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Next Article 
Volume 17, Number 10,
Issue of May 15, 1997
pp. 3379-3391
Copyright ©1997 Society for Neuroscience
Frequency-Dependent Inactivation of Mammalian A-Type
K+ Channel KV1.4 Regulated by
Ca2+/Calmodulin-Dependent Protein Kinase
Jochen Roeper,
Christoph Lorra, and
Olaf Pongs
Zentrum für Molekulare Neurobiologie, Martinistrasse 52, D-20246 Hamburg, Germany
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Ca2+/calmodulin dependent protein kinase (CaMKII) and
protein phosphatase 2B (calcineurin) are key enzymes in the regulation of synaptic strength, controlling the phosphorylation status of pre-
and postsynaptic target proteins. Here, we show that the inactivation
gating of the Shaker-related fast-inactivating
KV channel, Kv1.4 is controlled by
CaMKII and the calcineurin/inhibitor-1 protein phosphatase cascade.
CaMKII phosphorylation of an amino-terminal residue of
KV1.4 leads to slowing of inactivation gating and
accelerated recovery from N-type inactivated states. In contrast,
dephosphorylation of this residue induces a fast inactivating mode of
KV1.4 with time constants of inactivation 5 to 10 times
faster compared with the CaMKII-phosphorylated form. Dephosphorylated
KV1.4 channels also display slowed and partial recovery
from inactivation with increased trapping of KV1.4 channels
in long-absorbing C-type inactivated states. In consequence,
dephosphorylated KV1.4 displays a markedly increased
tendency to undergo cumulative inactivation during repetitive
stimulation. The balance between phosphorylated and dephosphorylated
KV1.4 channels is regulated by changes in intracellular
Ca2+ concentration rendering KV1.4 inactivation
gating Ca2+-sensitive. The reciprocal CaMKII and
calcineurin regulation of cumulative inactivation of presynaptic
KV1.4 may provide a novel mechanism to regulate the
critical frequency for presynaptic spike broadening and induction of
synaptic plasticity.
Key words:
voltage-activated K channels;
Shaker;
N-type
inactivation;
C-type inactivation;
phosphorylation;
CaMKII;
calcineurin;
protein phosphatase;
synapse
INTRODUCTION
The strength of synaptic connections within neuronal
circuits is flexible. This plasticity in neuronal excitability has been recognized as an important property underlying short- and long-term changes in the concerted activity of pre- and postsynaptic elements including ion channels (Kandel et al., 1991 ; Bliss and Collingridge, 1993; Zucker, 1993). It has been shown for a number of ligand- and
voltage-gated ion channels that their activity can be modulated by the
activation of protein kinases and phosphatases, which are regulated, in
turn, by second messenger systems, e.g., Ca2+ and cAMP
(Schulman, 1995). Potassium channels that constitute an extremely
diverse superfamily involved in the control of pre- and postsynaptic
excitability in this respect are particularly interesting. The
regulation of potassium channel activity by protein phosphorylation may
alter very distinctly neuronal excitability (Jonas and Kaszmarek,
1996).
Several recent in vitro studies have focused on the
Shaker superfamily of voltage-activated potassium
(KV) channels (Chandy and Gutman, 1994). It was shown that
phosphorylation by protein tyrosine kinase reduced the activity of
KV1.2 and KV1.3 channels (Huang et al., 1993;
Lev et al., 1995; Holmes et al., 1996). Also, cAMP-dependent protein
kinase A (PKA) phosphorylation upregulates the activity levels of
Kv1.2 (Huang et al., 1994) and KV2.1
(Wilson et al., 1994) and downregulates that of KV3.2
channels (Moreno et al., 1995). These KV channels belong to
the class of delayed-rectifier channels mediating currents that do not
inactivate rapidly. Other Shaker-related KV
channels, e.g., Shaker itself, KV1.4, and
KV3.4, express outward currents that rapidly inactivate
within milliseconds because of the presence of an amino-terminal
inactivation domain (Hoshi et al., 1990). It was shown that
phosphorylation of the amino-terminal inactivation domain of
KV3.4 channels by protein kinase C (PKC) completely
eliminated N-type inactivation (Covarrubias et al., 1994). Also,
dephosphorylation of a modulatory C-terminal site of Drosophila
Shaker channels considerably slowed the inactivation rate
(Drain et al., 1994).
Ca2+/calmodulin-dependent kinases (CaMKIIs) are
prominently expressed in mammalian brain, both pre- and
postsynaptically. It is well accepted that CaMKII, which phosphorylates
several pre- and postsynaptic proteins, has an important function in
regulating neuronal excitability and synaptic strength (Braun and
Schulman, 1995). It has been shown that members of the
Shaker KV channel family, e.g., KV
1.1, KV 1.2, and KV 1.4, are localized to pre- and postsynaptic compartments (Sheng et al., 1992, 1993; Veh et al.,
1995), making them possible targets for CaMKII phosphorylation. When we
screened the cytoplasmic regions of KV1.4, a rapidly
inactivating KV channel (Stühmer et al., 1989), for
possible CaMKII phosphorylation sites, we detected three CaMKII
consensus sequence motifs in the cytoplasmic amino-terminal sequence
(RXXS/T at serine 101/102 and 123, and threonine 191). We show here
that serine 123 of KV1.4 is a substrate for CaMKII
phosphorylation and that CaMKII-phosphorylated KV 1.4 channels are dephosphorylated by the Ca2+-regulated
calcineurin (protein phosphatase 2B)/inhibitor-1 protein phosphatase
cascade. This Ca2+-sensitive
phosphorylation/dephosphorylation of KV 1.4 has profound functional consequences for the inactivation properties of
KV 1.4-mediated A-type potassium currents.
MATERIALS AND METHODS
Construction of HEK 293-KV1.4 cell line.
Briefly, rat KV1.4 cDNA nucleotide (nt) 387-2658
(Stühmer et al., 1989) subcloned into pBluescript
pKS+ was combined with a blunt-end Ear I fragment of the
metallothionein IIA promotor (MT) [nt 30-830; (Karin et
al., 1984)] cloned into the SalI polylinker restriction
site of Bluescript pKS+. The resulting KV1.4
pKS+ clone was digested with
EcoRI/BglII. The isolated
EcoRI/BglII MT-KV1.4 restriction
fragment was ligated with EcoRI/BglII cut pML2 eucariontic expression vector, containing
polyadenylation and transcription termination signals from the SV40
late region. The KV1.4 pTMT-construct was checked by
restriction analysis. HEK 293 cells were transfected with
KV1.4 pTMT DNA after the protocol of Chen and Okayama (Chen
et al., 1987). HEK 293 cells were grown in DMEM:F12 (Life Technologies,
Gaithersburg, MD) supplemented with 10% FCS (Biother), 2 mM L-glutamine (Life Technologies) and penicillin-streptomycin (50 IU/ml-50 µg/ml; Life
Technologies). Stably transfected cells were selected with 1 mgl G418 (Life Technologies). Selected HEK
293-KV1.4 cell clones were analyzed by Northern blot analysis for KV1.4 RNA expression and by Western blot
analysis for KV1.4 protein expression.
In vitro Mutagenesis. Point mutations in the
KV1.4 amino terminus were introduced by a PCR-based
site-directed mutagenesis (Ho et al., 1989). For the mutation
KV1.4 S123A we used the following oligonucleotides: AAG ATC
CTT AGG GAG ATG GCC GAG GAG GAG (sense) and GTG GTA GAA AAT
AGT TAA A (antisense). The PCR products were digested with
SauI and ligated into KV1.4 pAKS2. The mutant
KV1.4 T191A was generated by an overlay PCR using the
oligonucleotides CTA CGC TTC GAA GCC CAA ATG AAA (sense) and
TTT CAT TTG GGC TTC GAA GCG TAG (antisense). The two PCR
fragments were digested with SauI and cloned into
KV1.4 pAKS2. The mutant Kv 1.4 SS101/102AA was generated by
using oligonucleotides CAC AGG CAG GCC GCT TTT CCT CAT TGC
(sense) and G AGG AAA AGC GGC CTG CCT GTG GTG GAG (antisense). The product from the overlap PCR was cut with
NcoI and cloned into Kv1.4 pAKS2. All mutants were verified
by sequencing (Sanger et al., 1977) before use. For mRNA synthesis
KV1.4 pAKS2, KV1.4 S123A pAKS2, and
KV1.4 T191A pAKS2 were linearized with EcoRI.
In vitro transcription was performed using the Sp6 Message Machine (Ambion, Austin, TX).
In vitro translation and in vitro
phosphorylation. The radioactive in vitro
translation of KV1.4 pAKS2 and KV1.4 S123A were performed using 1 µg cRNA in the FLEXI rabbit reticulocyte lysate system (Promega, Madison, WI) with 35S-methionine according
to protocol. The reactions (final volume, 50 µl) were incubated for 1 hr at 30°C in the presence of 7.2µ canine pancreatic microsomal
membranes. For in vitro phosphorylation, nonradioactive
in vitro translation was set up using a full amino acid
mixture and incubated under the same conditions as above. The
microsomal membranes were sedimented at 4°C with an Eppendorf (Madison, WI) tabletop centrifuge at 15,300 rpm and afterward resuspended in CAMKII reaction buffer (20 mM Tris/HCl, pH
7.5, 10 mM MgCl2, 2 mM
CaCl2, 0.5 mM dithiothreitol, 0.1 mM EDTA). For in vitro phosphorylation, 2.4 µM calmodulin, 100 µM ATP,
32P- -ATP (specific activity of 100 µCi/µmol) and 250 U CaMKII (New England Biolabs, Beverly, MA) were added to the mixture.
The reactions (final volume, 50 µl) were incubated at 30°C for 10 min in the presence of 1 mM Cyclosporin A and 100 µM okadaic acid. The reactions were stopped by addition
of 50 µl of prewarmed (56°C) 2× Laemmli buffer. Ten microliters of
the in vitro translation and 20 µl of the in
vitro phosphorylation reaction were loaded per lane on 10%
SDS-polyacrylamide gels. For control, in vitro translation and phosphorylation reactions without cRNA were performed to estimate the background of the in vitro phosphorylation reaction.
Electrophysiology and microinjection. Macroscopic currents
were obtained from perforated-patch (Rae et al., 1991) and standard whole-cell patch-clamp recording (Hamill et al., 1981) using HEK 293-KV1.4 cells and cRNA-microinjected 293 cells. They were
maintained with standard cell culture protocols and plated at a density
of 5 × 104/ml on poly-L-lysine (50 µg/ml)-coated cellocate grids (Eppendorf) 1 d before the
experiment. The bath solution for electrophysiological experiments
contained (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 5 HEPES, 10 glucose, 20 sucrose, pH = 7.4 with
NaOH. For perforated-patch recordings, the pipette solution contained
(in mM): 70 K2SO4, 10 KCl, 10 NaCl,
7 MgCl2, 10 HEPES, pH = 7.2 with KOH, and 0.25 mg/ml amphotericin-B. For standard whole cell, the pipette
solution contained (in mM): 95 K-aspartate, 20 KCl, 1 CaCl2, 11 EGTA, 10 HEPES, 2 glutathione, 2 Na2ATP, pH = 7.2 with KOH with a calculated (EQCAL,
Biosoft, Cambridge, UK) free Ca2+ concentration of 20 nM. Total CaCl2 concentrations were increased according to EQCAL calculations to obtain free Ca2+
concentrations between 100 and 1000 nM. Patch pipettes were
pulled from borosilicate glass with resistances between 2 and 4 M . Only standard whole-cell experiments with series
resistances <10 M and perforated-patch experiments with
<15 M were included in this study. Series resistance
compensation (0-80%) was used to obtain voltage errors <5 mV.
Currents were recorded with an EPC9 (HEKA Elektronik) patch-clamp
amplifier. The program package PULSE + PULSEFIT (HEKA Elektronik) was
used for data acquisition and analysis. Leakage and capacitive currents
were subtracted on-line using the P/4 subtraction method. P/4 pulses
were applied 2 sec after the test pulses and the interpulse intervals
were 30 sec. Records were digitized at 5 kHz and filtered with low-pass
Bessel characteristic of 1 kHz cut-off frequency. For quantification of
the time course of K currents, individual sweeps were fitted with a
Hodgkin-Huxley related formalism (PULSEFIT): I = I0m(t)4
ht) with m(t) = 1-exp
( t/tm) and
h(t) = h + a1a2
exp(t/th1) + (1a1a2)
exp(t/th2). The time constants
tm th1, and
th2 determine the activation and inactivation
kinetics, respectively, h the steady-state
inactivation. The two inactivation time constants are weighted by the
variable a1/a2. Steady-state activation and inactivation curves were fitted with Boltzmann functions and time courses of recovery with monoexponential functions using a
Marquardt-Levenberg algorithm in SIGMAPLOT (Jandel Scientific, San
Rafael, CA). All experiments were performed at 30 ± 0.5°C. Data
are given as mean ± SEM.
Autothiophosphorylation of purified CaMKII from rat brain (Calbiochem,
La Jolla, CA) was induced by 5 min incubation (25°C) of 2 µg/ml CaMKII with 30 mg/ml calmodulin, 50 mM ATP gm-S, and 1 mg/ml BSA in a solution
containing (in mM): 50 HEPES; 5 MgCl2; 0.3 CaCl2, pH 7.4. Aliqouts of CaMKII-solution were dialyzed on microdialysis membranes (Millipore, Bedford, MA) against a 150 mM KCl + 10 mM HEPES, pH 7.4, solution for 30 min at 4 C° and subsequently microinjected in 293 cells. Calcineurin
autoinhibitory peptide [(CNIP) Bachem, Torrance, CA) was dissolved in
water and microinjected. Stock solutions of KN-62 and KN-93, okadaic
acid (Calbiochem), and cylosporin A (Calbiochem) were dissolved in dimethylsulfoxide.
For microinjection of mRNA, CaMKII or CNIP, KV1.4-293 or
293 cells were plated on poly-L-lysine-coated cellocate
grids, and the Microinjector 5242 (Eppendorf) with an Automated
Injection System (AIS) from Zeiss (Thornwood, NY) was used with
solutions containing 50 ng/ml mRNA (Ikeda et al., 1992).
KV1.4 wild-type and mutant cDNAs were cloned into pAKS
expression vector for in vitro mRNA synthesis as described
(Stühmer et al., 1989). Appropriate filling of individual cells
was obtained with injection times of 200-300 msec and injection
pressures between 80 and 140 hPa. In initial experiments, the filling
of HEK 293-Kv1.4 cells was verified by co-injection
of 0.1% fluorescein isothiocyanate-Dextran [(Sigma, St. Louis, MO)
data not shown].
RESULTS
KV1.4 A-type currents in stably transfected
293 cells
Two hundred ninety-three cells were used as an in
vitro model system to characterize the modulation of
KV1.4 currents by CaMKII and protein phosphatases. The
cells were stably transfected with a rat KV1.4 cDNA
construct under control of the human metallothionin promoter
(Kv1.4-293). KV1.4-293 cells gave rise
to voltage-activated rapidly inactivating outward currents (Fig.
1A). They had properties similar to
KV1.4 currents expressed previously in the
Xenopus oocyte expression system (Stühmer et al.,
1989). In the perforated-patch whole-cell configuation, depolarization
of KV1.4-293 cells to +40 mV produced KV1.4
current amplitudes in the 1-10 nA range. For comparison, amplitudes of
slowly activating, noninactivating endogeneous outward currents were at
+40 mV in the 50-300 pA range in both control and
KV1.4-293 cells (data not shown). In our standard conditions, <5% of total outward current amplitude was attributable to endogenous 293 K channels. Activation of KV1.4 currents
in KV1.4-293 cells was fast (time rise to peak <5 msec)
and had a threshold at approximately 50 mV. KV1.4
currents inactivated rapidly with a residual steady-state current after
a 200 msec depolarizing test-pulse representing ~10% of the peak
current (Fig. 1A). Voltage-dependence of steady-state
KV1.4 current activation was described with a Boltzmann
function; mean half-maximal (V50) activation occurred at
22.6 ± 1.7 mV with a mean slope of 12.1 ± 1.2. mV
(n = 8; Fig. 1B). Mean half
inactivation was at 44.5 ± 0.6 mV with a mean slope of 2.3 ± 0.2 mV (n = 8; Fig. 1B).
Fig. 1.
Properties of KV1.4 currents in 293 cells. Recordings were obtained in the perforated-patch whole-cell
configuration from KV1.4-293 cells. A,
Currents were elicited by 200 msec depolarizing pulses of increasing
amplitudes in steps of 10 mV from a holding potential of 80 mV.
B, Mean steady-state activation (n = 8) and inactivation (n = 8) curves of
KV1.4 currents were fitted with Boltzmann functions to
normalized mean conductances
(g/gmax). Steady-state
inactivation of KV1.4 was determined using a 1 sec prepulse
to increasing potentials from 80 mV in steps of 5 mV, followed by a
200 msec test pulse to +40 mV. C, Voltage-dependence of
mean time constants of activation ( m;
n = 8) of KV1.4 is shown obtained from
fitting individual records of KV1.4 currents with a
Hodgkin-Huxley related formalism (see Materials and Methods). D, Voltage-dependence of mean time constants of the
dominant fast component (h1; n = 4)
and the minor slow component (h2; n = 4) of KV1.4 inactivation is shown as indicated. Data were
obtained from fitting individual records of KV1.4 currents
with a Hodgkin-Huxley related formalism with two inactivation time
constants (see Materials and Methods). E, Recovery from
inactivation of KV1.4 currents. KV1.4 recovery
was tested by a double-pulse protocol. Two 200 msec test pulses to +40
mV were separated by increasing the interpulse interval at 80 mV in
steps of 200 msec. F, Mean time course of initial
recovery from inactivation was fitted to normalized mean data
(I/I0; n = 5). The data
were fitted by a monoexponential function to determine time constant
(r1) and fraction (f) of initial fast recovery.
[View Larger Version of this Image (22K GIF file)]
A Hodgkin-Huxley related formalism (I = I0m(t)4
h(t); see Materials and Methods) with a
single time constant (m) for activation and two time
constants (h1,h2) for inactivation was
used to fit macroscopic KV1.4 currents. Within the studied
voltage range of 40 to +60 mV, m showed a marked
voltage dependency, decreasing from a mean of 3.6 ± 0.3 msec
(n = 8) at 40 mV to 0.7 ± 0.2 msec (n = 8) at +60 mV (Fig. 1C). In agreement
with previous single-channel data demonstrating the voltage
independence of transitions from the open to the inactivated state in
Shaker KV-channels (Zagotta and Aldrich, 1990),
time constants of KV1.4 inactivation showed no apparent
voltage dependency between 20 to +60 mV (n = 6; Fig. 1D). Throughout this range, >70%
(Ih1/I = 73.5 ± 4.1%;
n = 12) of a fast inactivation component
(h1= 16.6 ± 0.9 msec at +40 mV; n = 12) and <30% (Ih2/I = 26.5 ± 4.1%; n = 12) of a slower inactivation component
(h2 = 43.3 ± 2.3 msec at +40 msec;
n = 12) contributed to KV1.4 inactivation.
Two distinct molecular mechanisms for K+ channel
inactivation have been described: N-type inactivation, which depends on
an amino terminal inactivation domain acting rapidly as a tethered
intracellular K+ channel blocker (Hoshi et al., 1990), and
C-type inactivation, which seems to involve slower structural changes
at the extracellular mouth of the channel pore (Baukrowitz and Yellen,
1995, 1996). Deletion of the amino terminal of KV1.4
inactivation domain [KV1.4 110; (Rettig et al., 1994)]
removes rapid N-type inactivation of KV1.4 currents and a
pure C-type inactivation remains. Expression of KV1.4110
in 293 cells exhibited slowly inactivating currents (data not shown).
The inactivation time course was well described by a single time
constant in the range of 200 msec (h = 218 ± 16 msec; n = 5) that is significantly longer than the two
time constants for KV1.4 wild-type inactivation. It seems
that at least the dominant fast inactivation component of
KV1.4 represents pure N-type inactivation.
It has been shown that recovery from inactivation of Shaker
channels involves at least two processes, a fast one in the millisecond time range and a slow one, which takes several seconds for completion (Zagotta and Aldrich, 1990; Demo and Yellen, 1991; Baukrowitz and
Yellen, 1995). The fast component reflects recovery from N-type inactivation, the slow one that from C-type inactivated states (Baukrowitz and Yellen, 1995). Similarly, KV1.4 recovery
from inactivation, determined in the perforated-patch configuration by
a double pulse protocol of 200 msec depolarizing voltage steps to +40
mV separated by variable interpulse intervals at 80 mV (Fig.
1E), exhibited a fast and a slow component (Fig.
1F). Sixty-eight percent of the initial
KV1.4 current recovered from inactivation via the fast
recovery process [fraction (f) of fast
recovery = 0.68], which could be described by a single
exponential with a time constant r1 of 598 msec
(n = 5; Fig. 1F). More hyperpolarized interval pulse potentials accelerated the initial component of KV1.4 recovery from inactivation saturating at potentials
negative to 120 mV. The voltage dependence of the initial recovery
process was described with a Boltzmann function with a mean
V50 of 53.5 ± 1.5 mV and slope of
17.8 ± 11.2 mV (n = 3). Complete recovery of
KV1.4 from inactivation took up to 15 sec. The slow
recovery process apparently had a time constant in the range of several seconds. Recovery of KV1.4110 channels from C-type
inactivation was similarly slow (h = 2.4 ± 0.7 sec; n = 3). The slow recovery process showed no
apparent voltage dependence. Because of this slow component of
KV1.4 recovery, in all pulse protocols we used interval
times of 30 sec between test pulses to avoid cumulative inactivation of
KV1.4 currents.
Dephosphorylation accelerates KV1.4 inactivation
When calcineurin (protein phosphatase 2B) was inhibited in
KV1.4-293 cells by microinjecting the calcineurin
inhibitory peptide (CNIP, 10 µm) 1 hr before
KV1.4 current recordings in the perforated-patch configuration, a delayed KV1.4 inactivation time course
(Fig. 2B) was observed in comparison to
controls (Fig. 2A). In contrast, time constants of
activation were not affected by this treatment. Inactivation in the
presence of CNIP could be well described with only one time constant
(h). Its value, 48.1 ± 2.5 msec at + 40 mV
(n = 6), was similar to the slowly inactivating
component h2, which contributes <30% to the
inactivation time course of KV1.4 currents in controls.
Preincubation of KV1.4-293 cells with cyclosporin A, which
blocks calcineurin via cyclophillins, also resulted in a slowed
KV1.4 inactivation time course (46.1+ 1.9 msec;
n = 3) similar to those observed with microinjected
CNIP.
Fig. 2.
Modulation of KV1.4 by inactivation
protein phosphatases and CaMKII. A-E,
Recordings of KV1.4 currents were obtained in the perforated-patch whole-cell configuration. Current responses were elicited by a 200 msec depolarizing pulse to +40 mV from a holding potential of 80 msec from KV1.4-293 cells
(A) under control conditions, (B) after
microinjection of 10 µM CNIP,
(C) after 30 min preincubation with 100 nM
OA, (D) after 30 min preincubation
with 100 nM OA and 10 µM
KN-62, (E) after 30 min preincubation with 100 nM OA and 10 µM KN-93.
Inserts in A and C show
current responses to 1 sec depolarizing pulses. In B and
C, monoexponential fits of inactivation are
superimposed. For comparison, currents were normalized to peak.
F, Mean fitted time constants (h) of the
dominant (>70%) inactivation component of KV1.4 currents
recorded with the perforated-patch configuration under conditions shown
in A-E as well as after preincubation with 10 µM KN-93. Data were obtained from four to six
independent experiments.
[View Larger Version of this Image (19K GIF file)]
Calcineurin is able to initiate a calcineurin/inhibitor-1 protein
phosphatase cascade where active calcineurin, by means of dephosphorylating an inhibitor, upregulates the activity of the calcium-independent protein phosphatase 1 [(PP1); for review, see
Cohen, 1989]. PP1 activity is inhibited by nanomolar concentrations of
okadaic acid (OA). To determine whether this cascade was also active in
KV1.4-293 cells, they were preincubated with 100 nM OA for 30 min. In the presence of this protein
phosphatase blocker, KV1.4 inactivation kinetics were also
slow and similar to those observed with calcineurin inhibition (Fig.
2C). The inactivation time course of KV1.4
currents could again be well described by one time constant
h (51 ± 8 msec at + 40 mV; n = 6).
Fitting the inactivation time constant of KV1.4 current
responses to 1 sec depolarizing pulses gave similar time constants
(57 ± 10 msec at + 40 mV, n = 5). In addition to
PP1, 100 nM OA can also block PP2A, another
calcium-independent protein phosphatase. PP2A has the highest affinity
to this blocker and therefore can be selectively blocked with 1 nM OA (Cohen, 1989). Preincubation with 1 nM OA resulted in a less pronouced slowing of KV1.4 inactivation
kinetics compared with the control (h = 30.4 ± 2.8; n = 3) suggesting that in contrast with the
calcineurin/inhibitor-1 protein phosphatase cascade, PP2A plays a minor
role in regulation of KV1.4 inactivation kinetics.
When both the protein phosphatases and CaMKII were inhibited with
100 nM OA and 10 µM KN-62, respectively,
KV1.4 currents were indistinguishable from those in control
cells (Fig. 2A,D). This showed that the effect of
protein phosphatase inhibition on KV1.4 inactivation was
occluded completely by the simultaneous application of a CaMKII
inhibitor. Furthermore, incubation of KV1.4-293 cells with
100 nM okadaic acid and 10 µM KN-93, a more potent blocker of CaMKII compared with KN-62 (Sumi et al., 1991), yielded KV1.4 currents, which now inactivated approximately
threefold faster than KV1.4 control currents
(h1 = 4.4 ± 0.3 msec at + 40 mV; n = 5) (Fig. 2A,E). Similar to the results obtained in the simultaneous presence of OA and KN-93, the KV1.4
inactivation time course was dominated (OA+KN-93:
Ih1/I = 88.5 ± 1.5%;
n = 5) by a fast inactivation time constant
h1 in the presence of KN-93 alone (4.2 ± 0.3 msec;
Ih1/I = 92.9 ± 1.0%;
n = 6) (Fig. 2F). These data
suggested tentatively that KV1.4-293 cells contained a
steady-state equilibrium between dephosphorylated and phosphorylated KV1.4 protein altered by either blocking the
calcineurin/inhibitor-1 protein phosphatase cascade or CaMKII.
Apparently, an increase in the concentration of phosphorylated
KV1.4 protein induced by protein phosphatase blockers
caused a slowing of inactivation kinetics. Conversely, a decrease in
CaMKII-phosphorylated KV1.4 protein induced by CaMKII
blockers caused an acceleration of KV1.4 inactivation
kinetics. In contrast, the amplitude and kinetics of endogenous 293 currents were not effected by either inhibition of phosphatases (100 nM OA, n = 5) or CaMKII (10 µM KN-62; n = 5).
CaMKII phosphorylates KV1.4 channels
CaMKII might phosphorylate one or more of the three CaMKII
phosphorylation consensus sites present in the KV1.4 amino
terminus. Two of the motifs involving serine residues 101/102 and 123 are specific for KV1.4. They do not occur in other
KV1 subfamily members (Chandy and Gutman, 1994). The third
motif is related to threonine residue 191 located at equivalent
positions within the amino terminal tetramerization (T) domain of all
KV1 subfamily members (Shen and Pfaffinger, 1995). Serine
123, threonine 191, and serines 101/102 were replaced with alanine by
in vitro mutagenesis of KV1.4 cDNA
(KV1.4 S123A, KV1.4 T191A, KV1.4
SS101/102AA) to generate nonfunctional CaMKII consensus sites. The
cDNAs were used as templates for in vitro mRNA synthesis.
KV1.4 T191A mRNA did not express functional
KV1.4 channels after microinjection into 293 cells and
therefore was not analyzed further. KV1.4 SS101/102AA
expressed only small currents with properties that were similar to
wild-type KV1.4 (see below). KV1.4 S123A mRNA,
on the other hand, expressed large currents after microinjection into
293 cells. The combined biochemical and electrophysiological
characterization of KV1.4 S123A provided evidence that
strongly suggested that serine 123 was the CaMKII modulatory site of
KV1.4 (see below).
CaMKII-dependent phosphorylation of wild-type KV1.4 and
KV1.4 S123A mutants were assayed in vitro.
Wild-type KV1.4 and KV1.4 S123A mRNAs were
translated into protein using a reticulocyte lysate supplemented with
microsomes. The translated KV1.4 proteins were subsequently
incubated with purified CaMKII and 32P-ATP for
biochemical phosphorylation studies. The 32P-labeled
phosphorylated protein material was analyzed by denaturing SDS-PAGE
followed by autoradiography (Fig. 3A). The
results showed that CaMKII had phosphorylated wild-type and
KV1.4 S123A proteins to a different extent. Densitometric
analysis of 32P-labeled KV1.4 protein
normalized for protein concentration and averaged over seven
experiments showed that ~35% less 32P-phosphate had been
incorporated into KV1.4 S123A than into wild-type KV1.4 protein (Fig. 3A). Consistent with the
presence of three CaMKII phosphorylation consensus sites in the
KV1.4 amino terminus, the data suggested that CaMKII
in vitro phosphorylated more than one site on
KV1.4 protein and that one of the functional sites had been
eliminated in the S123A mutation.
Fig. 3.
CaMKII phosphorylates an N-terminal
KV1.4 residue and slows inactivation. A,
In vitro phosphorylation of KV1.4 and
KV1.4 S123A by CaMKII. KV1.4 and
KV1.4 S123A mRNA were translated in vitro in
the presence of microsomes. Translation products were incubated with
CaMKII and 32P--ATP for in vitro
phosphorylation. Products were separated by 10% SDS-PAGE. Gels were
dried and autoradiographed. Left panel shows
autoradiogram. Lanes 1-2, In vitro translated,
35S-labeled KV1.4 and KV1.4 S123A
probes as indicated. Lanes 3-5, In vitro phosphorylated,
32P-labeled control microsomal membranes (3); in
vitro phosphorylated, 32P-labeled Kv1.4 (4);
in vitro phosphorylated, 32P-labeled Kv1.4
S123 A (5). Right panel gives the mean
relative phosphorylation by CaMKII of in vitro
translated KV1.4 and KV1.4 S123A obtained from
seven independent experiments. Exposure time of the autoradiographs was
12 hr. Phosphorylation intensities were scanned by phosphoimager
(Fuji), analyzed by TINA 2.0 (raytest), and normalized to background.
B-G, Recordings of KV1.4 and
KV1.4 S123A currents were obtained in the perforated-patch
whole-cell configuration from 293 cells that had been microinjected
with 50 ng/µl KV1.4 or KV1.4 S123A mRNA as
indicated, 6 hr before recording. Current responses were elicited by a
200 msec depolarizing pulse to +40 mV from a holding potential of 80
msec from cells (B, C) under control conditions,
(D, E) after 30 min preincubation with 10 µM KN-93, and (F, G) 30 min after second
microinjection with autothiophosphorylated CaMKII (2 µg/ml). For
comparison, currents were normalized to peak.
[View Larger Version of this Image (20K GIF file)]
Wild-type KV1.4 and KV1.4 S123A mRNA were
microinjected into 293 cells and expressed currents were recorded in
the perforated-patch configuration. The wild-type KV1.4
currents were indistinguishable from those elicited in stably
transfected KV1.4-293 cells (compare Figs.
1A, 3B). The inactivation time course of
KV1.4 S123A currents, on the other hand, was faster than
wild type (Fig. 3B,C). It was well described with two time
constants (h1 = 10.1 ± 0.3 msec; h2 = 36.4 ± 3.7 msec;
Ih1/I = 88 ± 4%;
n = 6). Very similar time constants were obtained for
KV1.4 S123A currents inactivation in the presence of 1 µM cyclosporin A (h1 = 9.6 ± 1.4 msec; h2 = 37.5 ± 1.5 msec; n = 5), 100 nM OA (h1 = 10.5 ± 1.3 msec;
h2 = 32.0 ± 3.1 msec; n = 5), and
the CaMKII inhibitor KN-62 (h1 = 9.5 ± 0.4 msec;
h2 = 30.9 ± 1.6 msec, n = 3).
These data demonstrated that inhibition of the calcineurin/inhibitor-1
protein phosphatase cascade or of CaMKII with KN-62 did not affect
KV1.4 S123A inactivation kinetics in contrast to wild-type
KV1.4. In the presence of the CaMKII inhibitor KN-93,
wild-type KV1.4 (Figs. 2E, 3D)
(h1 = 4.2 ± 0.3 msec; h2 = 26.0 ± 0.6 msec; n = 6) and KV1.4
S123A (Fig. 3E) (h1 = 3.8 ± 0.4 msec;
h2 = 26.9 ± 1.6 msec; n = 4) inactivation kinetics were similar and most rapid. In contrast, when
the concentration of active CaMKII was increased by microinjecting the
Ca2+-independent autothiophosphorylated CaMKII (for review,
see Braun and Schulman, 1995) before electrophysiological experiments,
KV1.4 and KV1.4 S123A inactivation kinetics
were markedly different (Fig. 3F,G). Inactivation of
KV1.4 was delayed in comparison with controls (Fig.
3B,F) and was fitted by a single inactivation time constant (h = 38.3 ± 3.3 msec; n = 6). This h was similar to that obtained after blocking
the calcineurin/inhibitor-1 protein phosphatase cascade (Fig.
2B,C). In contrast, the time course of
KV1.4 S123A inactivation was not affected by microinjection of autothiophosphorylated CaMKII (Fig. 3G). The inactivation
time course was dominated by a fast inactivation time constant
(h1 = 7.9 ± 0.7 msec;
Ih1/I = 87.2 ± 3.3%;
n = 5) as in control recordings. The results shown in
Figure 3 demonstrated that KV1.4 is a substrate for CaMKII.
When KV1.4 was phosphorylated at the serine 123 modulatory site, KV1.4-mediated currents inactivated 5-10 times
slower than those mediated by dephosphorylated KV1.4. In
addition, a comparison of KV1.4 110 (Rettig et al.,
1994) and KV1.4 110 S123A-mediated currents showed that
they possessed very similar slow inactivation kinetics
(KV1.4 110: h = 218 ± 16 msec;
n = 5; KV1.4 110 S123A: h = 213 ± 33 msec; n = 5). This observation
suggested that the CaMKII phosphorylation at serine 123 apparently
modulated N-type and not C-type inactivation of KV1.4.
KV1.4 recovery from inactivation modulated
by CaMKII
KV1.4 currents that were recorded from
KV1.4-293 cells microinjected with autothiophosphorylated
CaMKII recovered from inactivation twofold faster and more completely
(Fig. 4A,B) than control (Fig. 1E,F). Eighty-seven percent
(f = 0.87) of the KV1.4 current
amplitude recovered from inactivation with a time constant
r1 = 309 msec (n = 5). This suggested
that most KV1.4 channels in the CaMKII-phosphorylated form
recovered directly from N-type inactivation and did not enter the long
absorbing C-type inactivated state. In the presence of KN-93, however,
initial KV1.4 recovery was twofold slower, with a time
constant similar to control (r1 = 601; n = 5) and recovered only to 49% with the initial fast component (Fig.
4C,D). Therefore, in the dephosphorylated form, ~50% of
the KV1.4 channels entered the long absorbing C-type
inactivated state via N-type inactivation. The mean voltage dependency
of the initial recovery component was not affected by CaMKII
phosphorylation. In CaMKII-microinjected cells, it was described with a
Boltzmann function with V50 at 52.9 ± 1.9 mV and a slope of 18.9 ± 1.6 mV (n = 4). With
KN-93, V50 was 55.2 ± 1.1 mV with a slope of
15.8 ± 0.8 mV (n = 3). Initial recovery of
KV1.4 S123A currents from inactivation was even slower
(r1 = 755 msec; n = 3), and less
completely (f = 0.41; n = 3)
suggesting that ~60% of the KV1.4 S123A channels recovered through a long absorbing state. These results indicated that
the modulation of KV1.4 inactivation kinetics by CaMKII
phosphorylation affected not only the rate of KV1.4
recovery from inactivation, but also the pathway of recovery. After
CaMKII phosphorylation, more KV1.4 channels apparently
returned directly from N-type inactivated state(s) and less entered
long absorbing C-type inactivated state(s).
Fig. 4.
CaMKII phosphorylation accelerates
KV1.4 recovery from inactivation. Recordings of
KV1.4 currents were obtained in the perforated-patch whole-cell configuration from KV1.4-293 cells. Recovery
from KV1.4 inactivation was determined by a double-pulse
protocol. Current responses to two 200 msec test pulses to +40 mV
separated by increasing the interpulse intervals at 80 mV in steps of
200 msec were recorded; A, microinjection with
autothiophosphorylated CaMKII (2 µg/ml); C,
after 30 min preincubation with 10 µM KN-93. Mean time
course of recovery from inactivation was fitted to normalized mean data (I/IO) obtained from five
independent experiments, (B) after microinjection with
autothiophosphorylated CaMKII, and (D) after 30 min
preincubation with 10 µM KN-93. The mean time course of
initial recovery was fitted by a monoexponential function to determine
time constant (r1) and fraction
(f) of initial recovery.
[View Larger Version of this Image (20K GIF file)]
Macroscopic kinetics of voltage-dependent activation and the
steady-state activation curve of KV1.4 were not affected by
CaMKII phosphorylation (Fig. 5). Steady-state activations
were described by Boltzmann functions and mean
V50 values and slopes obtained for
KV1.4 currents from both CaMKII-microinjected cells
(V50 = 20.6 ± 2.0 mV; slope = 12.5 ± 1.4 mV; n = 6) and KN-93 treated cells
(V50 = 23.7 ± 5.2 mV; slope = 6.4 ± 1.6 mV; n = 3) were similar to those obtained in control
recordings. CaMKII phosphorylation, however, affected the apparent
voltage dependence of KV1.4 steady-state inactivation.
Microinjection of CaMKII shifted the apparent voltage-dependence of
steady-state inactivation of KV1.4 currents by ~7 mV to
more positive membrane potentials (V50 at
37.8 ± 1.2 mV; n = 6; Fig. 5B)
compared with both control recordings (Fig. 1B) and
those in the presence of KN-93 (V50 was at
43.2 ± 1.8 mV; n = 4; Fig. 5D).
Similar to dephosphorylated KV1.4, the
V50 of KV1.4 S123A steady-state
inactivation was also at more negative potentials (V50 = 44.5 ± 0.6 mV; slope = 2.6 ± 0.5 mV; n = 4).
Fig. 5.
CaMKII phosphorylation shifts KV1.4
steady-state inactivation to positive potentials. Recordings of
KV1.4 currents were obtained in the perforated-patch
whole-cell configuration from KV1.4-293 cells (A,
B) microinjected with autothiophosphorylated CaMKII (2 µg/ml), or (C, D) 30 min preincubated with 10 µM KN-93. A-C, For
steady-state inactivation, current responses were elicited by 1 sec
prepulse to 80 and 40 mV (arrows) followed by a 200 msec test pulse to +40 msec. For comparison, currents were normalized to peak. B-D, Mean steady-state activation
(n = 3,6) and inactivation curves
(n = 4,6) of KV1.4 currents were fitted
with Boltzmann functions to normalized mean conductances
(g/gmax).
Dotted lines represent fitted steady-state inactivation
under control conditions.
[View Larger Version of this Image (21K GIF file)]
CaMKII phosphorylation modulates Kv1.4
cumulative inactivation
Standard voltage-clamp protocols reflect only poorly the
physiological patterns of excitation occurring in vivo;
i.e., repetitive discharge of short action potentials at varying
frequencies. Under these conditions, A-type KV channels
like KV1.4 are likely to undergo repetitive
activation-deactivation and inactivation-recovery cycles. This is
correlated with the occurrence of cummulative inactivation that
involves both N-type and C-type inactivation (Baukrowitz and Yellen,
1995). To approximate physiological consequences that may be correlated
with the CaMKII phosphorylation of KV1.4 channels, we
simulated physiological discharge patterns by stepping the membrane
potential of KV1.4-293 cells to +20 mV for 5 msec from
interpulse potentials of 60 mV at frequencies between 1 and 100 Hz.
Elicited KV1.4 current amplitudes were recorded in the
perforated-patch configuration. In CaMKII-microinjected
KV1.4-293 cells, 10 Hz stimulation induced only a small
cumulative inactivation (Fig. 6A). After
10 consecutive pulses, ~80% of the initial KV1.4 current
amplitude was still present. Throughout the studied frequency range
(1-100 Hz), an acceleration of cumulative inactivation with increasing
stimulation frequencies was apparent (Fig. 6B), but even with 100 Hz, >50% of the initial KV1.4 amplitude was
present after 10 pulses, demonstrating a relative resistence of
CaMKII-phosphorylated KV1.4 channels to cumulative
inactivation. In contrast, dephosphorylated KV1.4 channels
obtained by either KN-93 incubation of wild-type KV1.4
(Fig. 6C,D) or expression of KV1.4 S123 channels
(Fig. 6E,F) were sensitive to repetitive
stimulation. In both cases, 10 Hz stimulation induced a significant
cumulative inactivation after 10 pulses. A loss of ~80% of the
initial current amplitude was observed (Fig. 6C,E). The
increased sensitivity of dephosphorylated KV1.4 to
cumulative inactivation was already apparent at 1 Hz stimulation
frequencies and was accentuated at higher stimulation frequencies (Fig.
6D,F). After 10 pulses at 100 Hz, only ~10% of the initially available KV1.4 channels remained active.
Thus, the frequency-dependence of KV1.4 cumulative
inactivation is determined markedly by CaMKII phosphorylation of
KV1.4 at serine 123.
Fig. 6.
CaMKII phosphorylation reduces cumulative
inactivation of KV1.4 currents. Recordings of
KV1.4 currents were obtained in the perforated-patch
whole-cell configuration from KV1.4-293 cells (A,
B) microinjected with autothiophosphorylated CaMKII (2 µg/ml) (C, D) 30 min preincubated with 10 µM KN-93, and KV1.4 S123A cRNA microinjected 293 cells (E, F). A, C,
E, Current responses were elicited by 10 Hz stimulations of 5 msec depolarizations to +20 mV from a holding potential of 60
mV. For comparison, currents were normalized to peak of first current
response. B, D, F, Normalized mean current amplitudes
(I/I0; n = 6) elicited by 5 msec depolarizations to +20 mV from a holding potential of
60 mV with stimulation frequencies at 1, 5, 10, 50, and 100 Hz are
plotted versus pulse number. Symbols represent
stimulation frequencies as indicated.
[View Larger Version of this Image (23K GIF file)]
Inactivation kinetics of KV1.4 currents are
Ca2+ dependent
The activation of CaMKII and calcineurin is regulated by
intracellular Ca2+. In contrast with calcineurin, however,
CaMKII can be converted to a Ca2+-independent form after
autophosphorylation (Miller and Kennedy, 1986; Braun and Schulman,
1995). As we have shown, the phosphorylation status of the
KV1.4 modulatory site depends on the relative activities of
calcineurin and CaMKII and should thus depend on the intracellular Ca2+concentration. To examine this hypothesis directly, we
recorded from KV1.4-293 cells in the whole-cell
patch-clamp configuration to dialyze the cells with different free
Ca2+ concentrations. With 200 nM free
Ca2+ in the pipette solution, KV1.4 currents
were similar to those observed in the perforated-patch configuration
and showed no apparent change of inactivation kinetics throughout the
course of the experiment (Fig. 7A,E).
Accordingly, inactivation time courses could be described by double
exponential functions. A fast process with a time constant h1 of 10.4 ± 1.3 msec at +40 mV (n = 6) dominated inactivation (Ih1/I = 86.3 ± 3.1%)
and the minor component (>15%) had a mean time constant,
h2 of 34.4 ± 2.2 msec at +40 mV (n = 6) (Fig. 7F). Similar time constants were
determined for whole-cell patch-clamp recordings with free
Ca2+ concentrations between 0.1 and 1 µM in
the pipette solution (Fig. 2F). Also,
steady-state activation and inactivation curves were similar to
perforated-patch results (200 nM Ca2+:
activation, V50 = 23.1 ± 1.6; slope = 11.2 ± 0.6 mV; n = 8; inactivation:
V50 =48.7 ± 2.2 mV, slope = 2.8 ± 0.3 mV; n = 8). In contrast, when the free
Ca2+ concentration in the pipette solution was reduced to
20 nM, inactivation of KV1.4 currents
progressively slowed after whole-cell dialysis (Fig. 7B).
The first KV1.4 current response, which was recorded immediatedly after breaking into the cell, still exhibited fast inactivation comparable with those recorded with higher
Ca2+ concentrations. However, the KV1.4 current
responses, which were subsequently recorded at 30 sec intervals, showed
increasingly slower inactivation accompanied by an increase in
sustained current at the end of the 200 msec pulse and an increase in
total charge transfer (Fig. 7E). The
Ca2+-dependent modulation of KV1.4 inactivation
reached a steady-state 6-10 min after breaking into the cell for
standard whole-cell recording (Fig. 7B). During dialysis of
20 nM free Ca2+, the slowing of the
Kv1.4 inactivation time course could be described as
a continous increase of the contribution of the slowly inactivating component (h2 = 38.4 ± 1.6 msec, n = 5). This initially accounted for only ~12%
(h2/I = 11.8 ± 3.4%;
n = 5) of the inactivating Kv1.4
current and then rose to ~65% (h2/I = 65.0 ± 6.1%; n = 5). Accordingly, the
contribution of the fast inactivating component (h1 = 7.8 ± 1.1 msec; n = 5) had decreased during 20 nM Ca2+ dialysis from 88 to 35%. Preincubation
with KN-62 blocked the transition from fast to slow KV1.4
inactivation with dialysis of 20 nM free Ca2+
solutions (Fig. 7C,E). When similar whole-cell patch-clamp
experiments were performed with 293 cells expressing KV1.4
S123A currents, no Ca2+ sensitivity of the time course
of inactivation was observed, and the fast component of
inactivation was dominant (Ih1/I
>85%) with dialysis of both 20 nM (Fig. 7D,E)
and 200 nM free Ca2+ concentrations (20 nM: h1 9.2 ± 0.3 msec;
h2 37.2 ± 2.5 msec; Ih1/I = 90.4 +3.2%;
n = 10). In contrast, 293 cells expressing KV1.4 SS101/102AA showed similar slowing after dialysis
with 20 nM free Ca2+ concentrations compared
with KV1.4 wild-type (n = 3) indicating that serines 101/2 are not involved in Ca2+ dependend
modulation of KV1.4. These results suggested that the transition from fast to slow KV1.4 current inactivation
observed with 20 nM Ca2+ dialysis depended on
the presence of CaMKII-phosphorylated KV1.4 channels.
Accordingly, the transition was blocked by preincubation with the
CaMKII inhibitor KN-62 and apparently favored by the inhibition of the
calcineurin/inhibitor-1 protein phosphatase cascade (Fig.
2B,C). The most likely explanation for these
observations is that the balance between calcineurin and CaMKII
activitities in KV1.4-293 cells was shifted at 20 nM Ca2+ in favor of the Ca2+
independent autophosphorylated form of CaMKII, and consequently the
concentration of CaMKII-phosphorylated KV1.4 was increased. Our data provide strong evidence that the Ca2+ sensitivity
of KV1.4 inactivation kinetics is an expression of the
dynamic equilibrium between CaMKII-phosphorylated and
calcineurin/protein phosphatase 1 dephosphorylated
KV1.4.
Fig. 7.
KV1.4 inactivation kinetics are
modulated by intracellular Ca2+ concentration. Recordings
of KV1.4 currents were obtained in the standard whole-cell
configuration from KV1.4-293 cells with (A)
200 nM free Ca2+, (B) 20 nM Ca2+ in the pipette solution, and
(C), 20 nM Ca2+ in the pipette
solution after preincubation with 10 µM KN-62. D, Similar recordings were obtained from 293 cells
expressing KV1.4 S123A currents with 20 nM
Ca2+ in the pipette solution. Currents were elicited by 200 msec test pulse to +40 mV starting immediately after breaking into the
cell and subsequently in 30 sec intervals. Current responses at 0 min and 8 min as indicated (arrows). E,
Normalized mean charge transfer (n = 4) induced by
repetitive 200 msec test pulses from KV1.4- and
KV1.4 S123A-expressing cells to +40 mV with 20 nM and 200 nM free Ca2+ in the
pipette solution as indicated. F, Left panel, Mean
inactivation time constants of the fast (h1) and slow
component (h2) plotted against the free Ca2+
in the pipette solutions (n = 4-8) of
KV1.4 currents. Dotted lines represent mean
time constants at all Ca2+-concentrations tested. F,
Right panel, Mean normalized contribution (Ih1/I) of the fast
(h1) component to KV1.4 inactivating
currents. Ih1/I was
plotted against the free Ca2+ concentration in the pipette
solutions used for the whole-cell recordings (n = 4-8).
[View Larger Version of this Image (28K GIF file)]
DISCUSSION
CaMKII phosphorylation of a KV1.4 amino-terminal site
modulates inactivation kinetics
In this study, we have given biochemical and electrophysiological
evidence that the Ca2+/CaMKII phosphorylated an important
modulatory site of KV1.4 channels, located at serine 123 within the amino-terminal cytoplasmic domain. Furthermore, we have
shown that this site is dephosphorylated by the
Ca2+-dependent calcineurin/inhibitor-1 protein phosphatase
cascade. CaMKII-phosphorylation of Kv1.4 has several
functional consequences of KV1.4 activity. First, the time
course of inactivation of CaMKII-phosphorylated KV1.4
channels (h = 40-50 msec) (Fig. 3) is slowed by
approximately one order of magnitude in comparison to that of
dephosphorylated KV1.4 channels (h = 4-8
msec) (Fig. 3). Second, CaMKII-phosphorylated KV1.4 showed
an accelerated recovery from inactivation, a reduced propensity to
enter long absorbing inactivated states, and a reduced tendency to
undergo cumulative inactivation when stimulated with a train of action
potential-like short depolarizations. Third, the voltage-dependent
steady-state inactivation of CaMKII-phosphorylated KV1.4
channels was shifted to more positive potentials by ~7 mV. This might
increase a KV1.4 window current in the subthreshold range
of between 50 and 30 mV.
Under control conditions in the perforated-patch configuration,
KV1.4 currents had properties that were typical for
dephosphorylated KV1.4; i.e., a dominant fast inactivating
component, slow and incomplete initial recovery, and steady-state
inactivation with a V50 at 45 mV. This indicated that
KV1.4 was present in KV1.4-293 cells
predominantly in the dephosphorylated form. The concentration of
CaMKII-phosphorylated KV1.4 in 293 cells was increased by
either microinjection of autothiophosphorylated CaMKII or by inhibition of calcineurin/protein phosphatase 1. Dialysis of
KV1.4-293 cells with pipette solutions containing 20 nM free Ca2+ had a similar effect on
KV1.4 inactivation kinetics as pharmacological inhibition
of protein phosphatases. Both effects were occluded by the
preincubation with the CaMKII inhibitors KN-62 or KN-93. These results
suggest that in the 293 cellular enviroment KV1.4 channels
exist in a steady-state equilibrium of CaMKII-phosphorylated and
dephosphorylated forms. In aggreement with this notion are the
properties of the currents mediated by KV1.4 S123A, which cannot be phosphorylated at the serine 123 modulatory site. These channels were locked in the fast inactivating mode and were not affected by microinjection of autothiophosphorylated CaMKII, protein phosphatase inhibitors, or dialysis with 20 nM free
Ca2+ solutions.
CaMKII phosphorylation of KV1.4 modulates functional
coupling of N- and C-type inactivation and frequency-dependent
cumulative inactivation
Two principally different inactivation mechanisms have been
identified for Shaker-related A-type channels. One is
mediated by the amino-terminal inactivating "ball" domain acting as
a intracellular tethered blocker of the open channel and has been
termed N-type inactivation (Hoshi et al., 1990). The second mechanism,
named C-type, is mediated by C-terminal domains and involves
conformational changes at the extracellular mouth of the pore (Choi et
al., 1991; Hoshi et al., 1991; Lopez-Barneo et al., 1993). Both types
of inactivation can influence each other in a cooperative manner (Baukrowitz and Yellen, 1995, 1996). When the amino-terminal "ball" domain is deleted, e.g., in KV1.4 110 (Rettig et al.,
1994), KV1.4 channels inactivate only via C-type
inactivation (h = 213 msec). Comparison of the
inactivation kinetics of CaMKII-phosphorylated KV1.4
channels with that of KV1.4 110 shows that the former is still significantly faster than the latter. Also, the S123A mutation did not affect the time constant of C-type inactivation
(KV1.4 110 S123A: h = 218 msec). This
suggests that CaMKII-phosphorylation of the modulatory site at serine
123 may exert its effects on N-type inactivation of KV1.4.
It is likely, however, that CaMKII phosphorylation influences C-type
inactivation indirectly by reducing the functional coupling between
N-type and C-type inactivated states. As pointed out by Baukrowitz and
Yellen (1995), a short depolarizing pulse drives Shaker
A-type K channels into N-type inactivation. From this state, they have
two main pathways: they either recover directly via an open state
(Ruppersberg et al., 1991) to resting states with rate  or enter the
long absorbing C-type inactivated state (Demo and Yellen, 1991) with
rate µ. Therefore, the time constant for the fast phase of recovery
is r1 = [( + µ) 1 and the fraction
(f) of channels recovering via the fast route is f = /( + µ) (Baukrowitz and Yellen, 1995)].
In the dephosphorylated state of KV1.4, these parameters
are f = 0.5 and r1 = 600 msec (Fig.
4D), i.e., both pathways have an equal rate of ~0.8
sec1. In comparison, the rate of recovery from C-type
inactivation determined with the KV1.4110 mutant is
~0.4 sec1. This comparison suggests that
dephosphorylated KV1.4 channels are bound to accumulate in
the C-type inactivated state via N-type inactivation. In contrast, the
fraction of CaMKII-phosphorylated KV1.4 channels recovering
directly from N-type inactivation is almost 90%
(f = 0.87), with a time constant of 300 msec
(Fig. 4B). This predicts an accelerated rate of
KV1.4 recovery from N-type inactivation (2.8 sec1) and a reduced rate µ for entering the C-type
inactivated state from N-type inactivation (0.5 sec1).
The latter is only slightly faster than the rate of leaving the C-type
inactivated state (0.4 sec1). This suggests that only a
minor fraction of CaMKII phosphorylated KV1.4 channels can
accumulate in the C-type inactivated state via N-type inactivation.
Thus, CaMKII phosphorylation of KV1.4 is likely to
interfere with the functional interplay of N-type and C-type
inactivation. CaMKII phosphorylation of KV1.4 shifted the
frequency-dependence of cumulative inactivation, induced by short
repetitive depolarizations, toward higher stimulation frequencies (Fig.
6). Only dephosphorylated KV1.4 channels showed an
effective cumulative inactivation at low stimulation frequencies. This
is in agreement with previous results that cumulative inactivation of
Shaker A-type channels is most effective when N-type and
C-type inactivation are both present and strongly coupled (Baukrowitz and Yellen, 1995).
Different localizations of modulatory phosphorylation sites in
Shaker-related A-type channels
It has been proposed from detailed kinetic analysis of
Shaker channel inactivation combined with in
vitro mutagenesis that N-type inactivation follows a "ball and
chain" mechanism in which the ball is tethered to a flexible chain
(Hoshi et al., 1990). Most likely, long-range electrostatic
interactions are essential for the on-rate of inactivation
(Murrell-Lagnado and Aldrich, 1993). They are apparently dominated by
positive charges clustered within the inactivation domain and negative
charges near or at the ball receptor region (Isacoff et al., 1991; Yool
and Schwarz, 1995). Changes in net charge induced by phosphorylation in
the ball region or other channel domains therefore are likely to affect inactivation kinetics. Covarrubias et al. (1994) showed for the cloned
human A-type KV channel, hKV3.4 that
PKC-dependent phosphorylation within the inactivation domain completely
eliminates N-terminal inactivation. Conversely, Drain et al. (1994)
demonstrated for Shaker KV channels that the
phosphorylation of a C-terminal consensus site by PKA induces an
acceleration of the macroscopic and microscopic transition from the
open channel into the inactivated state. Therefore, the effect of
phosphorylation on inactivation kinetics might be critically dependent
on the localization of the phospho-acceptor in the channel protein. The
CaMKII phosphorylation site in KV1.4 is obviously not part
of the inactivation ball or ball receptor region, but rather is
situated in the putative chain domain. It is possible that the density
of negative charges near or at the CaMKII phosphorylation site might
affect the flexibility of the "chain" connecting the inactivation
ball with the core of the protein. Alternatively, phosphorylation at
serine 123 might weaken the electrostatic ball-receptor
interaction.
A role in synaptic frequency detection for CaMKII-dependent
phosphorylation of KV1.4?
It is likely that KV1.4 channels are an in
vivo substrate for CaMKII. In rat brain, KV1.4
channels may contribute, like Shaker channels in Drosophila,
to action potential repolarization (Pardo et al., 1992). They are
predominantly targeted to presynaptic compartments (Sheng et al., 1992,
1993; Veh et al., 1995) where CaMKII (Braun and Schulman, 1995) and
several protein phosphatases like calcineurin and protein phosphatase 1 are also abundant (Liu et al., 1994). The balance between CaMKII and
the calcineurin/inhibitor-1 protein phosphatase cascade activities is
known to regulate a number of pre- and postsynaptic key proteins (for
review, see Schulman, 1995), including AMPA receptors (McGlade-McCulloh
et al., 1993; Mulkey et al., 1994; Wyllie and Nicoll, 1994). It is an
attractive possibility that CaMKII phosphorylation of KV1.4 channels contributes to the described CaMKII-induced shift of frequency-dependent properties of central synapses to higher discharge rates (Bear, 1995; Chapman et al., 1995; Mayford et al., 1995). Although it is not clear how CaMKII is involved in synaptic frequency detection, several studies favor the calmodulin trapping mechanism by
autophosphorylated CaMKII as a good candidate (Meyer et al., 1992;
Dosemeci and Albers, 1996). CaMKII-dependent modulation of
KV1.4 inactivation kinetics might be an important
additional factor. A synapse that makes use of KV1.4
activity, would require higher firing frequencies to drive
CaMKII-phosphorylated KV1.4 channels into cumulative
inactivation and, in turn, to increase the presynaptic action-potential
duration facilitating Ca2+ entry (for review, see Byrne and
Kandel, 1996). Only functional analysis of synapses, however, will
clarify the relevance of CaMKII-dependent regulation of native
fast-inactivating K channels for neural excitability.
FOOTNOTES
Received Aug. 10, 1996; revised Feb. 18, 1997; accepted Feb. 24, 1997.
This work was supported by a Grant of the Deutsche
Forschungs-gemeinschaft (O. P.). We thank D. Clausen for excellent
graphical services, S. Sewing for help with in vitro
mutagenesis, and D. Kuhl and C. Stansfeld for critical reading of the
manuscript.
Correspondence should be addressed to Dr. Olaf Pongs, Zentrum für
Molekulare Neurobiologie, Martinistrasse 52, D-20246 Hamburg, Federal
Republic of Germany.
Dr. Lorra's present address: Institut für Neurobiologie; INF
364, 69120 Heidelberg, Germany.
REFERENCES
-
Baukrowitz T,
Yellen G
(1995)
Modulation of K+ current by frequency and external (K+): a tale of two inactivation mechanisms.
Neuron
15:951-960[Web of Science][Medline].
-
Baukrowitz T,
Yellen G
(1996)
Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel.
Science
271:653-656[Abstract].
-
Bear MF
(1995)
Mechanism for a sliding synaptic modification threshold.
Neuron
15:1-4[Web of Science][Medline].
-
Bliss TVP,
Collingridge GL
(1993)
A synaptic model of memory: long-term potentiation in the hippocampus.
Nature
361:31-39[Medline].
-
Braun AP,
Schulman H
(1995)
The multifunctional calcium/calmodulin dependent protein kinase: from form to function.
Annu Rev Physiol
57:417-445[Web of Science][Medline].
-
Byrne JH,
Kandel ER
(1996)
Presynaptic facilitation revisited: state and time dependence.
J Neurosci
16:425-435[Abstract/Free Full Text].
-
Chandy KG,
Gutman GA
(1994)
Voltage-gated potassium channels.
In: CRC Handbook of receptors and channels (North PA,
ed), pp 1-71. Boca Raton: CRC.
-
Chapman PF,
Frenguelli BG,
Smith A,
Chen CM,
Silva AJ
(1995)
The alpha-Ca2+/calmodulin kinase II: a bidirectional modulator of presynaptic plasticity.
Neuron
14:591-597[Web of Science][Medline].
-
Chen C,
Okayama H,
Cohen PT
(1987)
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol Cell Biol
7:2745-2752[Abstract/Free Full Text].
-
Choi KL,
Aldrich RW,
Yellen G
(1991)
Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels.
Proc Natl Acad Sci USA
88:5092-5095[Abstract/Free Full Text].
-
Cohen P
(1989)
The structure and regulation of protein phosphatases.
Annu Rev Biochem
58:453-508[Web of Science][Medline].
-
Covarrubias M,
Wei A,
Salkoff L,
Vyas TB
(1994)
Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate.
Neuron
13:1403-1412[Web of Science][Medline].
-
Demo SD,
Yellen G
(1991)
The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker.
Neuron
7:743-753[Web of Science][Medline].
-
Dosemeci A,
Albers RW
(1996)
A mechanism for synaptic frequency detection through autophosphorylation of CaM kinase II.
Biophys J
70:2493-2501[Web of Science][Medline].
-
Drain P,
Dubin AE,
Aldrich RW
(1994)
Regulation of Shaker K+ channel inactivation gating by the cAMP-dependent protein kinase.
Neuron
12:1097-1109[Web of Science][Medline].
-
Hamill OP,
Neher ME,
Sakmann B,
Sigworth F
(1981)
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:85-100[Web of Science][Medline].
-
Ho SN,
Hunt HD,
Horton RM,
Pullen JK,
Pease LR
(1989)
Site-directed mutagenesis by overlap extention using the polymerase chain reaction.
Gene
77:51-59[Web of Science][Medline].
-
Holmes TC,
Fadool DA,
Levitan IB
(1996)
Tyrosine phosphorylation of the Kv1.3 potassium channel.
J Neurosci
16:1581-1590[Abstract/Free Full Text].
-
Hoshi T,
Zagotta WN,
Aldrich RW
(1990)
Biophysical and molecular mechanisms of Shaker potassium channel inactivation.
Science
250:533-538[Abstract/Free Full Text].
-
Hoshi T,
Zagotta WN,
Aldrich RW
(1991)
Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region.
Neuron
7:547-556[Web of Science][Medline].
-
Huang X-Y,
Morelli AD,
Peralta EG
(1993)
Tyrosine kinase-dependent suppression of a potassium channel by the G protein-coupled m1 muscarinic receptor.
Cell
75:1145-1156[Web of Science][Medline].
-
Huang X-Y,
Morelli AD,
Peralta EG
(1994)
Molecular basis of a cardiac potassium channel stimulation by protein kinase A.
Proc Natl Acad Sci USA
94:624-628.
-
Ikeda SR,
Soler F,
Zuhlke RD,
Joho RH,
Lewis DL
(1992)
Heterologous expression of the human potassium channel Kv2.1 in clonal mammalian cells by direct cytoplasmic microinjection of cRNA.
Pflügers Arch
422:201-203[Web of Science][Medline].
-
Isacoff EY,
Jan YN,
Jan LY
(1991)
Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel.
Nature
353:86-90[Medline].
-
Jonas EA,
Kaszmarek LK
(1996)
Regulation of potassium channels by protein kinases.
Curr Opin Neurobiol
6:318-323[Web of Science][Medline].
-
Kandel ER,
Schwartz JH,
Jessell TM
(1991)
In: Principles of neural science, 3rd Ed. New York: Elsevier.
-
Karin M,
Haslinger A,
Holtgreve H,
Richards RI,
Krauter P,
Westphal HM,
Beato M
(1984)
Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene.
Nature
308:513-519[Medline].
-
Lev S,
Moreno H,
Martinez R,
Canoll P,
Peles E,
Musacchio JM,
Plowman GD,
Rudy B,
Schlessinger J
(1995)
Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions.
Nature
376:737-745[Medline].
-
Liu J-P,
Sim ATR,
Robinson PJ
(1994)
Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization.
Science
265:970-973[Abstract/Free Full Text].
-
Lopez-Barneo J,
Hoshi T,
Heinemann SH,
Aldrich RW
(1993)
Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels.
Receptors Channels
1:61-71[Web of Science][Medline].
-
Mayford M,
Wang J,
Kandel ER,
O'Dell TJ
(1995)
CaMKII regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP.
Cell
81:891-904[Web of Science][Medline].
-
McGlade-McCulloh E,
Yamamoto H,
Tan S-E,
Brickey DA,
Soderling TR
(1993)
Phosphorylation and regulation of glutamate receptors by calcium/calmodulin-dependent protein kinase II.
Nature
362:640-642[Medline].
-
Meyer T,
Hanson PI,
Stryer L,
Schulman H
(1992)
Calmodulin trapping by calcium-calmodulin dependent protein kinase.
Science
256:1199-1202[Abstract/Free Full Text].
-
Miller SG,
Kennedy MB
(1986)
Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch.
Cell
44:861-870[Web of Science][Medline].
-
Moreno H,
Kentros C,
Bueno E,
Weiser M,
Hernandez A,
Vega-Saenz de Miera E,
Ponce A,
Thornhill W,
Rudy B
(1995)
Thalamocortical projections have a K+ channel that is phosphorylated and modulated by cAMP-dependent protein kinase.
J Neurosci
15:5486-5501[Abstract].
-
Mulkey RM,
Endo S,
Shenolikar S,
Malenka RC
(1994)
Involvement of a calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term depression.
Nature
369:486-488[Medline].
-
Murrell-Lagnado RD,
Aldrich RW
(1993)
Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides.
J Gen Physiol
102:949-975[Abstract/Free Full Text].
-
Pardo LA,
Heinemann SH,
Terlau H,
Ludewig U,
Lorra C,
Pongs O
(1992)
Extracellular K+ specifically modulates a rat brain K+ channel.
Proc Natl Acad Sci USA
89:2466-2470[Abstract/Free Full Text].
-
Rae J,
Cooper K,
Gates P,
Watsky M
(1991)
Low access resistance perforated patch recordings using amphotericin B.
J Neurosci Methods
37:15-26[Web of Science][Medline].
-
Rettig J,
Heinemann SH,
Wunder F,
Lorra C,
Parcej DN,
Dolly JO,
Pongs O
(1994)
Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit.
Nature
369:289-294[Medline].
-
Ruppersberg JP,
Frank R,
Pongs O,
Stocker M
(1991)
Cloned neuronal IK(A) channels reopen during recovery from inactivation.
Nature
353:657-660[Medline].
-
Sanger F,
Nicklen S,
Coulson AR
(1977)
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA
74:5463-5467[Abstract/Free Full Text].
-
Schulman H
(1995)
Protein phosphorylation in neuronal plasticity and gene expression.
Curr Opin Neurobiol
5:375-381[Web of Science][Medline].
-
Shen V,
Pfaffinger P
(1995)
Molecular recognition and assembly seqeunces involved in the subfamily-specific assembly of voltage-gated K+ channel subunit proteins.
Neuron
14:625-633[Web of Science][Medline].
-
Sheng M,
Tsaur ML,
Jan YN,
Jan LY
(1992)
Subcellular segregation of two A-type K+ channel proteins in rat central neurons.
Neuron
9:271-284[Web of Science][Medline].
-
Sheng M,
Liao YJ,
Jan YN,
Jan LY
(1993)
Presynaptic A-current based on heteromultimeric K+ channels detected in vivo.
Nature
365:72-75[Medline].
-
Stühmer W,
Ruppersberg JP,
Schröter KH,
Sakmann B,
Stocker M,
Giese KP,
Perschke A,
Baumann A,
Pongs O
(1989)
Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.
EMBO J
8:3235-3244[Web of Science][Medline].
-
Sumi M,
Kiuchi K,
Ishikawa T,
Ishii A,
Hagiwara M,
Nagatsu T,
Hidaka H
(1991)
The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells.
Biochem Biophys Res Commun
181:968-975[Web of Science][Medline].
-
Veh RW,
Lichtinghagen R,
Sewing S,
Wunder F,
Grumbach IM,
Pongs O
(1995)
Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain.
Eur J Neurosci
7:2189-2205[Web of Science][Medline].
-
Wilson GG,
O'Neill CA,
Sivaprasadarao A,
Findlay JBC,
Wray D
(1994)
Modulation by protein kinase A of a cloned rat brain potassium channel expressed in Xenopus oocytes.
Pflügers Arch
428:186-193[Web of Science][Medline].
-
Wyllie DJA,
Nicoll RA
(1994)
A role for protein kinases and phosphatases in the Ca2+-induced enhancement of hippocampal AMPA receptor-mediated synaptic responses.
Neuron
13:635-643[Web of Science][Medline].
-
Yool AJ,
Schwarz TL
(1995)
Interactions of the H5 pore region and hydroxylamine with N-type inactivation in the Shaker K+ channel.
Biophys J
68:448-458[Web of Science][Medline].
-
Zagotta WN,
Aldrich RW
(1990)
Voltage-dependent gating of Shaker A-type potassium channels in Drosophila muscle.
J Gen Physiol
95:29-60[Abstract/Free Full Text].
-
Zucker RS
(1993)
Calcium and transmitter release.
J Physiol (Paris)
87:25-36[Web of Science][Medline].
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|
|
|
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|
|
|
|
|
|
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|
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|
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|
|
|
|
|
|
|
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[Full Text]
[PDF]
|
|
|
|
|
|
|
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|
|
|
|
|
|
|
S. A. Malin and J. M. Nerbonne
Molecular Heterogeneity of the Voltage-Gated Fast Transient Outward K+ Current, IAf, in Mammalian Neurons
J. Neurosci.,
October 15, 2001;
21(20):
8004 - 8014.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Bahring, L. M Boland, A. Varghese, M. Gebauer, and O. Pongs
Kinetic analysis of open- and closed-state inactivation transitions in human Kv4.2 A-type potassium channels
J. Physiol.,
August 15, 2001;
535(1):
65 - 81.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. A Greenwood, J. Ledoux, and N. Leblanc
Differential regulation of Ca2+-activated Cl- currents in rabbit arterial and portal vein smooth muscle cells by Ca2+-calmodulin-dependent kinase
J. Physiol.,
July 15, 2001;
534(2):
395 - 408.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Rusnak and P. Mertz
Calcineurin: Form and Function
Physiol Rev,
October 1, 2000;
80(4):
1483 - 1521.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. C. Sansom, R. Ma, P. K. Carmines, and D. A. Hall
Regulation of Ca2+-activated K+ channels by multifunctional Ca2+/calmodulin-dependent protein kinase
Am J Physiol Renal Physiol,
August 1, 2000;
279(2):
F283 - F288.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. McAnelly and H. H. Zakon
Coregulation of Voltage-Dependent Kinetics of Na+ and K+ Currents in Electric Organ
J. Neurosci.,
May 1, 2000;
20(9):
3408 - 3414.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Herzig and J. Neumann
Effects of Serine/Threonine Protein Phosphatases on Ion Channels in Excitable Membranes
Physiol Rev,
January 1, 2000;
80(1):
173 - 210.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Tessier, P. Karczewski, E.-G. Krause, Y. Pansard, C. Acar, M. Lang-Lazdunski, J.-J. Mercadier, and S. N. Hatem
Regulation of the Transient Outward K+ Current by Ca2+/Calmodulin-Dependent Protein Kinases II in Human Atrial Myocytes
Circ. Res.,
October 29, 1999;
85(9):
810 - 819.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Zhu, C. H. Gelband, P. Posner, and C. Sumners
Angiotensin II Decreases Neuronal Delayed Rectifier Potassium Current: Role of Calcium/Calmodulin-Dependent Protein Kinase II
J Neurophysiol,
September 1, 1999;
82(3):
1560 - 1568.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Peretz, A. Sobko, and B. Attali
Tyrosine kinases modulate K+ channel gating in mouse Schwann cells
J. Physiol.,
September 1, 1999;
519(2):
373 - 384.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Qian and P. Saggau
Activity-dependent modulation of K+ currents at presynaptic terminals of mammalian central synapses
J. Physiol.,
September 1, 1999;
519(2):
427 - 437.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. D. Koh, B. A Perrino, W. J Hatton, J. L Kenyon, and K. M Sanders
Novel regulation of the A-type K+ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II
J. Physiol.,
May 15, 1999;
517(1):
75 - 84.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. D. Wickenden, T. J. Jegla, R. Kaprielian, and P. H. Backx
Regional contributions of Kv1.4, Kv4.2, and Kv4.3 to transient outward K+ current in rat ventricle
Am J Physiol Heart Circ Physiol,
May 1, 1999;
276(5):
H1599 - H1607.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Leicher, R. Bahring, D. Isbrandt, and O. Pongs
Coexpression of the KCNA3B Gene Product with Kv1.5 Leads to a Novel A-type Potassium Channel
J. Biol. Chem.,
December 25, 1998;
273(52):
35095 - 35101.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Meiri, M.-K. Sun, Z. Segal, and D. L. Alkon
Memory and long-term potentiation (LTP) dissociated: Normal spatial memory despite CA1 LTP elimination with Kv1.4 antisense
PNAS,
December 8, 1998;
95(25):
15037 - 15042.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. J. Berman and L. Maler
Interaction of GABAB-Mediated Inhibition With Voltage-Gated Currents of Pyramidal Cells: Computational Mechanism of a Sensory Searchlight
J Neurophysiol,
December 1, 1998;
80(6):
3197 - 3213.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Peretz, I. Abitbol, A. Sobko, C.-F. Wu, and B. Attali
A Ca2+/Calmodulin-Dependent Protein Kinase Modulates Drosophila Photoreceptor K+ Currents: A Role in Shaping the Photoreceptor Potential
J. Neurosci.,
November 15, 1998;
18(22):
9153 - 9162.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. D. Whim and L. K. Kaczmarek
Heterologous Expression of the Kv3.1 Potassium Channel Eliminates Spike Broadening and the Induction of a Depolarizing Afterpotential in the Peptidergic Bag Cell Neurons
J. Neurosci.,
November 15, 1998;
18(22):
9171 - 9180.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Barros, D. Gomez-Varela, C. G Viloria, T. Palomero, T. Giraldez, and P. de la Pena
Modulation of human erg K+ channel gating by activation of a G protein-coupled receptor and protein kinase C
J. Physiol.,
September 1, 1998;
511(2):
333 - 346.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. L. Rasmusson, M. J. Morales, S. Wang, S. Liu, D. L. Campbell, M. V. Brahmajothi, and H. C. Strauss
Inactivation of Voltage-Gated Cardiac K+ Channels
Circ. Res.,
April 20, 1998;
82(7):
739 - 750.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. C. Cooper, A. Milroy, Y. N. Jan, L. Y. Jan, and D. H. Lowenstein
Presynaptic Localization of Kv1.4-Containing A-Type Potassium Channels Near Excitatory Synapses in the Hippocampus
J. Neurosci.,
February 1, 1998;
18(3):
965 - 974.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Bahring, J. Dannenberg, H. C. Peters, T. Leicher, O. Pongs, and D. Isbrandt
Conserved Kv4 N-terminal Domain Critical for Effects of Kv Channel-interacting Protein 2.2 on Channel Expression and Gating
J. Biol. Chem.,
June 22, 2001;
276(26):
23888 - 23894.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Wang, B. Yang, Y. Zhang, H. Han, J. Wang, H. Shi, and Z. Wang
Different Subtypes of alpha 1-Adrenoceptor Modulate Different K+ Currents via Different Signaling Pathways in Canine Ventricular Myocytes
J. Biol. Chem.,
October 26, 2001;
276(44):
40811 - 40816.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Compound via MeSH
