From the Max-Planck Institut für Experimentelle
Medizin, Molekulare Biologie Neuronaler Signale, Hermann-Rein
Strasse 3, 37075 Göttingen, Germany, the
§ Neurologische Universitätsklinik,
Robert-Koch-Strasse 40, 37075 Göttingen, Germany, the
Centro Internacional de Fisica, Edificio Manuel Ancizar, Ciudad
Universitaria, AA4948 Bogotà, Colombia, and the
** Wellcome Laboratory for Molecular Pharmacology, University
College London, Gower Street, London, WC1E 6BT, United Kingdom
Received for publication, December 23, 2002, and in revised form, March 13, 2003
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ABSTRACT |
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Voltage-gated potassium (Kv) channels containing
Voltage-gated potassium
(Kv)1 channels form the most
diverse class in the ion channel superfamily, giving rise to a large
variety of currents, the kinetics of which are shaped to the
requirements of their physiological function (1). They are composed of
four Kv2 Here we describe a pharmacological strategy to distinguish between
open- and closed-state inactivation of Kv2.1 and show that both
inactivation pathways lead to the same conformation. Moreover, we
investigate the structural determinants underlying the regulation of
Kv2.1 by the modulatory Site-directed Mutagenesis and cRNA Synthesis--
All of the DNA
manipulations were carried out using standard recombinant DNA
techniques (31). The cDNAs coding for Kv2.1 (DRK1) and Kv9.3 used
in this study were identical to the ones described previously (6, 14).
Point mutations were introduced into Kv2.1 cDNA using QuikChange
(Stratagene, La Jolla, CA). The chimera Kv2.1NRD was
generated by replacing amino acids 127-181 of Kv2.1 by the NRD domain
of Kv9.3 (amino acids 111-177). Amplification by low copy PCR (15 cycles) with the polymerase Pfu (Promega, Madison, WI) was
used to generate one fragment of both Kv2.1 and Kv9.3. These fragments
were then combined via a common introduced silent EcoRI
recognition site and ligated into an appropriately cut Kv2.1. For all
mutated or chimeric constructs, the sequence of the complete channel
subunit was verified by sequencing with a BigDye terminator cycle
sequencing kit and an ABI377 DNA sequencer (Applied Biosystems).
Following linearization capped cRNAs were synthesized in
vitro with mMessage mMachine (Ambion, Austin, TX). Isolation of
oocytes (stages V and VI) from Xenopus laevis and cRNA injection were performed as described previously (32).
Electrophysiological Characterization--
Whole cell currents
were recorded 1-4 days after injection under two-electrode
voltage-clamp control, using a Turbo TEC-10CD amplifier
(NPI-Elektronik, Tamm, Germany). Intracellular electrodes had
resistances of 0.3-0.8 M Statistical Analysis--
The data are given as the means ± S.E., with n specifying the number of independent
experiments. Statistical significance was evaluated using a two-tailed
Student's t test.
Open- and Closed-state Inactivation of Kv2.1 Can Be Distinguished
by Their Sensitivity to Intracellular TEA and Extracellular
K+--
The delayed rectifier potassium channel Kv2.1 (34)
has previously been suggested to inactivate from open and closed
states. This proposal was based on kinetic analyses (27) and on the observation that co-expressions of different modulatory
Throughout this study open- and closed-state inactivation were measured
using the pulse protocols PO inact and
PC inact, respectively. For
PO inact, the membrane was depolarized for
32 s to potentials of high open probability. Time constants (
Presently our knowledge about the molecular determinants of these
different inactivation pathways and the regulation by modulatory
Additionally, we tested the influence of elevated extracellular
[K+], a condition known to slow C-type inactivation of
other Kv channels (40-42). Elevation of the external
[K+] from 2.5 to 115 mM significantly
accelerated open-state inactivation. (Fig. 1B,
Vinact = +40 mV; F,
Taken together, these experiments demonstrate that the two inactivation
pathways for Kv2.1 can be separated pharmacologically. Open-state
inactivation is inhibited by intracellular TEA and accelerated by
elevated external [K+], whereas closed-state inactivation
is insensitive to intracellular TEA and inhibited by elevated external
[K+].
Open- and Closed-state Inactivation of Kv2.1 Lead to the Same
Conformation--
The different sensitivities of open- and
closed-state inactivation of Kv2.1 to internal TEA and external
[K+] could arise by either of two mechanisms. First,
inactivation from open and closed states could lead to different
inactivated conformations of Kv2.1 from which recovery should differ,
or second, they could represent different transitions that start from
open or closed channels, respectively, but lead to the same inactivated conformation. In this case recovery should be independent from the way
inactivation occurred. To discriminate between the two possibilities,
recovery from open- and closed-state inactivation of Kv2.1 was
analyzed. As illustrated in the Fig.
2A, following a 300-ms pulse
to +40 mV (P1) channels were inactivated by clamping the voltage for
40 s at either +40 mV (open-state inactivation) or Identification and Characterization of the NRD of Kv9.3--
We
previously characterized the modulatory
In conclusion, the NRD of Kv9.3, rather than shifting the maximum of
inactivation from open to closed states, inhibits both inactivation
pathways. Hence, another region of Kv9.3 must participate in the
regulation of Kv2.1.
The Involvement of S6 in the Modulatory Effect of Kv9.3 on Channel
Gating--
To find such alternative regions we compared the amino
acid sequences of all of modulatory Preferential Closed-state Inactivation of Kv2.1-P410T Is Confirmed
by Its Pharmacological Profile--
The voltage dependence of
Kv2.1-P410T inactivation suggested that these channels inactivate
preferentially from closed states. Therefore, if the inactivation
mechanism is preserved, inactivation of Kv2.1-P410T should be slowed by
high extracellular [K+] but remain unchanged upon
application of internal TEA, which selectively slowed open-state
inactivation of Kv2.1 (Fig. 1, A, C, and
E). Indeed, injection of intracellular TEA blocking
approximately 50% of the maximal current (47.2 ± 2.9%,
n = 8) was without significant effect on inactivation
of Kv2.1-P410T measured at voltages between The Effect of Kv2.1-P410T Prevails upon Co-expression with
Kv2.1--
The functional properties of Kv2.1-P410T channels resembled
those of Kv2.1/Kv9.3 heteromers in all aspects that we have analyzed. To further assess the importance of this residue in the regulatory function of Kv9.3, Kv2.1-P410T was co-expressed with Kv2.1. We assumed
a random assembly of both subunits, because the region guiding this
process, the T1 domain, is identical for both subunits. Therefore, to
favor a 2:2 stoichiometry that has been suggested to predominate in the
assembly of Kv2.1 with modulatory
Thus, Kv2.1/Kv2.1-P410T heteromers inactivate faster from closed than
open states, thereby recapitulating the shift in the state preference
of inactivation that is central to the modulation of Kv2.1 by Kv9.3.
The point mutation P410T therefore not only induces all of the gating
characteristics conferred by Kv9.3 when the corresponding subunit
(Kv2.1-P410T) is expressed alone, but it also dominates the functional
properties of heteromeric channels resulting from its co-expression
with Kv2.1.
In addition to this gain of function mutation, transferring the
regulatory properties of Kv9.3 to Kv2.1, we attempted a loss of
function mutation of Kv9.3 Ion channels containing Kv2.1 Different proteins have evolved that contribute to the functional
diversity of Kv channels. For example, The effect of the modulatory Conserved among all of the The mechanism of Kv2.1 inactivation, which has been called U-type
inactivation for its nonmonotonic voltage dependence (27), is unknown.
It has been suggested to involve a narrowing of the selectivity filter,
allowing Na+ to permeate inactivated channels (63), similar
to what has been observed for C-type inactivation of Shaker channels
(64). However, these data remain controversial because the apparent Na+ conductance could be accounted for by changed
intracellular K+ concentrations and thus changed
K+ reversal potential under the particular experimental
conditions (65). Here we show that mutation of the second proline of
the S6 PXP motif, thought to comprise the activation gate,
accelerates closed-state inactivation and inhibits open-state
inactivation, which in turn are suggested to lead to the same
inactivated conformation. Interestingly, mutations in the two residues
surrounding the second proline of the S6 PXP motif of Kv4.1
have been shown to disrupt closed-state inactivation of the respective
channels, which was suggested to occur at the internal vestibule of the
pore (66). However, internal TEA and extracellular
K+, which are thought to bind on either side of the
selectivity filter (33, 39), as well as disruption of the intracellular T1 domain and T1-S1 linker affect Kv2.1 inactivation. Thus, further experiments delineating which of the above manipulations affects the
inactivation gate directly and which can be accounted for by allosteric
effects on a distant gate are needed.
-subunits of the Kv2 subfamily mediate delayed rectifier currents in
excitable cells. Channels formed by Kv2.1
-subunits inactivate from
open- and closed states with both forms of inactivation serving
different physiological functions. Here we show that open- and
closed-state inactivation of Kv2.1 can be distinguished by the
sensitivity to intracellular tetraethylammonium and
extracellular potassium and lead to the same inactivated conformation.
The functional properties of Kv2.1 are regulated by its association
with modulatory
-subunits (Kv5, Kv6, Kv8, and Kv9). For instance,
Kv9.3 changes the state preference of Kv2.1 inactivation by
accelerating closed-state inactivation and inhibiting open-state
inactivation. An N-terminal regulatory domain (NRD) has been suggested
to determine the function of the modulatory
-subunit Kv8.1. However,
when we tested the NRD of Kv9.3, we found that the functional
properties of chimeric Kv2.1 channels containing the NRD of Kv9.3
(Kv2.1NRD) did not resemble those of Kv2.1/Kv9.3
heteromers, thus questioning the role of the NRD in Kv9 subunits. A
further region of interest is a PXP motif in the sixth
transmembrane segment. This motif is conserved among all
-subunits
of the Kv1, Kv2, Kv3, and Kv4 subfamilies, whereas the second proline
is not conserved in any modulatory
-subunit. Exchanging this proline
in Kv2.1 for the corresponding residue of Kv9.3 resulted in channels
(Kv2.1-P410T) that show all hallmarks of the regulation of Kv2.1 by
Kv9.3. The effect prevailed in heteromeric channels following
co-expression of Kv2.1-P410T with Kv2.1. These data suggest that the
alteration of the PXP motif is an important determinant of
the regulatory function of modulatory
-subunits.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits, each containing six transmembrane segments (S1-S6), arranged around a central potassium-selective pore (2). The ability of
different
-subunits to form heteromeric channels increases the
diversity of K+ currents in native cells (3-5). Modulatory
-subunits constitute a group of proteins that are unable to build
functional channels by themselves. They associate with Kv2
-subunits
forming heteromeric channels that activate, deactivate, inactivate, and
recover from inactivation differently from homomeric Kv2 channels
(6-12). The group of mammalian modulatory
-subunits so far consists
of Kv5.1, Kv6.1-6.4, Kv8.1-2, and Kv9.1-9.3 (7, 10-15). Their
selective association with Kv2
-subunits is guided by an
intracellular N-terminal domain (15, 16) initially identified in
-subunits of the subfamilies Kv1-Kv4 and named T1 for its role in
tetramerization and subunit segregation (17-19). We previously
described the functional properties of heteromeric channels arising
from the co-expression of Kv2.1 with Kv9.3 (Kv2.1/Kv9.3) (6). Channel
activation and deactivation were slowed, and their equilibrium shifted
to hyperpolarized potentials when compared with homomeric Kv2.1
channels. Moreover, Kv9.3 changed the state dependence of Kv2.1 inactivation.
-subunits have been identified as an important component of
delayed rectifier currents in a variety of excitable cells where they
participate in action potential repolarization, regulation of the
firing frequency, and setting the resting membrane potential (20-25).
Inactivation of Kv2 channels reduces currents through these channels
and thus regulates membrane excitability. Based on a model adapting the
Monod-Wyman-Changeux model for allosteric proteins (26) to ion
channels, Kv2.1 has been suggested to inactivate from open and closed
states (27). The maximum of inactivation in this description was
assigned to the last of five closed states passed by opening channels,
which is linked with the open state through a transition with a
voltage-independent on rate and a voltage-dependent off
rate (27). Here we refer to inactivation from this last closed and from
open states as open-state inactivation. The term closed-state
inactivation indicates inactivation from proximal states in the
activation pathway that are separated from open states by
voltage-dependent transitions. For Kv2.1 open-state inactivation is fast, and closed-state inactivation slow. On the contrary, heteromeric Kv2.1/Kv9.3 channels exhibit slow open- and fast
closed-state inactivation. This shift to preferential closed-state
inactivation has been referred to as a change in the state dependence
of inactivation (6). Preferential closed-state inactivation has been
suggested to participate in the control of membrane excitability by
modulating repetitive firing and back-propagation of action potentials
in neurons as well as the repolarization of cardiac action potentials
(28, 29).
-subunit Kv9.3. An N-terminal regulatory domain (NRD) originally characterized in Kv8.1 (30) and suggested to
govern its function was identified also in Kv9.3. However, the
functional properties of chimeric Kv2.1 channels containing the NRD of
Kv9.3 differed from those of Kv2.1/Kv9.3 heteromers, thereby
questioning the importance of NRD in Kv9 subunits. On the contrary, we
show that a single amino acid in the distal part of the pore-lining S6
determines the regulatory properties of Kv9.3.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
when filled with 2 M KCl. The
standard bath solution was normal frog Ringer containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM
CaCl2, 10 mM HEPES-NaOH (pH 7.2). In
experiments in which KCl concentrations were raised, NaCl
concentrations were lowered so that the sum of KCl an NaCl remained
constant. Recordings testing the effect of internal TEA could not be
performed in inside out patches because of fast rundown of Kv2.1
currents in this configuration
(33).2 We therefore injected
TEA into oocytes using a glass pipette filled with 105 mM
KCl, 10 mM TEACl, 2.5 mM NaCl, 1.8 mM CaCl2, 10 mM HEPES-NaOH (pH
7.2). The currents were low pass filtered at 0.7-1 kHz (
3dB) and
sampled at 3-5 kHz. All of the experiments were carried out at room
temperature (20-22 °C). Data acquisition and analysis were
performed with the Pulse+PulseFit software package (HEKA Elektronik,
Lambrecht, Germany), EXCEL (Microsoft), and IGOR (Wavemetrics).
Boltzman functions of the type
PO/PO,max = Offset + 1/(1 + exp(V1/2
Vm)/a) were used to fit steady-state activation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits with Kv2.1 have opposite effects on inactivation occurring at high and
low open probabilities (6, 10).
inact) derived from mono-exponential functions fit to
the decay of the resulting outward currents were used to assess
open-state inactivation (
and
in Figs.
1, E and F,
4D, and 5, E and F). For
PC inact, 300-ms test pulses to +40 mV were
given at the beginning (P1) and end (Pn) of conditioning
pulses of increasing length to potentials of low open probability. The
ratio of currents elicited by the test pulses
(IPn/IP1) is proportional
to the number of channels that did not inactivate during the
conditioning pulse and declines with increasing length of the
conditioning pulse. Time constants (
inact) derived from
mono-exponential functions fit to this decline were used to quantify
closed-state inactivation (
and
in Figs. 1, E and
F, 4D, and 5, E and F). For
potentials of intermediate open probability both pulse protocols were
used to measure inactivation occurring from both open and closed states (
and
in Figs. 1, E and F, 4D,
and 5, E and F).
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Fig. 1.
TEAi and
[K+]o can be used to distinguish open- and
closed-state inactivation of Kv2.1. A, Kv2.1 currents
were elicited by a 32-s depolarizing pulse to +40 mV
(PO inact) in the presence
(+TEAi) or absence ( TEAi) of
intracellular TEA. For easier comparison, the traces are
scaled to their maximum. Inset, to determine the voltage
dependence of activation, Kv2.1 channels were challenged by a 300-ms
pulses to potentials ranging from
80 to +70 mV, in 10-mV increments.
Subsequently, the voltage was clamped to
40 mV, and the initial
current was estimated from a mono-exponential fit to its decay. The
relative open probabilities
(PO/PO, max) derived
from the initial currents were plotted against the voltages of the
depolarization and fit with a Boltzmann function. The data are given as
the means ± S.E. (n = 9). B, Kv2.1
currents were evoked as in A with 2.5 or 115 mM
K+ in the extracellular solution. C, Kv2.1
closed-state inactivation evoked by PC inact at
an inactivating potential of
30 mV (Vinact =
30 mV). Recordings were performed either in the presence
(+TEAi) or absence (
TEAi) of
intracellular TEA. D, measurements as in C, with
2.5 or 115 mM K+ in the extracellular solution.
E, time constants derived from mono-exponential functions
fit to the inactivation processes measured by
PO inact (
and
),
PC inact (
and
), or both (
and
)
in the presence (filled symbols) or absence (open
symbols) of intracellular TEA were plotted against the voltage of
the inactivating pulse. F, plot of time constants of
inactivation measured by PO inact (
and
), PC inact (
and
), or both (
and
) with 2.5 mM (open symbols) or 115 mM (filled symbols) K+ in the
extracellular solution. The data in E and F
represent the means ± S.E. (n = 4-10).
-subunits is scarce. To have an additional tool for their analysis, we first investigated whether open- and closed-state inactivation can
be separated pharmacologically. Kv2.1 channels were expressed in
Xenopus oocytes, and currents were measured under
two-electrode voltage clamp conditions. The quaternary ammonium ion
TEA, which blocks Kv2.1 channels (34, 35), has been shown to slow C- (36, 37) and N-type inactivation (38, 39) of other Kv channels when
applied to the extra- or intracellular side of the channels,
respectively. Therefore, we tested whether TEA was affecting Kv2.1
inactivation when applied from either side of the membrane. After the
current response was stable for at least 5 min, TEA (10 mM
solution in high K+ Ringer) was injected through a third
pipette impaled into the oocyte until approximately 50% of the initial
current was blocked (55.3 ± 1.2%, n = 16). This
intracellular application of TEA markedly slowed the rate of open-state
inactivation (Fig. 1A, Vinact = +40
mV; E,
and
; p < 0.02). On the other
hand, closed-state inactivation was not altered significantly by
internal TEA (Fig. 1C; Vinact =
30
mV; E,
and
; p > 0.6). Accordingly,
Fig. 1E shows that the effect of intracellular TEA increases
with more pronounced depolarizations, corresponding to increasing open
probabilities of the channels. In agreement with previous publications,
extracellular TEA did not affect open- or closed-state inactivation of
Kv2.1 (data not shown and Ref. 27).
and
;
p < 0.01). On the contrary, closed-state inactivation
was decelerated when external [K+] was increased (Fig.
1D, Vinact =
30 mV; F,
and
;
p < 0.02).
40 mV
(closed-state inactivation) and a second 300-ms pulse to +40 mV (P2)
was given to measure the degree of inactivation. Channels were then
recovered from inactivation at hyperpolarized potentials (
80 to
120
mV) for increasing time intervals before applying another 300-ms pulse
to +40 mV (Pn). The number of channels that recover during
this period is proportional to the ratio of currents elicited by the
short pulses (IPn
IP2/IP1
IP2) and increases with increasing time spent at
the recovery potential. Time constants derived from mono-exponential
functions fit to this process were used to quantify recovery. They were indistinguishable between recovery from open- (Rec
Io) and closed-state inactivation (Rec
Ic) at all potentials tested (Fig.
2B, p > 0.3). Moreover, elevating the
extracellular [K+] accelerated recovery to a rate that
was identical for open- and closed-state inactivation (Fig.
2C, p > 0.2). These observations support
the idea that open- and closed-state inactivation of Kv2.1 are separate
transitions leading to the same inactivated conformation.
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Fig. 2.
Recovery of Kv2.1 channels is identical for
open- and closed-state inactivation. A, representative
traces of recovery from open- (Rec Io)
and closed-state inactivation (Rec
Ic)
at a recovery potential of
100 mV (Vrecovery =
100 mV). As the pulse protocol illustrates, the channels were
activated from a holding potential of
100 mV by a 300-ms pulse to +40
mV (P1) before inactivation was induced by clamping the voltage for
40 s (*) at either +40 mV (Rec
Io)
or at
40 mV (Rec
Ic). This period is
omitted in the presentation of the current traces (*). A second 300-ms
pulse to +40 mV (P2) was applied to determine the extent of
inactivation. Afterward, the channels were allowed to recover from
inactivation for increasing time intervals at recovery potentials
between
80 and
120 mV before the fraction of the recovered current
(IPn
IP2/IP1
IP2) was determined by another 300-ms pulse to
+40 mV (Pn). B, time constants of recovery from
open- (
, Rec
Io) and closed-state
(
, Rec
Ic) inactivation plotted
against the recovery potentials are indistinguishable
(n = 5-6; p > 0.3). C,
time constants of recovery from open- (black bars,
Io) and closed-state (white bars,
Ic) inactivation at a recovery potential of
90
mV with 2.5 or 115 mM K+ in the extracellular
solution as indicated. Kv2.1 channels recover substantially faster in
high extracellular [K+], yet there was no difference in
this effect between recovery from open- or closed-state inactivation
(external [K+] 115 mM, Rec
Io
rec = 0.49 ± 0.04 s; Rec
Ic
rec = 0.55 ± 0.02 s; n = 4-5; p > 0.2).
-subunit Kv9.3, which forms
heteromeric channels with Kv2.1 and changes the preferred state from
which inactivation occurs (6). Although Kv2.1 inactivates fast from
open and slowly from closed states, heteromeric Kv2.1/Kv9.3 channels
show little open- but fast and complete closed-state inactivation
(Fig. 3, A and B)
(6). The molecular determinants of this regulation are unknown. The
inhibition of open-state inactivation of Kv2.1 by the modulatory
-subunit Kv8.1 has been attributed to a segment of 59 amino acids
preceding the first transmembrane segment (30). A comparison of this
region, termed NRD (30), with the respective region in Kv9.3 showed
little sequence identity. To test whether the proposed function of this
domain was conserved, chimeric Kv2.1 channels containing the NRD of
Kv9.3 (Kv2.1NRD) were constructed (Fig. 3C).
Steady-state activation of Kv2.1NRD was shifted in the
depolarized direction with respect to Kv2.1 (Kv2.1,
V1/2 = 3 ± 1 mV (n = 9); Kv2.1NRD, V1/2 = 12 ± 2 mV,
(n = 12) (Fig. 3D) in contrast to what has
been observed for Kv2.1/Kv9.3 heteromers, which opened at more
hyperpolarized potentials than Kv2.1 (6). For Kv2.1NRD, as
for Kv2.1/Kv9.3 heteromers, the kinetics of activation and deactivation
were slowed (data not shown), and open-state inactivation was reduced.
Accordingly, at the end of 32-s depolarizing pulses to +40 mV only
34 ± 0.4% (n = 6) of the Kv2.1NRD
current was inactivated, compared with 84.9 ± 1.8%
(n = 24) of the Kv2.1 current (Fig. 3, E and
G). However, Kv2.1NRD channels did not show the
prominent closed-state inactivation that is the hallmark of Kv2.1/Kv9.3
heteromers (Fig. 3B) (6). On the contrary, they display less
closed-state inactivation than Kv2.1. Thus, following 32 s at
30
mV, 84 ± 1.1% (n = 6) of the initial
Kv2.1NRD current was left compared with 59.7 ± 5.5%,
(n = 16) for Kv2.1 (Fig. 3, F and
G).
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Fig. 3.
The NRD of Kv9.3. A, Kv2.1
and Kv2.1/Kv9.3 currents elicited by 10-s pulses to +40 mV. Kv2.1 shows
pronounced inactivation from the open state, whereas Kv2.1/Kv9.3 does
not. B, closed-state inactivation elicited by
PC inact at an inactivating potential of 30
mV (Vinact =
30 mV). The noninactivated
fraction of the current
(IPn/IP1) through Kv2.1
(
) and Kv2.1/Kv9.3 (
) is plotted as a function of the time spent
at the inactivating potential. Closed-state inactivation is slow and
incomplete for Kv2.1, whereas it is fast and complete for Kv2.1/Kv9.3.
C, schematic drawing of the chimera Kv2.1NRD
indicating the region of Kv2.1 that has been replaced by the
corresponding amino acids (111-177) of Kv9.3. D, to analyze
the voltage dependence of activation, Kv2.1 (
) and
Kv2.1NRD (
) were activated by a 300-ms pulse to
potentials ranging from
80 to +70 mV, applied in increments of 10 mV.
Subsequently, the voltage was clamped at
40 mV, and the initial
current in this segment was estimated from a mono-exponential fit to
its decay. The relative open probabilities
(PO/PO max) derived from
the initial currents were plotted against the voltage of the
depolarization and fit with a Boltzmann function. The data are given as
the means ± S.E. (n = 9-12). E,
representative scaled currents of Kv2.1 and Kv2.1NRD
elicited by a 32-s depolarizing pulse to +40 mV showing decreased
open-state inactivation for K2.1NRD compared with Kv2.1.
F, closed-state inactivation of Kv2.1 and
Kv2.1NRD was measured by PC inact
at an inactivation potential of
30 mV (Vinact =
30 mV). G, bar diagram showing the fraction of currents
(I32s/I0s) through Kv2.1
(white bars) and Kv2.1NRD channels (black
bars) that did not inactivate from closed (
30 mV) or open states
(+40 mV) during a 32-s pulse to the respective potential. The
error bars represent the means ± S.E.
(n = 6-24). The current traces in A and
E are scaled to their maximum.
-subunits (Kv5.1, Kv6.1-6.4,
Kv8.1-8.2, and Kv9.1-9.3) with the sequences of Kv1, Kv2, Kv3, and
Kv4
-subunits and thus identified the sixth transmembrane segment
(S6) as a region of interest. Fig.
4A shows an alignment of the
S6 in which one representative member of each subfamily was included.
The second proline of a PXP motif (Fig. 4A,
arrow) is conserved among all 17
-subunits of the
Kv1-Kv4 subfamilies and is replaced by serine, threonine, alanine, or
histidine in all 10 modulatory
-subunits. To test the hypothesis
that the alteration of this PXP motif participates in the
regulatory function of Kv9.3, we mutated the proline at position 410 in
Kv2.1 to the corresponding threonine of Kv9.3. The resulting
-subunit (Kv2.1-P410T) formed functional channels in
Xenopus oocytes. Steady-state activation of Kv2.1-P410T was
shifted 12 mV toward hyperpolarized potentials compared with Kv2.1
(Kv2.1, V1/2 = 3 ± 1 mV, n = 9;
Kv2.1-P410T, V1/2 =
9 ± 2 mV, n = 10;
Fig. 4B, inset), and activation and deactivation kinetics decelerated, similar to what has been observed for heteromeric Kv2.1/Kv9.3 channels (6). Moreover, like Kv2.1/Kv9.3 heteromers, Kv2.1-P410T channels showed little open-state inactivation (Fig. 4B, Vinact = +40 mV, and
D) while inactivating fast from closed states (Fig.
4C, Vinact =
40 mV, and
D). Plotting the time constants of inactivation as a
function of the voltage for both Kv2.1 and Kv2.1-P410T shows their
inverse state preference for inactivation (Fig. 4D). The
curves cross in the intermediate voltage range where channels start to
open. At voltages negative to the activation threshold (closed-state
inactivation), Kv2.1 inactivation is slow and incomplete, whereas
Kv2.1-P410T channels inactivate in a fast and complete manner. At
potentials positive to the activation threshold (open-state
inactivation) Kv2.1 inactivation is fast, whereas only a few
Kv2.1-P410T channels inactivate. Recovery from inactivation, as for
Kv2.1/Kv9.3 heteromeric channels, was accelerated for Kv2.1-P410T and
showed less pronounced voltage and K+ dependence compared
with Kv2.1 (data not shown). Thus, the single amino acid exchange P410T
in Kv2.1-P410T conveys all of the functional properties that Kv9.3
brings to Kv2.1/Kv9.3 heteromers.
View larger version (22K):
[in a new window]
Fig. 4.
The involvement of S6 in the regulatory
function of Kv9.3. A, alignment of the last
transmembrane segment (S6) for one representative member from each Kv
subfamily. Kv subunits were assigned into subfamilies following the
guidelines of the HUGO Gene Nomenclature Committee
(www.gene.ucl.ac.uk/nomenclature/genefamily/KCN.shtml) B,
currents through Kv2.1 and Kv2.1-P410T elicited by a 32-s depolarizing
pulse to +40 mV (PO inact). To determine
voltage dependence of activation (inset) for Kv2.1 ( ) and
Kv2.1-P410T (
), 300-ms pulses from
80 to +70 mV were applied in
10-mV increments. Subsequently, the voltage was clamped to
40 mV, and
the initial current in this segment was estimated from a
mono-exponential fit to its decay. The relative open probabilities
(PO/PO, max) derived
from the initial currents were plotted against the voltage of the
conditioning pulses and fit with a Boltzmann function
(n = 9-10). C, closed-state inactivation of
Kv2.1 and Kv2.1-P410T measured by the pulse protocol illustrated
(PC inact, Vinact =
40
mV). The currents in B and C were scaled to their
maximum. D, time constants of inactivation of Kv2.1
(open symbols) and Kv2.1-P410T (filled symbols)
measured by PO inact (
and
),
PC inact (
and
), or both (
and
),
plotted as a function of the voltage of the inactivating pulse. The
data in D are represented as the means ± S.E.
(n = 4-10).
60 mV and +60 mV (Fig.
5, A, C, and
E; p > 0.1). When the extracellular K+ concentration was increased to 115 mM,
closed-state inactivation was inhibited (Fig. 5, D and
F; p < 0.01) as for Kv2.1, and open-state inactivation was not significantly affected (Fig. 5, D and
F; p > 0.15). These data corroborate the
idea that Kv2.1-P410T channels inactivate by the same mechanism as
Kv2.1 but preferentially from closed states.
View larger version (25K):
[in a new window]
Fig. 5.
The pharmacological profile of Kv2.1-P410T
corroborates preferential closed-state inactivation. A,
currents through Kv2.1-P410T elicited by 32-s depolarizing pulse to +40
mV (PO inact) in the presence
(+TEAi) or absence ( TEAi) of
intracellular TEA. The currents were scaled to their maximum.
B, currents evoked as in A with 2.5 or 115 mM K+ in the extracellular solution.
C, closed-state inactivation of Kv2.1-P410T measured by
PC inact (Vinact =
40
mV) in the presence (+TEAi) or absence
(
TEAi) of intracellular TEA. D,
recording as in C, with 2.5 or 115 mM
K+ in the extracellular solution. E and
F, time constants of inactivation evoked by
PO inact (
and
),
PC inact (
and
), or both (
and
),
plotted as a function of the voltage of the inactivating pulse.
E, measurements in the presence (filled symbols)
or absence (open symbols) of internal TEA. F,
measurements with 2.5 mM (open symbols) or 115 mM (filled symbols) K+ in the
extracellular solution. The data in E and F are
presented as the means ± S.E. (n = 4-10).
-subunits (30), Xenopus
oocytes were co-injected with cRNA dilutions that gave a current ratio
of 1:1 (Fig. 6A, at +40 mV
Kv2.1 = 20.1 ± 2.6µA; Kv2.1-P410T = 19.4 ± 3µA; Kv2.1/Kv2.1-P410T = 18.1 ± 0.5µA, n = 7-10). Steady-state activation of channels resulting from the
co-expression of Kv2.1 with Kv2.1-P410T was intermediate when compared
with the respective homomeric channels (Fig. 6B;
Kv2.1/Kv2.1-P410T V1/2 =
4 ± 1.5 mV
n = 7). Open-state inactivation of Kv2.1, evaluated as
the fraction of the initial current that is left at the end of 32 s depolarizations to +40 mV
(I32s/I0s), was inhibited
by co-expression of Kv2.1-P410T (Fig. 6, C and F,
Kv2.1 current left = 15.1 ± 1.8%, n = 24;
Kv2.1/Kv2.1-P410T current left = 42 ± 1.6%,
n = 7). On the contrary, closed-state inactivation of
Kv2.1 was accelerated by co-expression of Kv2.1-P410T (Fig. 6,
E and F, after a 32-s pulse to
30 mV Kv2.1,
current left = 59.7 ± 5.5%, n = 16;
Kv2.1/Kv2.1-P410T current left = 30 ± 1.8%,
n = 6).
View larger version (18K):
[in a new window]
Fig. 6.
Co-expression of Kv2.1 with Kv2.1-P410T.
A, mean amplitude of outward currents evoked by 300-ms
pulses to +40 mV for Xenopus oocytes injected with cRNA
encoding Kv2.1 (white bar), Kv2.1-P410T (black
bar), or a 1:1 mix of the respective dilutions, Kv2.1/Kv2.1-P410T
(gray bar). The data are given as the means ± S.E.
(n = 10). B, to analyze steady-state
activation of Kv2.1 (open circle), Kv2.1-P410T (black
circle), and Kv2.1/Kv2.1-P410T (shaded circle) 300-ms
conditioning pulses from 80 to +70 mV were applied in 10-mV
increments. Subsequently, the voltage was clamped to
40 mV, and the
initial current in this segment was estimated from a mono-exponential
fit to its decay. The data are given as the means ± S.E.
(n = 7-10). C, representative scaled traces
recorded during 32-s depolarizations to +40 mV
(PO inact) for Kv2.1 and Kv2.1/Kv2.1-P410T.
D, representative traces scaled of closed-state inactivation
of Kv2.1 and Kv2.1/Kv2.1-P410T measured by the pulse protocol
illustrated (PC inact,
Vinact =
30 mV). E, inactivation
depicted as the fraction of the initial current left at the end of a
32-s pulse to the indicated potentials
(I32s/I0s). The
bars represent the means ± S.E. (n = 6-24).
-subunits by exchanging the threonine at
position 403 for a proline, thus introducing a PXP motif.
When cRNA coding for this subunit (Kv9.3-T403P) was co-injected with Kv2.1 cRNA in the ratios of Kv2.1 to Kv9.3-T403P of 1:1, 1:5, or 1:10,
no effect on closed-state inactivation was observed (e.g. 1:5 after a 32-s pulse to
30mV Kv2.1/Kv9.3-T403P current left = 69.9 ± 0.5%, p > 0.06, n = 7),
whereas open-state inactivation was slightly slower (e.g.
1:5 after a 32-s pulse to +40mV Kv2.1/Kv9.3-T403P current left = 30.7 ± 0.2%, p < 0.0001, n = 9). cRNA of wild type Kv9.3 displayed complete regulatory effect at a
ratio of 1:3 and 1:5 (6, 15). However, when a ratio of Kv2.1 to
Kv9.3-T403P of 1:50 was used, the resulting currents showed both an
increased closed-state inactivation (after a 32-s pulse to
30mV
Kv2.1/Kv9.3-T403P current left = 23.5 ± 0.1%,
p < 0.0001, n = 7) and a reduced
open-state inactivation (after a 32-s pulse to +40mV Kv2.1/Kv9.3-T403P
current left = 54.5 ± 0.1% p < 0,0001, n = 7). These experiments show that although
Kv9.3-T403P subunits retain a regulatory effect, their potency is
reduced when compared with Kv9.3, accounting for a partial loss of
function by this mutation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits mediate delayed
rectifier K+ currents in a variety of cells, contributing
to such diverse physiological processes as the repolarization of
neuronal (20-22, 43) and cardiac (23, 25) action potentials, hypoxic
vasoconstriction of pulmonary arteries (7, 24), and insulin secretion
of pancreatic
cells (44, 45). Open and closed Kv2.1 channels can
inactivate (27), reducing the pool of available channels in a
voltage-dependent manner. We show that both inactivation
pathways can be selectively antagonized by intracellular TEA and
extracellular K+, respectively, and lead to the same
conformation. The pharmacological protocol introduced in this study
should facilitate further analysis of the role of open- and
closed-state inactivation in native currents.
-subunits of the Kv1 and Kv4
subfamily associate with Kv
-subunits (46) and Ca2+-binding proteins (47), respectively. The interaction
with these intracellular proteins changes the surface expression
(47-50) as well as the gating characteristics of the resulting channel
complexes (46, 47, 51, 52). Kv2
-subunits form heterotetrameric channels with modulatory
-subunits. These transmembrane proteins are
the biggest group of Kv channel modifying proteins, with 10 members so
far (7, 10-15), and their malfunction has been implied in the
pathogenesis of such diverse conditions as epilepsy (53) and pulmonary
hypertension (7). We previously characterized the modulatory
-subunit Kv9.3, which changes the state dependence of Kv2.1
inactivation, inhibiting open-state inactivation and accelerating
closed-state inactivation. Such preferential closed-state inactivation
has been suggested to participate in the control membrane excitability
by modulating repetitive firing and back-propagation of action
potentials in neurons as well as the repolarization of cardiac action
potentials (28, 29). In this study we wanted to identify the structural
determinants of this regulation.
-subunit Kv8.1 was assigned to the 59 amino acids preceding S1 (30). This region called NRD contains the
T1-S1 linker and layer 4, the membrane-facing surface, of the T1
domain, with part of its Zn2+ coordination site (54). The
NRD of Kv8.1 in chimeric Kv2.1 channels caused slowing of activation
and deactivation kinetics. Inactivation at potentials of maximal open
probability was inhibited, whereas closed-state inactivation was not
analyzed (30). Transferring the equivalent region of the Kv9.3 to Kv2.1
(Kv2.1NRD) had the same effect (Fig. 3). Activation,
deactivation and open-state inactivation were slowed. However, unlike
Kv2.1/Kv9.3 heteromers, Kv2.1NRD channels show no
significant closed-state inactivation. To our understanding, the effect
of NRD on channel gating results from a disruption of the normal T1
function of Kv2.1 that has been observed following a number of
different manipulations. For instance, deletion of a part of the N
terminus (55), application of methylmethanesulfonate attaching a
thiomethyl group to native N-terminal cysteines involved in
Zn2+ coordination (56), as well as replacing part of the
Kv2.1 N terminus by corresponding regions of Kv1.5 (57), Kv8.1 (30), and Kv9.3 (Fig. 3) all result in channels with slow activation, deactivation, and open-state inactivation. Where investigated (Kv9.3
and Kv1.5), closed-state inactivation of the corresponding channels was
inhibited (Fig. 3, C and D) (57). This implies that NRD participates in the regulation of Kv2.1 channel gating by
modulatory
-subunits but is alone insufficient to explain the effect
of Kv9 subunits.
-subunits from the subfamilies Kv1-Kv4
is a PXP motif in the distal part of S6. This region
projects to the bundle crossing in the KcsA structure (58) and was
proposed to cause a bend in the S6 of Kv channels based on sequence
analysis, as well as blocker protection and Cd2+ bridging
of introduced cysteines (59, 60). The intracellular activation gate of
Kv channels is thought to coincide with this bend (61). Proline
residues, which are potent helix breakers in aqueous solutions, are
commonly found in transmembrane
-helices, where multiple but not
single prolines cause bends (62). In modulatory
-subunits the second
proline of the PXP motif is replaced by threonine, serine,
alanine, or histidine, predicting the S6 to be straight. To investigate
whether this exchange participates in the regulation of Kv2.1 by Kv9.3,
the second proline of the PXP motif in Kv2.1 was mutated to
the corresponding threonine of Kv9.3. The resulting channels,
Kv2.1-P410T, show all hallmarks of Kv2.1/Kv9.3 heteromers (6).
Steady-state activation is shifted to hyperpolarized potentials, and
activation and deactivation kinetics are slowed. In particular, the
state dependence of inactivation is reversed, causing Kv2.1-P410T to
inactivate in a fast and complete manner from closed states, whereas
open-state inactivation is inhibited. The inactivation mechanism
appears to be conserved, because closed-state inactivation remains
sensitive to extracellular K+ and insensitive to
intracellular TEA. In addition, like for Kv2.1/Kv9.3 heteromers,
recovery from inactivation is accelerated and displays reduced voltage
and K+ dependence. These effects on channel gating prevail
upon co-expression of Kv2.1-P410T with Kv2.1 in conditions favoring a
2:2 stoichiometry, which is supposed to be the preferred assembly of
modulatory and Kv2
-subunits (30). Introducing a PXP
motif in Kv9.3 subunits by exchanging the threonine at position 403 for
a proline results in subunits (Kv9.3-T403P) that retain a regulatory
effect but require roughly 10-fold higher amounts of cRNA to exert it,
thus showing a reduced potency and a partial loss of function. Taken together, these data suggest that replacement of the second proline in
modulatory
-subunits is an important structural determinant of their
regulatory function.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Walter Stühmer for generous support. We thank the technical personnel in the Research Group of Molecular & Cellular Neuropharmacology and S. Schäpermeier for help with the oocyte preparation and R. Schliephacke for computer support. We thank Drs. Florentina Soto and Paola Pedarzani for critically reading of the manuscript. We thank Dr. E. Posada, Dr. M. Camacho, Dr. E. Rey, and I. Rugeles for support.
![]() |
FOOTNOTES |
---|
* This work was supported by the Max-Planck Society and the Wellcome Trust.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: Max-Planck Institut für Exp. Medizin, Abt. Mol. Biol. Neuronaler Signale, Hermann-Rein Str. 3, 37075 Göttingen, Germany. Tel.: 49-551-3899624; Fax: 49-551-3899644; E-mail: dkersch@gwdg.de.
Wellcome Trust Senior Research Fellow.
Published, JBC Papers in Press, March 17, 2003, DOI 10.1074/jbc.M213117200
2 D. Kerschensteiner, F. Monje, and M. Stocker, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Kv, voltage-gated potassium; NRD, N-terminal regulatory domain; TEA, tetraethylammonium.
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