig. 7) were obtained by stepping the membrane potential
from
120 mV to potentials between
80 and 60 mV (10-mV increment)
for 250 ms. The current amplitude was divided by the driving force to yield channel activation as a function of membrane potential.
120 or
80 mV for a variable period. 3) The final step
used to determine the fraction of channels that recovered during the
hyperpolarizing pulse was to
20 mV.
40-50) mutant channels, the
measured steady-state activation and inactivation curves and the
recovery from inactivation as well as the time constants for activation
and inactivation on a broad voltage range (
100 to 80 mV) were used to
determine the Hodgkin-Huxley parameters of channel gating (33). The
following rates were used to model the inactivation gating of Kv1.4 WT
(
h(V) = 0.000025608·exp(
V/45.4217),
h(V) = 0.0330402/exp(
(V + 45.6599)/2.30235 + 1)) and of Kv1.4-(
40-50)
(
h(V) = 0.000169537·exp(
V/223.943),
h(V) = 0.0090891/exp(
(V + 43.7084)/2.63186 + 1). The voltage-gated Na+ conductance
was 0.12 S/cm2, and the values for voltage-gated
K+ and leakage K+ conductance were 0.036 and
0.0003 S/cm2, respectively. The membrane capacitance was
0.7 microfarad/cm2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-proton of
one residue and the amide (d
N(i,i+3)) or
-proton (d
(i,i+3))
of the third amino acid following were observed together with weak
sequential contacts (d
N(i,i+1)) and strong amide-amide NOEs
(dNN(i,i+1)), a NOE pattern usually seen with
-helices. In line with
this interpretation, all of the 3JHNH
coupling constants determined for residues 20-50 were between 4 and
5.6 Hz (Fig. 1B), values regarded diagnostic for helical
conformation (34), whereas the scalar couplings for the residues N- and
C-terminal to this "core region" of Kv1.4N-(1-75) were between 6 and 8 Hz, as typically seen for random coil peptides (35).
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Fig. 1.
Functional domains of Kv1.4 and NOE contacts
observed for Kv1.4N-(1-75) in aqueous solution.
A, Cartoon illustrating trans-membrane topology and
functional domains identified in Kv1.4 channels. ID1 and ID2 represent
inactivation domains as identified earlier (9, 20), T1 (50) and NAB
(51) are domains involved in subunit assembly and binding of Kv
subunits, respectively. B, NOE connectivities and
J-coupling constants observed for Kv1.4N-(1-75) in aqueous
solution. Sequential and medium-range NOEs are shown as a function of
the amino acid sequence. Intensity of NOEs is represented by the line
thickness. Filled circles represent
3JHNH
coupling constants between 3.9 and 6 Hz.
-protons as well as the
backbone dynamics. Thus, most of the
-protons within the core region
are shifted upfield by 0.1 ppm or more with respect to random coil
chemical shifts (Fig. 2, upper
panel). The backbone dynamics on the picosecond to nanosecond time
scale as determined from 15N relaxation and the
1H-15N heteronuclear NOE (36) strongly
suggested an ordered core region flanked by flexible N and C termini.
As illustrated in Fig. 2 (middle and lower
panels), the termini displayed very short transverse relaxation
rates (R2) and negative values for the heteronuclear NOE,
whereas residues 20-50 exhibited positive values of the
1H-15N NOE and relatively high R2
rates. Moreover, the spectral density functions J(0) and
J(
H) demonstrated reduced mobility of the protein
backbone between residues 20 and 50 together with high mobility in the
rest of the polypeptide (data not shown) (24).
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Fig. 2.
Dynamics of Kv1.4N-(1-75) reveal an
ordered core flanked by flexible N and C termini. Upper
panel, deviation of H chemical shifts from random coil values.
H
was calculated as the difference between the random coil
H
and the experimentally determined
H
. Middle
panel, ratio of transversal (R2) and longitudinal
(R1) relaxation rates. Lower panel,
1H-15N heteronuclear NOEs (measured at 500 MHz). Increasing positive values indicate decreasing flexibility of the
backbone (36).
chemical shifts, the scalar couplings, and the relaxation data suggest
that the helical conformation may not extend throughout amino acids
20-50. Rather, there may be two helices with a different stability:
the more stable one formed by amino acids Tyr-21 to Ala-38 (average
deviation of 3JHNH
from random coil
values of
1.7 ± 0.6 Hz); the second one involving the
alanine-rich stretch between residues 40 and 50 (average deviation of
3JHNH
from random coil values of
0.8 ± 0.3 Hz).
Structural statistics for the family of 25 Kv1.4N-(1-75) structures
-helix between residues 21 and 38 (Fig.
4A). This structure is similar to the 37 amino acid piece we reported earlier (18) with the exception
that the
-helix is extended by 2 turns and bent in its center. The
C-terminal domain of Kv1.4N-(1-75) equivalent to the stretch harboring
ID2 (20) consists of a 2.5-turn
-helix between amino acids 41 and 49 connected to a flexible domain formed by residues 50-75 (Fig.
4A).
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Fig. 3.
Solution structure of Kv1.4N-(1-75).
A, backbone traces (N, C , and C' atoms) of the
family of best structures of Kv1.4N-(1-75) (with lowest target
function) between amino acids 15 and 53 superimposed between residues
21 and 37 (highlighted in red). B,
backbone traces as in A, but superimposed between residues
41 and 49 (highlighted in green). Superpositions
depict 22 of the 25 best structures of Kv1.4N-(1-75) (see "Solution
Structure of Kv1.4N-(1-75)" under "Results"). N and C termini
are indicated.
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Fig. 4.
Structure-function relation of fast
inactivation in Kv1.4. A, N-terminal 53 residues of the best
Kv1.4N-(1-75) structure in ribbon representation emphasizing the
secondary structural elements between residues Gly-20 and Ala-38 and
alanine residues 40 and 50. B, deletion analysis
illustrating the functional significance of the structural domains
shown in A. Traces are current responses to a voltage step
from 80 to 20 mV recorded in Chinese hamster ovary cells injected
with Kv1.4 WT or the deletion mutants indicated. Scaling is 100 ms and
1 nA for all of the traces. Note that fast inactivation is only
observed with either residues 1-38 (ID1) or 39-50 present at the N
terminus of the channel.
80 to 20 mV. Similar to earlier reports, rapid and
complete inactivation was observed with WT channels bearing ID1 at its
N terminus as well as with the
2-38 mutant in which the ID1 stretch
was deleted (20). In either case, the inactivation time constant
(
inact) at 20 mV was ~16 ms (
inact of
16.1 ± 2.6 ms (n = 6) and 15.8 ± 2.7 ms
(n = 7) for WT and
2-38, respectively). More
detailed analysis showed that in WT, rapid inactivation was largely
impaired by deleting either the flexible domain of ID1 formed by
residues 1-20 or the 5-turn
-helix (Fig. 4B, left
panel). In contrast, fast inactivation in the
2-38 mutant
predominantly required the
-helical domain between amino acids 40 and 50. Its deletion completely abolished inactivation, whereas further
deletion had no additional effect (Fig. 4B, right
panel).
2-38 mutant fulfilled another
hallmark of the ball-and-chain mechanism: inactivation was competed by
the open channel blocker TEA (16, 37, 38). As shown in Fig.
5A, 1 mM TEA
slowed down the inactivation time course in either channel perfectly by
the amount that would be predicted, assuming direct competition between
TEA and the respective N-terminal ID for an overlapping receptor site
within the open channel pore (39).
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Fig. 5.
Distinct inactivation properties of WT
and 2-38 mutant channels. A,
inactivation of both WT and the
2-38 deletion mutant is competed by
1 mM TEA applied to the cytoplasmic side of the channels.
Left panel, current response to voltage steps from
80 to
20 mV recorded in the absence (gray) or presence
(black) of TEA. Current and time scales are 1 nA and 50 ms,
respectively. Right panel, relative increase in inactivation
time constant (mean ± S.D.; n = 3) induced by 1 mM TEA. Line represents theoretical dependence
of
inact on the fraction of channels blocked by TEA,
assuming competition between TEA and the inactivation domain
(
inact = 1/(1
x), where
x denotes the fraction of blocked channels (39)).
B, representative recovery from inactivation recorded at a
membrane potential of
120 mV, with a conditioning pulse of 1 s
to 60 mV (for protocol see "Materials and Methods").
Line represents fit of a monoexponential to the recovery
time course. Values for the time constant were 2.8 and 195.9 s for WT
and
2-38, respectively.
120 mV. The time course of
recovery was well described by a monoexponential with a time constant
of 2.75 ± 0.52 s (n = 6). In contrast, the
unbinding of ID2 was roughly one hundred-fold slower than that of ID1,
and recovery from inactivation could hardly be achieved in the
2-38
mutant channels, even with hyperpolarizing pulses of >4.5-min duration
(Fig. 5B; the respective time constant as extrapolated from
such experiments was 295 ± 141 s (n = 3)).
Such slow recovery, which by far exceeds the value obtained with any
other Kv channel, reflects very slow dissociation of ID2 from its
receptor site and may result from the particular "sticky" character
of the polyalanine stretch in the ID2
-helix.
-helix or changed two consecutive residues of the
ID2
-helix to proline to induce a helix break at defined positions.
The resulting mutants were then investigated for their gating
properties. As illustrated by the current response in Fig. 6A, deletion of the
polyalanine helix considerably slowed down channel inactivation, while
the activation process remained unaltered. More detailed analysis
showed that
inact was increased by more than a factor of
4 on the voltage-range
0 mV where the inactivation time course is
independent of channel activation and steady-state inactivation is
complete (Fig. 6B). A similar
4-fold increase of
inact was observed when proline residues were introduced
at either the N-terminal end or the middle of the ID2
-helix
(A42/A43P and A45/A46P), whereas only a small increase was seen
with prolines replacing amino acids 49 and 50 at the C-terminal end of
the ID2 helix (Fig. 6C). Moreover,
inact
remained unchanged when the stretch between amino acids 50 and 60 (
50-60) was deleted (Fig. 6C).
View larger version (17K):
[in a new window]
Fig. 6.
Time course of inactivation is accelerated by
the ID2 helix. A, current responses as in Fig. 5 for WT and
40-50 mutant channels (scales as indicated). Inset,
first 60 ms of the current response. Note the significant slowing in
inactivation mediated by the deletion of the helical domain between
residues 40 and 50. B, inactivation time constant (mean ± S.D. of seven experiments) as a function of membrane potential. Line
is fit of equation A0 + A1 × exp(
bV) to the
data (values for A0 and b were 15.8 ms and 9.6 mV for WT and 66.0 ms and 7.7 mV for
40-50 mutant channels).
Inset, steady-state inactivation of WT and
40-50 mutant
channels. Values are mean ± S.D. of five experiments. Line is fit
of a Boltzmann function to the data with values for
V1/2 and the slope factor of
58.3 and 4.2 mV for
WT and
47.7 and 4.3 mV for
40-50. C, relative change
in the inactivation time constant observed with a proline-scan of the
ID2 helix. Inactivation time constants obtained with the mutants
indicated (6-8 experiments each) were divided by the mean value of the
inact of WT channels. Relative changes are given as
mean ± S.D. Note that deletion of amino acids 50-60 had no
effect on
inact. D, recovery from
inactivation at a membrane potential of
80 mV. Line represents fit of
a monoexponential to the recovery time course with values for the time
constants of 6.5 and 5.3 s for WT and
40-50, respectively.
Note the close match in recovery for both channels.
2-38 mutant, recovery from inactivation in the
40-50 channels was basically unchanged with respect to WT. Thus, a
monoexponential fit to the recovery time course at
80 mV yielded mean
values of 5.3 ± 0.7 s (n = 7) and 5.9 ± 0.5 s (n = 3) for
40-50 and WT channels,
respectively (Fig. 6D). Consequently, the 4-fold increase in
inact did not result from a change in dissociation of
ID1 from its receptor. Instead, the rate of association between ID and
receptor must be decreased. In the "sequential-step" model of
inactivation (13, 17, 40), such association actually implies two
separate steps: first, the approach of the ID toward the channel
entrance (preinactivation) and, second, the binding of the ID to its
receptor (inactivation). In this context, the alanine-rich helix in ID2
may be expected to work as a "docking domain" that binds the N
terminus to the surface of the channel vestibule and thus promotes
rapid insertion of ID1 into its receptor, which finally occludes the
pore and inactivates the channel (see also "Discussion").
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix, and the C-terminal half is a 2.5-turn
-helix (ID2)
followed by a disordered C-terminal tail (Fig. 4A). This
structural bipartitioning correlates with distinct functional
properties determined for the two domains in the context of the whole
Kv1.4 protein. ID1 occludes the ion pathway consistent with the
function of a classical ball domain, whereas ID2 most probably serves
as a docking domain that pins ID1 to the inner surface of the channel
and promotes its rapid access to its receptor site in the open channel.
2-38 mutant, occurred
on a time scale >100-fold slower than that observed with ID1 (Fig.
5B). Second, channel inactivation was largely reduced by
deleting either of the two distinct structural domains in ID1 with ID2
left unchanged in either molecule (Fig. 4B). Third, the
removal of the ID2 helix or perturbation of its structural integrity
(proline mutants) slowed the onset of inactivation ~4-fold
(Fig. 6, B and C) but did not change the recovery
from inactivation with respect to WT channels (Fig. 6D).
These observations are most consistent with ID1 working as the
"inactivation particle" and ID2 speeding the access of ID1 to its
receptor without acting itself as a pore-occluding domain. As an
alternative that is not completely ruled out by our experiments, ID2
might act as the actual ball domain whose high affinity is impaired
100-fold by ID1.
40-50)
channels (for details see "Material and Methods").
View larger version (25K):
[in a new window]
Fig. 7.
Functional significance of the docking domain
probed by simulations with a model neuron. A,
differential spiking behavior of a neuron equipped with either
non-inactivating Kv channels (upper panel), rapidly inactivating Kv1.4
channels (middle panel) or Kv1.4 channels lacking the docking domain
( 40-50). Simulations were performed with the Neuron simulation
environment (Neuron 4.3.1) for a spherical neuron exhibiting
voltage-dependent Na+ and K+
conductances, as well as a leakage K+ conductance. Note
cessation of spiking because of cumulative inactivation of Kv1.4
channels that is absent in non-inactivating Kv channels or the
40-50 mutant. B, number of APs triggered by stimulation
with current of varying amplitude from simulations as in A; filled
symbols refer to Kv1.4 WT (squares) and Kv1.4(
40-50) (circles),
open circles refer to non-inactivating Kv channels. Note the decrease
in AP number observed for rapidly inactivating Kv1.4 channels at higher
stimulus amplitudes. C, memory effect of rapidly
inactivating Kv channels envisaged by double-pulse excitation is
largely impaired by deletion of the docking domain. Note the difference
in response to the second excitation observed between Kv1.4 WT and
mutant channels. Single pulse excitation of a Kv1.4 containing neuron
is shown for comparison.
40-50) displayed a spiking pattern very similar to that
observed with non-inactivating Kv channels (Fig. 7A,
lower panel). As shown in Fig. 7B, cumulative
inactivation and, concomitantly, the effect of the docking domain were
dependent on the stimulus amplitude. The larger the stimulus amplitude,
the more prominent it appeared.
40-50 channels spike regularly with no obvious difference. However,
the following excitation revealed marked differences between both
channels. Kv1.4 WT cells "remembering" the preceding pulse only
fire very few APs and then remain depolarized throughout the rest of
the stimulus, whereas Kv1.4-(
40-50) cells responded with continuous
spiking as if no preceding pulse occurred. Thus, the docking domain
appears to be crucial for the molecular memory generated by rapidly
inactivating Kv1.4 channels.
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ACKNOWLEDGEMENTS |
---|
We thank Otogene AG (Tübingen) for providing access to the NMR spectrometer. We are particularly indebted to Dr. P. Jonas for help with Neuron and the simulations of the neuronal input-output relations shown in Fig. 7.
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FOOTNOTES |
---|
* This work was supported in parts by grants of the Deutsche Forschungsgemeinschaft (Fa332/3-1) and the Interdisciplinary Center of Clinical Research (IZKF) Tübingen (Project IA4) (to B. F.).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 on-line version of this article (available at
http://www.jbc.org) contains Supplemental Fig. 1.
To whom correspondence should be addressed. Tel.:
49-761-203-5175; Fax: 49-761-203-5191; E-mail:
bernd.fakler@physiologie.uni-freiburg.de.
Published, JBC Papers in Press, February 16, 2003, DOI 10.1074/jbc.M210191200
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ABBREVIATIONS |
---|
The abbreviations used are: Kv, voltage-activated K+ channel; AP, action potential; ID, inactivation domain; TEA, tetraethylammonium; NOE, Nuclear Overhauser Enhancement; GFP, green fluorescent protein; CMV, cytomegalovirus; WT, wild type; HSQC, heteronuclear single quantum coherence; NOESY, NOE spectroscopy.
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