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The Journal of Neuroscience, January 1, 2003, 23(1):84-95
Subunit-Specific Modulation of KCNQ Potassium Channels by Src
Tyrosine Kinase
Nikita
Gamper,
James D.
Stockand, and
Mark S.
Shapiro
Department of Physiology, University of Texas Health Science Center
at San Antonio, San Antonio, Texas 78229
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ABSTRACT |
We studied regulation by c-Src tyrosine kinase (Src) of
KCNQ1-5 channels heterologously expressed in Chinese hamster ovary (CHO) cells and of native M current in rat sympathetic neurons. Using
whole-cell patch clamp, we found that Src modulates currents from
KCNQ3, KCNQ4, and KCNQ5 homomultimers, KCNQ2/3 heteromultimers and
native M current, but not currents from KCNQ1 or KCNQ2 homomultimers. Src overexpression had two effects: a decrease of current amplitude (4- to 15-fold for cloned channels and ~3-fold for M current) and a
slowing of activation kinetics by 2-fold. Both Src actions were mostly
reversed by bath application of the Src inhibitors erbstatin (20 µM) and PP2 (200 nM), and mimicked by
the tyrosine phosphatase inhibitor sodium vanadate (100 µM). Immunoprecipitation and immunoblot analysis showed
Src-dependent phosphotyrosine signals associated with KCNQ3, KCNQ4, and
KCNQ5 but not with KCNQ1 or KCNQ2 that may be tyrosine phosphorylation
of the channel subunits. Expression of a dominant negative Src that
cannot phosphorylate substrates had no effect on the current and did
not induce phosphotyrosine signals associated with KCNQ3-5 subunits,
further indicating that Src actions on KCNQ currents are mediated by
tyrosine phosphorylation. Immunostaining and confocal analysis showed
no effect of Src overexpression on the abundance of KCNQ3 protein in
CHO cells. Finally, experiments using cloned KCNQ2/3 channels, Src and
M1 muscarinic receptors, and sympathetic neurons
demonstrated that the actions on KCNQ channels by Src and by muscarinic
agonists use distinct mechanisms.
Key words:
tyrosine kinase; Src; K+
channel; patch-clamp; ion channel modulation; M current; KCNQ channel; signaling; muscarinic receptor
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Introduction |
Although identified only recently,
the family of KCNQ K+ channels has
generated great interest because of its clear physiological correlates
to important K+ currents in several types
of tissues and its significance in human disease. The five known
members of the KCNQ family, KCNQ1-5, are widely expressed in neurons,
inner ear, heart, and intestine. KCNQ2, KCNQ3, and KCNQ5 underlie
neuronal M currents (Wang et al., 1998 ; Schroeder et al., 2000; Roche
et al., 2002); KCNQ1 contributes to the cardiac
IKs current (Sanguinetti et
al., 1996) and to a K+ current in
intestine (Warth et al., 2002), and KCNQ1 and KCNQ4 underlie
K+ currents in the inner ear critical to
auditory function (Holt and Corey, 1999; Kubisch et al., 1999).
Mutations within these KCNQ channels produce inherited syndromes of
cardiac arrhythmia, epilepsy, and deafness (Wang et al., 1996; Shalaby
et al., 1997; Biervert et al., 1998; Charlier et al., 1998; Singh et
al., 1998; Coucke et al., 1999; Kharkovets et al., 2000). Thus, the
elucidation of the mechanisms of KCNQ channel regulation is being
vigorously pursued.
Although the motif of regulation of excitable cells via signaling
pathways acting on ion channels has become a theme of physiology (Hille, 2001), KCNQ channels seem to be particularly strong modulatory targets. Several members of the KCNQ family are modulated by their interaction with auxiliary subunits of the KCNE family (Sanguinetti et
al., 1996; Tinel et al., 2000). KCNQ1 and KCNQ2 currents are regulated
by intracellular cAMP (Yang et al., 1997; Schroeder et al., 1998). In
neurons, the M current is so named for its strong modulation by
muscarinic acetylcholine receptors (mAchRs; Brown and Adams, 1980;
Constanti and Brown, 1981). Even after considerable study, the
transduction mechanism linking mAchR stimulation to suppression of the
M current is still unclear.
Intriguingly, the Src family of nonreceptor tyrosine kinases is also
regulated by G-protein pathways (Igishi and Gutkind, 1998; Ma and
Huang, 2002), making the synergy between G-protein- and tyrosine
kinase-mediated signaling particularly interesting. The possibility
that Src could play a role in KCNQ- and M-type channel regulation
seemed attractive, because inhibition of Kv1.2 K+ channels by M1
mAchRs is mediated by tyrosine phosphorylation by Pyk2 kinase
(Huang et al., 1993; Felsch et al., 1998), and Src family kinases
regulate several different K+ channels of
the Kv1 family (Holmes et al., 1996a,b; Fadool et al., 1997; Cayabyab
et al., 2000). In addition, BK-type calcium-activated (Ling et al.,
2000) and HERG K+ channels
(Cayabyab and Schlichter, 2002) have been shown to be modulated by Src.
Here we show that three of five cloned KCNQ channels are modulated by
Src kinase. We demonstrate Src effects in both a heterologous expression system using cloned channels and in primary sympathetic neurons. Our data indicate that the Src-mediated and muscarinic pathways of KCNQ channel modulation are distinct. It is suggested that
modulation of KCNQ channels by Src may be important for control of
neuronal excitability.
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Materials and Methods |
cDNA constructs. Plasmids encoding human KCNQ1, human
KCNQ2, rat KCNQ3, human KCNQ4, and human KCNQ5 (GenBank accession
numbers NM000218, AF110020, AF091247, AF105202, and AF249278, respectively) were kindly given to us by Michael Sanguinetti
(University of Utah, Salt Lake City, UT; KCNQ1), David McKinnon (State
University of New York, Stony Brook, NY; KCNQ2 and KCNQ3), Thomas
Jentsch (Zentrum für Molekulare Neurobiologie, Hamburg, Germany;
KCNQ4), and Klaus Steinmeyer (Aventis Pharma, Frankfurt am Main,
Germany; KCNQ5). A plasmid containing mouse M1
receptor cDNA was given to as by Neil Nathanson (University of
Washington, Seattle, WA). The proto-oncogene c-Src (Src) was previously
cloned from rat testis (GenBank accession number AF130457; Al-Khalili
et al., 2001). K298M mutant Src (kinase-dead Src) was generated by
using the Quikchange mutagenesis kit (Stratagene, La Jolla, CA)
according to the instructions of the manufacturer. KCNQ1 was subcloned
into pCEP4 (Invitrogen, San Diego, CA) using HindIII and
XbaI. KCNQ2 and KCNQ3 were subcloned into pcDNA3
(Invitrogen) as described previously (Shapiro et al., 2000). KCNQ4 and
KCNQ5 were subcloned into pcDNA3.1zeo+ and pcDNA3.1zeo (Invitrogen)
using XhoI-HindIII and
XbaI-EcoRI, respectively. Myc-tagged KCNQ2-5
were generated by subcloning each channel in-frame into
cytomegalovirus-myc plasmid (Clontech, Palo Alto, CA) behind the
myc epitope. Rat wild-type and K298M Src were subcloned into
pcDNA3.1zeo using EcoRI.
Cell culture and transfections. Chinese hamster ovary (CHO)
cells were a kind gift of Feng Liu (Department of Pharmacology, University of Texas Health Science Center at San Antonio). Cells were
grown in 100 mm tissue culture dishes (Falcon; Becton Dickinson, Mountain View, CA) in DMEM with 10% heat-inactivated fetal bovine serum and 0.1% penicillin and streptomycin in a humidified incubator at 37°C (5% CO2) and passaged every 3-4 d.
Cells were discarded after ~30 passages. For transfection, cells were
plated onto poly-L-lysine-coated coverslip chips and
transfected 24 hr later with Polyfect reagent (Qiagen, Hilden, Germany)
according to the instructions of the manufacturer. For
electrophysiological and biochemical experiments, cells were used
48-96 hr after transfection. As a marker for successfully transfected
cells, cDNA encoding green fluorescent protein (GFP) was cotransfected
together with the cDNAs of the genes of interest. We found that >95%
of green-fluorescing cells expressed KCNQ currents in control experiments.
Superior cervical ganglia sympathetic neuron culture and
transduction. Sympathetic neurons were isolated from the superior cervical ganglia (SCG) of 2- to 6-week-old male rats (Sprague Dawley)
and cultured for 2-4 d. Rats were anesthetized with halothane and
decapitated. Neurons were dissociated using methods of Bernheim et al.
(1991), plated on 4 × 4 mm glass coverslips (coated with poly-L-lysine), and incubated at 37°C (5%
CO2). Fresh culture medium containing nerve
growth factor (50 ng/ml) was added to the cells 3 hr after plating. For
exogenous expression of Src in SCG neurons, we used the Sindbis
 -viral expression system (Invitrogen). To construct the appropriate
vectors, Src cDNA was subcloned into the multiple cloning site of
pIRES2-enhanced GFP (EGFP; Clontech) using XhoI and
BamHI and nonmethylated DNA extracted from SCS110
Escherichia coli, (Stratagene). The Src-IRES-EGFP coding
region flanked by XbaI-XbaI was then subcloned
into the pSinRep5 vector (Invitrogen) using NheI. Proper
directional cloning was verified with an XhoI digest.
Pseudovirions were generated in baby hamster kidney cells according to
the Sindbis expression system manual using constructed vector and
DH(26S) helper RNAs (mMessage mMachine; Ambion). Infection of cells
with these pseudovirions leads to expression of Src and EGFP as
separate proteins from a common promoter, allowing us to identify
transduced cells with EGFP fluorescence. Recordings from transduced
cells were made between 12 and 18 hr after exposure to pseudovirions.
Electrophysiology. The whole-cell configuration of the
patch-clamp technique was used to voltage clamp and dialyze cells at room temperature (22-25°C). Pipettes were pulled from borosilicate glass capillaries (1B150F-4; World Precision Instruments) using a
Flaming-Brown P-97 micropipette puller (Sutter Instruments, Novato, CA)
and had resistances of 2-3 M when filled with internal solution and
measured in Ringer's solution. Membrane current was measured under
whole-cell clamp with pipette and membrane capacitance cancellation,
sampled at 5 msec, and filtered at 200 Hz by an EPC-9 amplifier
(HEKA, Lambrecht, Germany). Data acquisition and analysis were
performed by Pulse software (HEKA) and ITC-16 Interface (Instrutech,
Port Washington, NY). The whole-cell access resistance was typically
5-10 M. Cells were placed in a 500 µl perfusion chamber through
which solution flowed at 1-2 ml/min. Inflow to the chamber was by
gravity from several reservoirs, selectable by activation of solenoid
valves (VaveLink 8; Automate Scientific, Inc.). Bath solution exchange
was complete by <30 sec. To observe GFP fluorescence, the polychrome
IV monochromater (TILL Photonics, Martinsreid, Germany) was used
in combination with an Eclipse TE300 inverted microscope (Nikon,
Melville, NY).
Several voltage protocols were used to study KCNQ current in CHO cells.
To evaluate the kinetics of current activation and deactivation as well
as voltage dependence, CHO cells were held at -60 mV, and a family of
800 msec test voltage pulses were applied starting from -80 to 40 mV
in 10 mV increments every 3 sec. Each test pulse was followed by a 500 msec step to -60 mV, and tail currents were fit by exponential
functions using PulseFit software. In experiments with oxotremorine
(oxo-M) and in some erbstatin, PP2, and PP3 experiments, cells
were held at 0 mV, and 500 msec hyperpolarizing steps to -60 mV,
followed by 650 msec pulses back to 0 mV, were applied every 3 sec. The
amplitude of the current in CHO cells was usually defined as the
maximal outward current at a given depolarizing potential. In some
experiments, XE991 (50 µM), a selective blocker of
KCNQ channels, was used to verify current identity. CHO cells have
negligible endogenous macroscopic K+
currents under our experimental conditions, and 50 µM
XE991 completely blocked K+ current in
KCNQ-transfected CHO cells, having no effect on nontransfected ones.
Cells exhibited variable "run-down" in the amplitude of KCNQ
currents and usually stabilized within several minutes of whole-cell
dialysis. Cells in which the rundown exceeded 3%/min were
discarded. In all experiments with pipette solutions containing 20 mM 1,2-bis(2-aminophenoxy)ethane
N,N,N',N'-tetraacetic acid (BAPTA), we waited at least 5 min
after whole-cell formation before starting the experiment to allow for
dialysis of BAPTA and other ingredients into the cell. M currents in
SCG cells were studied by holding the membrane potential at 25 mV and
applying a 500 msec hyperpolarizing pulse to 60 mV every 3 sec. The M
current amplitude was measured at 60 mV from the decaying time course of deactivating current as the difference between the average of a 10 msec segment, taken 20-30 msec into the hyperpolarizing step, and the
average during the last 50 msec of that step. All results are reported
as mean ± SEM. Time constants for current activation and
deactivation were calculated by fitting individual current traces by
monoexponential functions using PulseFit software. Channel voltage
dependence was evaluated by fitting the individual activation curves to
a Boltzmann equation:
I/Imax = Imax/{1 exp[(V1/2 V)/k]}, where
V1/2 is the voltage that produces
half-maximal activation of the conductance, and k is the
slope factor.
Immunoprecipitations and immunoblotting. Cells were grown in
100 mm culture dishes and transfected with KCNQ1 and GFP or myc-tagged KCNQ2-5 and GFP. After 48 hr, cells were harvested with a rubber policeman in radioimmunoprecipitation assay (RIPA) lysis buffer (10 mM NaPO4, 150 mM NaCl,
1% Na deoxycholate, 1% Triton X-100, and 0.1% SDS) plus a mixture of
protease (1 µM
N--p-tosyl-L-lysine chloromethyl ketone, 1 µM
N-tosyl-L-phenylalanine chloromethyl ketone, 1 µM 4-(2-aminoethyl)-benzene
sulfonylfluoride HCl, 1 µM E-64, 1 µg/ml leupeptin, and 1 µM pepstatin; all from
Sigma, St. Louis, MO) and tyrosine phosphatase (in
µM: 100 ZnCl2, 100 Na2MoO4, 500 NaF, 100 Na
pyrophosphate, and 40 Na3VO4; all from Sigma)
inhibitors, and lysate proteins were quantified with a BCA assay
(Pierce, Rockford, IL). Proteins (400 µg/reaction) were immunoprecipitated overnight at 4°C using 2 µg of
anti-phosphotyrosine antibodies (Upstate Biotechnology, Lake Placid,
NY) and 40 µl of protein A/G beads (Santa Cruz Biotechnology, Santa
Cruz, CA). Immunoprecipitated proteins bound to pelleted protein A/G
beads were washed thoroughly in RIPA buffer, denatured in Laemmli
sample buffer, separated using SDS-PAGE, and electroblotted onto
nitrocellulose membranes. Immunoblots were probed with mouse anti-myc
(KCNQ2-5; Clontech) or anti-KCNQ1 (Santa Cruz Biotechnology) primary
antibodies (1:1000 dilution, overnight at 4°C) in a blocking solution
containing 5% nonfat dry milk (Carnation) in TBS and Tween 20 and
subsequently treated with goat anti-mouse horseradish
peroxidase-conjugated secondary antibodies (1:25,000 dilution, 45 min,
room temperature; Jackson ImmunoResearch, West Grove, PA). Blots were
developed with enhanced chemiluminescence (Supersignal; Pierce) and
exposed on x-ray film (Biomax).
Immunostaining and confocal analysis. Cells were transfected
with myc-tagged KCNQ3 and GFP or myc-tagged KCNQ3, Src, and GFP, grown
on poly-L-lysine-coated coverslips, fixed in 4%
paraformaldehyde, washed twice with 100 mM sodium phosphate
buffer (PB), pH 7.4, and three times with PBS, and blocked with 5%
goat serum and 0.1% saponin in PBS (PBS + GS). The cells were
incubated for 3 hr at room temperature with primary anti-myc antibody
(Clontech) diluted 1:1000 in PBS+GS. Cells were washed six times with
PBS and then incubated with goat rhodamine red-conjugated anti-mouse
secondary antibody (1:150; Jackson ImmunoResearch) in PBS + GS for 1 hr. Cells were then washed three times with PBS, twice with PB, and three times with water. Air-dried slides were mounted on a drop of
Vectashield (Vector Laboratories, Burlingame, CA) and sealed with nail
polish. Stained cells were viewed with an Olympus Optical (Tokyo,
Japan) FV-500 confocal microscope in the Optical Imaging Core Facility
at the University of Texas Health Science Center using the lasers and
excitation and emission filters appropriate for GFP and rhodamine red.
Because GFP was used as a reporter for successful transfection, images
were collected in "sequential" mode to avoid bleed-through of the
GFP (green) and rhodamine (red) signals. Single images were collected
3-5 µm above the surface of the coverslip. The fluorescence
intensity was quantified using TotalLab software (Nonlinear Dynamics,
Newcastle, UK).
Solutions and materials. The external solution used to
record KCNQ currents in CHO cells contained (in mM): 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4 with NaOH. The
regular pipette solution contained (in mM): 160 KCl, 5 MgCl2, 5 HEPES, 0.1 BAPTA, 3 K2ATP, and 0.1 NaGTP, pH 7.4 with KOH. In
oxotremorine experiments, a Ca2+-clamping
mixture was used, which contained (in mM): 20 BAPTA, 10 CaCl2, 110 KCl, 5 MgCl2, 5 HEPES, 3 K2ATP, and 0.1 NaGTP and 100 µg/ml
pentosan polysulfate, pH 7.4 with KOH. Reagents were obtained as
follows: oxotremorine methiodide, Research Biochemicals (Natick, MA);
BAPTA, Molecular Probes (Eugene, OR); DMEM, fetal bovine serum, nerve
growth factor, penicillin, and streptomycin, Invitrogen; ATP, GTP,
pentosan polysulfate, and sodium orthovanadate, Sigma; erbstatin, PP2,
and PP3 (Calbiochem); and XE991, a kind gift from Michael E. Schnee
(DuPont Pharmaceuticals, Billerica, MA).
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Results |
c-Src suppresses current amplitudes and slows activation of cloned
KCNQ2/3 heteromultimers
We first studied the effect of rat c-Src (Src) on cloned KCNQ2/3
K+ channels using a heterologous
expression system. CHO cells were cotransfected with cDNA coding for
KCNQ2 and KCNQ3 subunits with or without the cDNA for Src. Previous
work has shown that coexpression of KCNQ2 and KCNQ3 recapitulates
heteromeric KCNQ2/3 channels with the biophysical, pharmacological, and
modulatory properties of the M current of sympathetic neurons (Wang et
al., 1998; Selyanko et al., 2000; Shapiro et al., 2000). We used
coexpression of GFP as a reporter for successful transfection, and only
cells that fluoresced green were chosen for study using whole-cell
clamp. CHO cells transfected with KCNQ2 and KCNQ3 expressed
voltage-gated K+ currents with slow
activation kinetics typical of KCNQ channels (Fig.
1A), whereas
nontransfected CHO cells had negligible macroscopic K+ currents (data not shown).
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Figure 1.
Src suppresses the amplitude and slows activation
of currents from KCNQ2/3 heteromultimers expressed in CHO cells.
A, Superimposed current traces recorded from a cell
cotransfected with KCNQ2 and KCNQ3 (Control) or a
cell cotransfected with KCNQ2, KCNQ3, and rat c-Src
(Src). Currents were evoked by a family of 500 msec
voltage pulses from 80 to 40 mV in 10 mV increments from a holding
potential of -60 mV. B, Superimposed current traces at
0 mV from the current families similar to those shown in
A, shown at an expanded scale. The trace
from the cell cotransfected with Src is also shown
scaled up (gray) to match the control trace to
more clearly show the effect of Src on activation kinetics.
C, Summarized data for the current density
(left) and activation  act
(right) for the currents recorded at 0 mV (as in
B), from control (n = 45) and Src
cotransfected (n = 42) cells.
*p  0.05; **p 0.01;
***p 0.001 in this and subsequent figures.
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We compared the properties of KCNQ2/3 currents in cells only
transfected with the channels to cells also transfected with Src.
Immunoblot analysis showed that Src-transfected CHO cells highly
express Src protein and that non-Src-transfected cells contain a modest
level of endogenous Src (data not shown). Src cotransfection had two
main actions: (1) a strong suppression of current amplitudes and (2) a
slowing of activation kinetics. Shown in Figure 1A
are families of currents from a cell without (left) and with
(right) Src cotransfection. The Src-transfected cell
displayed profoundly reduced KCNQ2/3 current amplitudes at all voltages
tested, relative to the control cell. Figure 1B shows that Src overexpression also slowed activation kinetics. Shown are
current traces evoked by 800 msec voltage pulse from 60 to 0 mV. The current trace from the Src-transfected cell is
also shown scaled up to match the trace from the control cell, clearly showing that the activation kinetics in this cell are slower than in
control. Src overexpression also induced modest acceleration of current
deactivation (estimated using tail currents similar to that shown in
Fig. 1B). Such data are summarized in Figure 1C and Table 1. Coexpression
of Src with KCNQ2/3 resulted in a 4.5-fold reduction of current density
and a 2-fold slowing of activation kinetics at 0 mV.
c-Src actions are reversed by tyrosine kinase inhibitors and
mimicked by a tyrosine phosphatase inhibitor
To investigate the mechanism by which Src acts on KCNQ2/3
channels, we first asked whether pharmacological inhibitors of tyrosine kinases could acutely reverse Src actions. The broad-spectrum tyrosine
kinase inhibitor erbstatin and the Src family-specific inhibitor PP2
were used in patch-clamp experiments on CHO cells expressing KCNQ2/3
channels together with Src (Fig.
2A-C). Families of
KCNQ2/3 currents were obtained before and 15 min after addition of
drugs to the bathing solution. Application of 20 µM erbstatin to the bath increased current
amplitudes and accelerated activation kinetics from a CHO cell
transfected with KCNQ2/3 and Src (Fig. 2A).
The effect reached its maximum within 15 min of erbstatin application. Figure 2B shows experiments in which the
specific Src inhibitor PP2 or its inactive analog PP3 (both at 200 nM) were used. In 5 of 6 experiments, PP2,
similar to erbstatin, increased current amplitude and accelerated
activation kinetics. In contrast, application of PP3 had no effect
(n = 6). These data are summarized in Figure
2C. Erbstatin increased current amplitudes at 0 mV by 2.3 ± 0.2-fold (p 0.002;
n = 7) and the activation time constants (act) at 0 mV were decreased to 46.6 ± 4.9% (p 0.001; n = 7) of
their initial values.
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Figure 2.
Src inhibitors reverse Src effects on KCNQ2/3
currents. A, Currents recorded from a cell cotransfected
with KCNQ2, KCNQ3, and Src as in Figure 1 before and after (15 min)
erbstatin application. B, Experiment similar to that in
A, but the specific Src inhibitor PP2 (200 nM; top panel) or its inactive
analog, PP3 (200 nM; bottom panel)
was applied instead of erbstatin. C, Summary of the
erbstatin, PP2, and PP3 actions on current amplitude
(left) and act (right),
expressed as a percentage of the initial values (before inhibitor
application). D, Current ampli tudes at 0 mV during voltage pulses given every 3 sec
consisting of a 500 msec step to -60 mV, followed by a 650 msec step
back to the holding potential of 0 mV, in a cell cotransfected with
KCNQ2 and KCNQ3 but not Src. Erbstatin and XE991 (50 µM)
were applied during the times indicated by the bars.
Inset, Currents at the indicated times during the
experiment. The dotted line indicates the zero current
level.
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To test the effect of Src inhibitors on KCNQ2/3 currents in cells not
overexpressing Src, we repeated the experiments shown in Figure
2A-C in non-Src-overexpressing CHO cells. We found
that both erbstatin and PP2 induced small but reliable "run-up" of the KCNQ2/3 current within 10-15 min of perfusion with erbstatin (15 ± 7%, four of five cells) or PP2 (13 ± 6%, four of
five cells; p 0.05; paired Student's t
test), a time course similar to that found in experiments with
Src-overexpressing cells. One such experiment is shown in Figure
2D. However, PP3 did not increase the current (n = 3). These data are consistent with modest
endogenous Src-like activity in CHO cells.
Phosphorylation states represent a balance between the activities of
protein kinases and phosphatases. Indeed, we expect that the effects of
Src in CHO cells shown in Figure 1 are attributable to tonic Src
activity that is greatly augmented by overexpressing Src, resulting in
the balance between phosphorylation and dephosphorylation being biased
toward the former. Thus, we tested whether the effects of Src
overexpression would be mimicked by treatment of non-Src-overexpressing cells with sodium vanadate, an inhibitor of protein tyrosine
phosphatases (Fig. 3). We first compared
CHO cells transfected with KCNQ2/3, but not Src, which had or had not
been preincubated with vanadate (100 µM) in the culture
medium for 1 hr. Figure 3, A and B, shows representative experiments. In the vanadate-treated cell, the current
amplitude was profoundly suppressed, and activation kinetics was
slowed, mimicking the effect of Src overexpression. Such data are
summarized in Figure 3C. Measured at 0 mV, the current
density and act in control cells (measured
during the same days as the vanadate experiments) were 89.9 ± 27.8 pA/pF (n = 9) and 195 ± 17 msec
(n = 9), respectively, but in vanadate-treated cells
they were 16.8 ± 5.1 pA/pF (n = 9;
p 0.01) and 418 ± 55 msec (n = 9; p 0.001).
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Figure 3.
A tyrosine phosphatase inhibitor mimics Src
actions. A, Current traces recorded from a cell
cotransfected with KCNQ2 and KCNQ3 (Control) and
from a similar cell preincubated for 1 hr with 100 µM
sodium vanadate (Vanadate). Currents were recorded as in
Figure 1A. B, Superimposed currents at 0 mV similar to
that shown in A are shown at an expanded scale. The
trace from the cell incubated in Vanadate
is also shown scaled up (gray) to match the
Control trace to more clearly show the effect of
vanadate treatment on activation kinetics. C, Summarized
data for the current density (left) and
act (right) for the currents recorded at
0 mV (as in B) from control (n = 9)
and vanadate-treated (n = 9) cells.
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The decrease of KCNQ2/3 current density and slowing of the current
activation in Src-cotransfected cells was accompanied by a shift of
channel voltage dependence toward more depolarizing potentials (Fig.
4A). We estimated the
voltage dependence of activation of KCNQ2/3 heteromultimers using tail
current amplitudes recorded at -60 mV after a family of prepulses from
-80 to 40 mV. The half-maximal voltages of current activation
(V1/2) in cells transfected with KCNQ2/3 alone
and those transfected with KCNQ2/3 together with Src were -21.1 ± 1.5 mV (n = 19) and -11.8 ± 2.6 mV
(p 0.01; n = 19)
respectively. Erbstatin completely reversed this effect (Fig.
4B). The V1/2 of KCNQ2/3
channels in Src-transfected cells after erbstatin application was
-23.6 ± 4.9 mV (p 0.05;
n = 5). In contrast, vanadate treatment mimicked the
effect of Src on the V1/2 of current activation
(Fig. 4C), shifting the V1/2 of cells
transfected with KCNQ2/3 channels (but not Src) to -8.0 ± 3.8 mV
(p 0.001; n = 6). Because the
voltage dependence of the activation of the conductance of
voltage-gated channels is the sum effect of the rates of activation and
deactivation, the shift of the voltage dependence induced by Src is
biophysically consistent with (and expected from) a slowing of
act. It should be pointed out that the effect
of Src on current amplitude (greater than fourfold for KCNQ2/3
heteromultimers) is much more profound than would be expected from just
a 10 mV shift in voltage dependence.
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Figure 4.
Src overexpression shifts the voltage dependence
of KCNQ2/3 currents. Shown are activation curves generated from
normalized tail currents
(I/Imax) recorded by
500 msec voltage steps to 60 mV after a family of test potentials
from 80 to 40 mV in 10 mV increments. A, Activation
curves for cells transfected with KCNQ2 and KCNQ3 only
(Control, filled circles;
n = 19) or for those also cotransfected with Src
(Src, open circles; n = 19). B, Activation curves for cells transfected with
KCNQ2, KCNQ3, and Src before (Src, open
circles; n = 19) and 15 min after
application of 20 µM erbstatin
(Src+Erbstatin, filled triangles;
n = 5). C, Activation curves for
cells transfected with KCNQ2 and KCNQ3 either without
(Control, filled circles;
n = 19) or after 1 hr preincubation with 100 µM sodium vanadate (Vanadate, open
triangles; n = 6). The
curves shown in A-C were fit to
Boltzmann equations (see Materials and Methods), and the parameters for
each fit are stated in the text.
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c-Src suppresses the M current in SCG neurons
We then tested whether Src overexpression in rat sympathetic
neurons would have an effect on endogenous M current similar to that on
currents from cloned KCNQ2/3 channels. Dissociated neurons from rat SCG
were cultured overnight and exogenously expressed with Src and EGFP or
just EGFP as a control, using the Sindbis -viral expression system
(see Materials and Methods). Figure 5A shows transmitted light
(left) and fluorescent (right;
 exit = 470 nm) micrographs of a successfully
transduced SCG cell. We compared the M current density in
Src-transduced and control neurons, quantified as the amplitude per
picofarad of the time-dependent deactivating current at -60 mV, using
a classical M current voltage protocol. Figure 5B shows M
current traces from a cell transduced with Src and EGFP or only EGFP
(Control). The M current amplitude in the
Src-transduced neuron is much smaller than in the control neuron. Such
data are summarized in Figure 5B (right). The M
current density was reduced from 1.8 ± 0.1 pA/pF
(n = 7) in cells expressing EGFP alone to 0.6 ± 0.2 pA/pF (p 0.001; n = 8) in
cells expressing EGFP together with Src. Thus, Src suppresses native M
currents in neurons as well as from heterologously expressed KCNQ2/3
channels. Although we qualitatively observed slowing of M current
activation kinetics by Src overexpression, we could not quantify this
effect because of interference from other endogenous SCG
K+ currents.
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Figure 5.
Src suppresses M current in cultured SCG neurons.
A, Transmitted light (left) and
fluorescent (right; excitation = 470 nm)
micrographs of a cultured rat SCG neuron, successfully transduced by
Sindbis pseudovirions containing Src and EGFP constructs in a
bicistronic expression vector. The arrow indicates a
typical neuron chosen for study. B, Left,
Superimposed current traces recorded from an SCG neuron transduced with
only EGFP (black trace, Control)
and from a cell transduced with EGFP together with Src
(gray trace, Src). Currents were
evoked by a 500 msec voltage pulse to -60 mV, followed by a 650 msec
pulse back to the holding potential of -25 mV. The dotted
line indicates the zero current level. M current was quantified
as the amplitude of the time-dependent deactivating relaxation at -60
mV. Right, Summarized data for current density, recorded
as on the left, from the control
(n = 7) and Src cotransduced (n = 8) neurons. C, Effect of sodium vanadate on M current
in SCG neurons. Left, Currents recorded as in
B from a control neuron (black trace,
Control) and from a neuron preincubated for 1 hr
with 100 µM sodium vanadate (gray
trace, Vanadate). Right,
Summarized data for the current density, recorded as on the
left from the control (n = 15) and
vanadate-treated (n = 13) neurons.
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We then investigated the effect of vanadate on the M current in
nontransduced neurons. Similar to KCNQ2/3 currents in CHO cells,
pretreatment of SCG cells with 100 µM vanadate for 1 hr suppressed the M current density (Fig. 5C). The effect is
summarized in Figure 5C (right). M-current
density was reduced from 0.9 ± 0.1 pA/pF (n = 15)
in control cells to 0.4 ± 0.1 pA/pF in vanadate-treated cells
(p 0.05; n = 13). Thus, for
both cloned KCNQ2/3 heteromultimers in CHO cells and native M current
in SCG neurons, blockade of tyrosine phosphatases mimics Src
overexpression, suggesting that Src acts by phosphorylating target proteins.
A kinase-dead c-Src is without effect
To further test whether the effects of Src are dependent on
phosphorylation, we compared the effect of overexpression of wild-type Src on KCNQ2/3 heteromultimers with that of a kinase-dead mutant Src that has a point mutation in the ATP binding site (K298M), which
completely abolishes kinase activity (Miller et al., 2000). The results
of these experiments are shown in Figure
6. Immunoblots with an anti-Src antibody
showed similar levels of expression of wild-type and K298M Src (Fig. 6,
inset). Cotransfection of K298M Src had little effect on
current amplitudes or on activation kinetics of the KCNQ2/3 current
(Fig. 6A,B). The current density and
act at 0 mV in cells cotransfected with
KCNQ2/3 and K298M Src were 50.6 ± 8.8 pA/pF (n = 8) and 141 ± 18 msec (n = 8). In control cells
transfected only with KCNQ2/3 (same days as when experiments with K298M
Src were performed), the current density and
act at 0 mV were 61.1 ± 18.7 pA/pF
(n = 8) and 140 ± 12 msec (n = 8). These values in both control and K298M Src-cotransfected cells were
also not significantly different from the pooled values of control
cells (Student's t test; p 0.05; Table
1).
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Figure 6.
A kinase-dead mutant Src does not affect the
KCNQ2/3 current. A, Currents recorded from a cell
transfected with KCNQ2 and KCNQ3 alone (left,
Control) or together with kinase-dead (K298M) Src
(right, K298MSrc). Currents were recorded
as in Figure 1A. Inset,
Immunoblots from lysates of cells transfected with wild-type or K298M
Src. Lysate proteins were separated by SDS-PAGE and transferred to
nitrocellulose, and the immunoblot was probed with anti-Src antibodies.
C, Summarized data of the current density
(left) and act (right) at
0 mV for control (n = 8) and K298M
Src-cotransfected (hatched columns;
n = 7) cells.
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Subunit specificity of c-Src action
We investigated the sensitivity of the five different KCNQ
channels to modulation by Src. All five cloned channels of the KCNQ
family (KCNQ1-5) were individually expressed in CHO cells with or
without Src. The data from these experiments are summarized in Table 1
and Figure 7. Cotransfection of Src with
KCNQ1 or KCNQ2 had no effect on the current density or on activation
kinetics of the currents. In contrast, current amplitudes from KCNQ3,
KCNQ4, and KCNQ5 homomultimers were dramatically reduced by Src
cotransfection (Fig. 7A,B, Table 1). The largest effect of
Src was observed for KCNQ3, for which the current density at 0 mV was
15 times lower in Src-overexpressing cells compared with cells
expressing KCNQ3 alone. The activation time constants of KCNQ3 currents
were also twofold larger in Src-cotransfected cells. In cells
transfected with KCNQ4 or KCNQ5, cotransfection of Src decreased the
current density at 0 mV by 4.0- and 5.5-fold and increased
act by 2.3- and 2.1-fold, respectively. These
data are summarized in Figure 7B and Table 1. Among
KCNQ1-5, Src acts on KCNQ3, KCNQ4, and KCNQ5 but spares KCNQ1 and
KCNQ2. The lack of effect of Src on KCNQ2 homomultimers suggests that
its actions on KCNQ2/3 heteromultimers and on SCG M current localize to
the KCNQ3 subunits in the tetrameric channel.
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Figure 7.
Src action is subunit-specific. A,
Current traces recorded as in Figure 1A from
cells individually transfected with KCNQ1-5 (as indicated) without
(left traces) or together with wild-type Src
(right traces). B, Mean suppression of
KCNQ current density at 0 mV by Src cotransfection expressed as a
percentage of the appropriate control current
[(ISrc/Icontrol) × 100%]. The data for pooled KCNQ2/3 are also shown for comparison.
The number of experiments for each condition is given in Table 1.
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Modulation of KCNQ channels by c-Src is via
tyrosine phosphorylation
The experiments with tyrosine kinase and phosphatase inhibitors
and kinase-dead Src strongly suggested that the effect of Src on KCNQ
channels involves tyrosine phosphorylation. To test whether the five
different KCNQ channels are associated with tyrosine phosphorylation by
Src, we used a strategy involving immunoprecipitations, followed by
immunoblotting. KCNQ2-5 subunits were epitope-tagged by introduction
of the myc epitope to their N termini (see Materials and Methods) and
individually expressed in CHO cells. Current properties of KCNQ2-5
channels were not affected by introduction of the myc epitope (data not
shown). We were not able to construct myc-tagged KCNQ1 and so used the
wild-type channel. In the immunoprecipitation and immunoblot
experiments presented next, we used myc-tagged KCNQ2-5 channels in
combination with anti-myc antibodies and wild-type KCNQ1 with an
anti-KCNQ1 antibody. In immunoblots prepared from whole-cell lysates of
CHO cells individually transfected with the five different KCNQ
channels, these antibodies specifically labeled KCNQ1-5 at their
appropriate molecular weights of ~60 kDa for KCNQ1, 100 kDa for
KCNQ2, 110 kDa for KCNQ3, 80 kDa for KCNQ4, and 125 kDa for KCNQ5 (Fig.
8A).
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Figure 8.
KCNQ3, KCNQ4, and KCNQ5 but not KCNQ1 or KCNQ2
display tyrosine phosphorylation in the presence of Src.
A, Immunoblot detection of myc-tagged KCNQ2, KCNQ3,
KCNQ4, and KCNQ5 and wild-type KCNQ1 proteins in CHO cells. Lysate
proteins were separated by SDS-PAGE, transferred to nitrocellulose, and
probed with anti-myc or anti-KCNQ1 antibodies. B, Top
panel, Immunoprecipitation and detection by immunoblots of
phosphotyrosine signals associated with KCNQ proteins. CHO cells were
transfected with myc-tagged KCNQ2-5 or wild type KCNQ1 with or without
(as indicated in the table at top)
wild-type or K298M Src. Lysate proteins were immunoprecipitated with
anti-phosphotyrosine antibodies; the immunoprecipitates were run as
Western blot gels; and the resulting immunoblots were probed with
anti-myc or anti-KCNQ1 antibodies. The bands for each
channel with or without Src are taken from the same original film.
Bottom panel, Immunoblots with anti-myc (or anti-KCNQ1)
antibodies from the same lysates as in the top panel
before immunoprecipitation.
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CHO cells were cotransfected with myc-tagged KCNQ2-5 or with
wild-type KCNQ1 channels with or without wild-type or K298M Src. Tyrosine-phosphorylated proteins were then immunoprecipitated with
anti-phosphotyrosine antibodies; the immunoprecipitates were separated
using SDS-PAGE; and the immunoblots were probed with anti-myc
(or anti-KCNQ1) antibodies. Consistent with the patch-clamp data
(Fig. 7), neither KCNQ1 nor myc-tagged KCNQ2 was precipitated by the
phosphotyrosine antibodies from control cells or from cells overexpressing Src. In contrast, proteins of the appropriate molecular weight for KCNQ3-5 were immunoprecipitated from cells expressing myc-tagged KCNQ3 and Src, myc-tagged KCNQ4 and Src, and myc-tagged KCNQ5 and Src (Fig. 8B, top panel),
suggesting phosphorylation associated with these channels by Src
activity. KCNQ4 showed a phosphotyrosine signal even without Src
cotransfection, consistent with the ability of vanadate to mimic Src
overexpression (Fig. 3), with the detection of modest endogenous Src
using immunoblots from non-Src-transfected CHO cells and with the
modest run-up of KCNQ2/3 currents in non-Src-transfected
cells (Fig. 2D). No subunit-specific phosphotyrosine
signal was detected when KCNQ3, KCNQ4, or KCNQ5 was coexpressed with
kinase-dead K298M Src. Figure 8B, bottom
panel, shows immunoblots performed using the same whole-cell lysates as those used in the top panel but without any
immunoprecipitation. Proteins specific for KCNQ1-5 were always
strongly detected in such experiments. The experiments shown in Figure
8 were reproduced three to five times for each individual KCNQ subunit.
Thus, we detect a phosphotyrosine signal associated with KCNQ3, KCNQ4, and KCNQ5 but not KCNQ1 and KCNQ2. The phosphorylation is more profound
if Src is overexpressed in the cells and wholly absent if a kinase-dead
Src is overexpressed instead, strongly suggesting that the tyrosine
phosphorylation observed is Src-dependent.
The suppression of KCNQ current amplitude by Src overexpression
described in this work could be attributable to a reduction of channel
activity (i.e., open probability) or a reduction in channel number.
Although the rapid time course of the reversal of the current
suppression by Src inhibitors suggests the former, we tested the
possibility that Src suppresses KCNQ currents by altering channel
expression. We used immunofluorescence to measure the expression of a
myc-tagged KCNQ3 protein in CHO cells with or without Src
cotransfection. Cells were immunostained with anti-myc antibodies (see
Materials and Methods), and confocal images were taken. Densitometry of
the confocal micrographs was used to semiquantitatively evaluate
channel abundance. Shown in Figure
9A are examples of such
micrographs obtained with myc-tagged KCNQ3 transfected cells (top) and myc-KCNQ3- and Src-transfected cells
(bottom). Figure 9B shows the summary of such
densitometry data from control (myc-KCNQ3; n = 9) and Src-cotransfected (myc-KCNQ3+Src;
n = 7) cells. These experiments revealed no difference
in KCNQ3 protein abundance between control and Src-overexpressing
cells. Although the approach used cannot precisely distinguish between
KCNQ channels located in the plasma membrane and those localized
subcellularly (nor can it rule out the theoretical possibility that Src
action alters the single-channel conductance of the channels), these
data are more consistent with the Src effect on current amplitude being caused by suppression of channel activity.
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Figure 9.
Src does not suppress KCNQ3 currents by inhibiting
KCNQ3 protein abundance. A, Confocal images of cells
transfected with myc-tagged KCNQ3 without (KCNQ3) or
together with (KCNQ3+Src) Src and
immunostained with anti-myc antibodies. B, Summarized
densitometry data from the confocal images as in A.
Fluorescence is expressed as mean pixel intensity. Black
boxes in A indicate areas where pixel intensity
was calculated. For myc-KCNQ3, n = 9; for myc-KCNQ3+Src,
n = 7.
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Does c-Src mediate muscarinic modulation of KCNQ channels?
Several members of the KCNQ channel family are best known for
their modulation by muscarinic receptor agonists. Such modulation requires Gq/11 subunits and an as yet
unidentified diffusible cytosolic messenger (Selyanko et al., 1992,
2000). Thus, it was important to determine whether Src might be the
unidentified messenger and whether Src action is a part of the
muscarinic signal. To test such involvement of Src, KCNQ2 and KCNQ3
were coexpressed in CHO cells together with M1
muscarinic receptors and wild-type or kinase-dead K298M Src, and the
ability of a muscarinic agonist to inhibit the KCNQ2/3 current was
assayed. As for cells not cotransfected with the
M1 receptor, Src suppressed current amplitudes
and slowed activation kinetics, and K298M Src was without effect. For
cells transfected with KCNQ2, KCNQ3, and M1
receptors but not Src, the current density and
act (at 0 mV) were 55.3 ± 12.3 pA/pF and 140 ± 21 msec (n = 7); for cells cotransfected
with Src, they were 13.4 ± 4.9 pA/pF (p 0.01) and 293 ± 26 msec (p 0.001; n = 6); and for cells transfected with K298M Src, they
were 54.1 ± 11.0 pA/pF and 148 ± 24 msec (n = 7).
Shown in Figure
10A are experiments
demonstrating muscarinic modulation of KCNQ2/3 currents by bath
application of the muscarinic agonist oxo-M (10 µM). We used the
Ca2+-clamping mixture pipette solution in
these experiments to exclude an influence of cytosolic
Ca2+ on the current (Shapiro et al.,
2000). After 1-2 min of recording of the control current, oxo-M was
bath-applied, and the inhibition of the current was observed. We found
that oxo-M suppressed the KCNQ2/3 current equally well in cells not
cotransfected with Src (Fig. 10A, left
panel) or cotransfected with wild-type Src (Fig. 10A, middle panel) or K298M
kinase-dead Src (Fig. 10A, right
panel). In these three groups, the muscarinic inhibition
values of the KCNQ2/3 current by oxo-M were 79 ± 9%
(n = 7), 83 ± 8% (n = 6), and
92 ± 6% (n = 7), respectively.
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Figure 10.
Src action is distinct from muscarinic modulation
of KCNQ2/3 and M currents. A, CHO cells were
cotransfected with KCNQ2, KCNQ3, and M1 muscarinic
receptors, either alone (left panel) or together
with wild-type (middle panel) or K298M
(right panel) Src. Plotted are the current
densities measured at 0 mV using a 500 msec voltage pulse to -60 mV,
followed by a 650 msec pulse back to the holding potential of 0 mV,
given every 3 sec. Oxotremorine (oxo; 10 µM) and XE991 (XE; 50 µM)
were bath-applied during the times shown by the bars.
Insets, Currents at the indicated times during the
experiments. The dotted lines indicate the zero current
level. B, C, Src inhibitors do not block or reverse
muscarinic modulation of KCNQ2/3 and M currents. B,
Plotted are current densities at 0 mV from the voltage protocol as in
A from CHO cells cotransfected with KCNQ2, KCNQ3, and
M1 receptors (but not Src). Oxo (10 µM), PP2
(200 nM), and XE991 (50 µM) were bath-applied
during the periods shown by their respective bars.
Insets, currents at the indicated times during the
experiments. C, M current densities measured from the
amplitude of the time-dependent deactivating relaxations at 60 mV in
an uninfected SCG neuron. The cell was held at 25 mV and a 500 msec
step to -60 mV, followed by a 650 msec step back to 25 mV, given
every 3 sec under continuous bath perfusion. Oxo (10 µM),
PP2 (200 nM), and XE991 (50 µM) were
bath-applied during the periods shown by their respective
bars.
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To further test for involvement of Src in mAchR-mediated
suppression of KCNQ2/3 currents, we tested whether Src inhibitors could
prevent or reverse muscarinic modulation. Shown in Figure 10B, left panel, is an experiment in which
a CHO cell transfected with KCNQ2/3 but not with Src was pretreated
with PP2 and then oxo-M was applied. Similar to the effect of PP2 on
the non-Src-transfected cell shown in Figure 2D, we
observed a modest run-up of the current during PP2 application,
consistent with blockade of endogenous Src-like activity on the
channels. However, PP2 was unable to prevent subsequent inhibition of
KCNQ2/3 current by oxo-M. Application of oxo-M to such CHO cells
pretreated with PP2 (10-15 min) resulted in the inhibition of KCNQ2/3
current by 60 ± 7% (n = 5), a value indistinguishable from that of cells preincubated with PP3 (63 ± 8%; n = 3; data not shown). We also asked whether PP2
or erbstatin could reverse muscarinic modulation. Neither PP2 (Fig.
10B, right panel; n = 3)
nor erbstatin (n = 3; data not shown) reversed
muscarinic modulation of the KCNQ2/3 current after oxo-M had been
applied. We also performed experiments of this kind on M current in SCG neurons. SCG cells were preincubated with erbstatin or with PP2, and
the ability of oxo-M to subsequently inhibit M current was assayed. A
representative experiment using PP2 is shown in Fig. 10C,
demonstrating unaltered modulation of M current by the Src inhibitor.
For SCG cells pretreated with erbstatin or with PP2, oxo-M subsequently
inhibited the M current by 86 ± 3% (n = 3) and
76% (n = 2), respectively, inhibitions
indistinguishable from those in control cells (82 ± 3%;
n = 6). As for the experiments on KCNQ2/3 channels in
CHO cells, neither PP2 (n = 3) nor erbstatin (n = 3) reversed muscarinic modulation of M current in
SCG neurons after application of oxo-M (data not shown). Thus, we
conclude that Src and muscarinic receptors use different pathways for
KCNQ and M current modulation.
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Discussion |
In this work, we describe a novel pathway of KCNQ and M-type
K+ channel regulation by Src-dependent
tyrosine phosphorylation. We show that Src acts both in a heterologous
expression system using cloned channels and on native M channels in
primary sympathetic neurons. The actions of Src were subunit-specific.
Thus, Src strongly suppressed current amplitudes from, and slowed
activation kinetics of, KCNQ3, KCNQ4, and KCNQ5 homomultimers, as well
as KCNQ2/3 heteromultimers and M current, but wholly spared KCNQ1 and
KCNQ2 homomultimers. The strongest effect of Src was on KCNQ3 channels, whose activity is profoundly inhibited by Src overexpression (Table 1,
Fig. 7). The lack of action on KCNQ2 channels suggests that Src effects
on KCNQ2/3 heteromultimers and on M current are likely attributable to
an action solely on the KCNQ3 subunits of the channel tetramers. We
found that suppression of the current amplitude in all Src-sensitive
channels was always accompanied by slowing of the activation kinetics,
possibly indicating that both effects localize to the same site of Src
action on the channels.
Several lines of evidence demonstrate that the effect of Src is
mediated by tyrosine phosphorylation: (1) all the observed effects of
Src (suppression of current amplitude, slowing of activation, and shift
of voltage dependence) were mostly reversed by the tyrosine kinase
inhibitors erbstatin and PP2; (2) the tyrosine phosphatase inhibitor
vanadate fully mimicked all the effects of Src; (3) use of the Src
K298M point mutant that abolishes kinase activity, but not Src binding
to substrates, was without any effect on KCNQ2/3 currents; and (4)
KCNQ3, KCNQ4, and KCNQ5 (the subunits that were modulated by Src in our
patch-clamp experiments) were associated with phosphotyrosine signals
when coexpressed with Src. In contrast, KCNQ1 and KCNQ2, which did not
respond to Src overexpression in patch-clamp experiments, were never
associated with a phosphotyrosine signal. Interestingly, KCNQ4
displayed a phosphotyrosine signal even without Src overexpression
(Fig. 8B). The phosphorylation signal became stronger
in Src-overexpressing cells and was absent when the kinase-dead K298M
Src (which serves as dominant negative in this case) was overexpressed
instead of wild-type Src (Fig. 8B). Such experiments
further support our interpretation of the effect of Src inhibitors and
of vanadate on the KCNQ2/3 currents in non-Src-transfected cells as
being attributable to modest tonic Src-like activity in CHO cells.
Taken together, these data suggest that the effects of Src on KCNQ3-5
channels are attributable to tyrosine phosphorylation of the channel
proteins, although we cannot rule out that Src acts via phosphorylation
of physically associated adaptor proteins that can be
coimmunoprecipitated with the channels.
There are several examples of Src actions on
K+ channels known from the literature. The
closest analogy to this study is with Src modulation of
Shaker family (Kv1) channels. It has been shown that Src
family tyrosine kinases strongly modulate Kv1.3 (Holmes et al., 1996a),
Kv1.4, and Kv1.5 (Holmes et al., 1996b; Nitabach et al., 2001)
channels. For the case of Kv1.3, overexpression of constitutively
active viral Src phosphorylates, suppresses current amplitudes, and
alters kinetics (Fadool et al., 1997). For the case of Kv1.4 and Kv1.5,
Src appears to have dual actions: suppression of current amplitudes via
binding (without requisite phosphorylation) to Kv1.5 subunits as
homomultimers, or as heteromultimers with Kv1.4, and modulation of
kinetics of Kv1.4 homomultimers via tyrosine phosphorylation (without
requisite binding; Nitabach et al., 2001; Nitabach et al., 2002). Our
results with KCNQ2/3 and M current are similar in that our data suggest
that Src acts on the heteromeric channel by acting on only one type of
subunit (KCNQ3). For Kv1.4 and Kv1.5 channels, however, the effects on current amplitudes and on kinetics seem to be attributable to distinct
actions on the channels, the former mostly from Src binding to
proline-rich Src homology 3 (SH3)-binding domains of Kv1.5 and the
latter apparently by direct tyrosine phosphorylation of Kv1.4 (Nitabach
et al., 2001; Nitabach et al., 2002). This does not seems to be the
case here for KCNQ channels, because the tyrosine kinase and
phosphatase inhibitors affected both current amplitude and kinetics,
and the kinase-dead Src had none of these effects. There has been a
study implying tyrosine kinase-mediated augmentation of KCNQ2 currents
(kinase inhibitors decreased the current; Jow and Wang, 2000), but the
mechanism underlying their data are unclear.
Sequence gazing of KCNQ3-5 does not reveal the existence of preferred
proline-rich SH3-binding domains (RPLPXXP), preferred SH2-binding domains (pYEEI), or the optimum tyrosine substrate sequence
(EEEIYG/EEFD; for
review, see Tatosyan and Mizenina, 2000). However, all three channels
contain the minimum PXXP sequence for binding to SH3
domains, and the sequence around Src-phosphorylated tyrosines can be
highly variable (Hubbard and Till, 2000). Future work will identify the
molecular determinants of the Src actions described here and whether
they suggest novel Src-interacting motifs in KCNQ3-5 channels or
variants of those already described.
Modulation of KCNQ3-5 by Src seems to be unrelated to the well studied
pathway of M-type channel modulation by mAchRs. This conclusion comes
from our results showing that muscarinic agonists inhibit Src-modulated
channels, and that expression of the kinase-dead Src had no effect on
muscarinic modulation. In addition, Src inhibitors failed to prevent or
reverse such modulation (Fig. 10). Also arguing against involvement of
Src in muscarinic modulation of KCNQ channels is that KCNQ1 and KCNQ2
are both well modulated by muscarinic stimulation (Selyanko et al.,
2000; Shapiro et al., 2000) but not by Src (Table 1, Fig. 7). It is
important to note that we were able to reproduce our data obtained with
the heterologous expression system on native M current in SCG neurons.
It is now thought that combinations of heteromeric KCNQ2/3 and KCNQ3/5
and homomeric KCNQ3-5 channels can underlie the heterogeneity of
M-type currents (Wang et al., 1998; Cooper et al., 2000; Lerche et al., 2000; Robbins, 2001; Roche et al., 2002). The modulation of M-type K+ channels by Src reported here might
therefore be an important mechanism of regulation of neuronal
excitability. Thus, not only do mutations in KCNQ2 and KCNQ3 genes lead
to a form of inherited epilepsy (for review, see Jentsch, 2000), but
also, an increase in Src kinase activity was found on induction of
spontaneous epileptiform activity in rat hippocampus (Sanna et al.,
2000). Indeed, in that study, the epileptiform activity could be
strongly reduced by the Src inhibitor PP2.
What physiological signals are we observing by using Src overexpression
or tyrosine phosphatase blockade? The Src family of nonreceptor
tyrosine kinases has been shown to be activated by both
G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases
(RTKs), to be a signaling intermediary between both GPCRs and RTKs, and
to be involved in downstream activation of the MAP kinase signaling
cascade by both types of receptors (Diverse-Pierluissi et al., 1997;
Luttrell et al., 1997, 1999; Pierce et al., 2001). Indeed, of strongest
relevance to sympathetic neurons, both mAchRs and the nerve growth
factor (NGF) receptor TrkA activate Src (for review, see Abram and
Courtneidge, 2000). NGF, which is required for growth, survival, and
differentiation of sympathetic neurons, is a member of the neurotrophin
family that has both acute effects on synaptic transmission and
plasticity (Schinder and Poo, 2000) as well as the long-term effects
common among growth factor receptors (Barbacid, 1993). Thus, our
effects of Src overexpression may constitute a "shortcut" in both
acute and long-lasting signals triggered by growth factors or GPCR
agonists, and the relatively rapid actions on M-type channels described
here may constitute one mechanism by which tyrosine kinases acutely
regulate neuronal excitability. Future work will ask whether Src
overexpression indeed mimics stimulation of receptor tyrosine kinases
and will seek to probe the biophysical mechanisms of the modulation of this family of K+ channels by tyrosine
kinases in general.
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FOOTNOTES |
Received June 13, 2002; revised Oct. 15, 2002; accepted Oct. 15, 2002.
This work was supported by a new faculty startup grant from the Howard
Hughes Medical Institute, an American Heart Association (Texas
Affiliate) research award, and National Institutes of Health Grant
NS43394 (M.S.S.). We thank Pamela Martin and Emily McDermott for expert
technical assistance.
Correspondence should be addressed to Mark S. Shapiro, Department of
Physiology, University of Texas Health Science Center at San Antonio,
7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail:
shapirom{at}uthscsa.edu.
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References |
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Abram CL,
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(2000)
Src family tyrosine kinases and growth factor signaling.
Exp Cell Res
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TOP
ABSTRACT
Introduction
