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The Journal of Neuroscience, August 15, 1998, 18(16):6126-6137
Modulation of Olfactory Bulb Neuron Potassium Current by Tyrosine
Phosphorylation
Debra A.
Fadool and
Irwin B.
Levitan
Biochemistry Department and Volen Center for Complex Systems,
Brandeis University, Waltham, Massachusetts 02254
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ABSTRACT |
Insulin causes a suppression of whole-cell voltage-dependent
outward current in cultured neurons from the rat olfactory bulb. This
suppression is time-dependent; it is mimicked by application of Src
tyrosine kinase inside the cell via the whole-cell patch electrode or
by treatment of the olfactory bulb neurons with the tyrosine
phosphatase inhibitor pervanadate. The C-type inactivation properties
of the outward current in olfactory bulb neurons resemble those of the
cloned Kv1.3 potassium channel. In addition, at picomolar concentrations at which it is specific for Kv1.3, the scorpion toxin
margatoxin blocks most of the olfactory bulb neuron outward current.
Immunocytochemical analysis demonstrates that Kv1.3 is prominent in the
cultured olfactory bulb neurons. To identify specific amino acid
residues that might be important for potassium current modulation, we
examined the effects of pervanadate and insulin on wild-type and mutant
Kv1.3 channels expressed in human embryonic kidney (HEK 293) cells. As
shown previously, treatment with either pervanadate or insulin
suppresses Kv1.3 current in these cells. Mutational analysis
demonstrates that at least two distinct tyrosine residues are required
for current suppression by pervanadate. Insulin treatment stimulates
the tyrosine phosphorylation of Kv1.3 in HEK 293 cells, and a different
combination of tyrosine residues is required for the current
suppression by insulin. The results suggest that complex patterns of
phosphorylation may be involved in the modulation of neuronal potassium
current by receptor and nonreceptor tyrosine kinases.
Key words:
tyrosine kinase; tyrosine phosphatase; potassium channel; insulin receptor; modulation; mutational analysis; olfactory bulb; neuron; Kv1.3
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INTRODUCTION |
Modulation of membrane ion channels
is a fundamental mechanism in neuronal plasticity. The molecular
mechanism of ion channel modulation that is best understood is
modulation by protein phosphorylation. Ion channels, like many other
proteins, are substrates for protein kinases and phosphoprotein
phosphatases, and phosphorylation can influence a variety of channel
functional properties. Although serine-threonine phosphorylation has
been most thoroughly studied (Levitan, 1994 ), more recently the role of
tyrosine phosphorylation in channel modulation has begun to be
elucidated (Jonas and Kaczmarek, 1996). Both ligand-gated and
voltage-dependent ion channels are subject to modulation by tyrosine
phosphorylation, and different channel properties are modulated
depending on the specific type of channel and tyrosine kinase involved
(Hopfield et al., 1988; Huang et al., 1993; Wang and Salter, 1994; Yu
et al., 1997). In the case of voltage-dependent potassium (Kv)
channels, a consistent pattern has begun to emerge, in that the
activity of several different cloned Kv channels is suppressed after
phosphorylation by both receptor and nonreceptor tyrosine kinases
(Huang et al., 1993; Peralta, 1995; Holmes et al., 1996a,b; Bowlby et
al., 1997; Fadool et al., 1997).
The physiological significance of channel modulation by tyrosine
phosphorylation is studied best by examining the modulation of
native ion channels in neurons, either in situ or in cell
culture. On the other hand, the expression of cloned ion channels in
heterologous host cells provides a convenient means of analyzing the
molecular details of channel modulation. We have attempted to marry
these two approaches for the Kv1.3 potassium channel, a prominent
potassium channel in specific brain regions, including the olfactory
bulb and olfactory cortex (Kues and Wunder, 1992). The insulin receptor tyrosine kinase is also expressed at high levels in the olfactory bulb
(Gupta et al., 1992; Folli et al., 1994), and it is becoming evident
that insulin can modulate neuronal ion channels (Jonas et al., 1996)
and may be important for certain higher brain functions (Wickelgren,
1998). We show here that a substantial portion of the outward current
in cultured olfactory bulb neurons (OBNs) is carried through Kv1.3
channels and that the outward current in these neurons is suppressed
markedly within minutes after activation of the insulin receptor
tyrosine kinase. Incubation of OBNs with the tyrosine phosphatase
inhibitor pervanadate or internal perfusion of the tyrosine kinase Src
also suppress the outward current with a similar time course. We have
used mutational analysis of cloned Kv1.3 to identify specific
combinations of tyrosine residues that are important for current
suppression by pervanadate and insulin. Interestingly, a different
combination of residues is required for suppression of Kv1.3 by Src
(Fadool et al., 1997), and yet another tyrosine residue is involved in
the suppression of Kv1.3 by activation of the epidermal growth factor
receptor (EGFr) tyrosine kinase (Bowlby et al., 1997). The study of a
modulatory phenomenon in neurons, in combination with mutational
analysis in a heterologous expression system, provides an approach to
elucidating the molecular details of a physiological response. The data
are consistent with the idea that the modulation of neuronal
potassium current may require complex patterns of protein tyrosine
phosphorylation.
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MATERIALS AND METHODS |
Solutions and reagents. Human embryonic kidney (HEK
293) cell patch pipette solution contained (in mM):
30 KCl, 120 NaCl, 10 HEPES, and 2 CaCl2, pH 7.4. OBN
patch pipette solution contained (in mM): 145 KCl, 10 HEPES, 10 EGTA, 2 MgCl2, and 0.20 NaATP, pH 7.3. HEK
293 cell bath recording solution contained (in mM): 150 KCl, 10 HEPES, 1 EGTA, and 0.5 MgCl2, pH 7.4. OBN
bath recording solution contained (in mM): 150 NaCl, 5 KCl,
2.6 CaCl2, 2 MgCl2, 10 HEPES, and
100 nM tetrodotoxin (TTX), pH 7.3. Protease and phosphatase
inhibitor (PPI) solution contained (in mM): 25 Tris, 250 NaCl, 5 EDTA, 1 Na3VO4, 1 phenylmethylsulfonyl fluoride, and 1% Triton X-100, 1 µg/ml each
leupeptin and pepstatin, and 2 µg/ml aprotinin, pH 7.5. All salts
were purchased from Sigma, St. Louis, MO. Tissue culture and
transfection reagents were purchased from Life Technologies
(Gaithersburg, MD). TTX and nerve growth factor (NGF) 7S were purchased
from Calbiochem (La Jolla, CA). Human recombinant insulin was purchased
from Upstate Biotechnology (Lake Placid, NY) or Boehringer Mannheim
(Indianapolis, IN). Margatoxin (MgTx) was a generous gift from Dr. Reid
Leonard, Merck Research Laboratories (Rahway, NJ).
Pervanadate (Na3VO4) (Sigma) was
prepared from a 1 mM orthovanadate stock solution as
described previously (Fantus et al., 1989). Immediately before use, the
orthovanadate stock was mixed with 0.003% H2O2
for 15 min at room temperature, followed by the addition of 54 µM catalase to remove residual
H2O2. This protocol generated the peroxidized
form of vanadate, pervanadate, which was stable for 2 hr.
cDNA constructs and antibodies. Kv1.3 channels were
expressed transiently in HEK 293 cells using the Invitrogen (San Diego, CA) vector pcDNA3. Kv1.3 was subcloned into
pcDNA3 at the unique HindIII site of the
multiple cloning region, placing the channel coding region downstream
from a cytomegalovirus promoter. The insulin receptor (IR) cDNA
was generously provided by Richard Roth (Stanford University, Stanford,
CA) in the pECE vector. The entire IR coding region was removed using
the unique restriction sites SalI and XbaI and
subcloned into pcDNA3 between the XhoI and
XbaI sites in the multiple cloning region.
A rabbit polyclonal antiserum, raised against a MalE fusion protein
(New England BioLabs, Beverly, MA) containing an extracellular sequence
specific to Kv1.3 (Cai and Douglass, 1993), was generously provided by
Dr. James Douglass (Vollum Institute, Portland, OR). This antibody was
used for immunocytochemical analysis of OBNs and of HEK 293 cells
transfected with Kv1.3 channel cDNA, and for immunoprecipitation and
Western blot detection of Kv1.3. Tyrosine-phosphorylated proteins were
immunoprecipitated and detected on Western blots with the mouse
monoclonal antibody 4G10 (Upstate Biotechnology), which recognizes
phosphotyrosine.
Primary cell culture. Olfactory bulbs were harvested from
24-hr-old Sprague Dawley rats, and neuronal primary cultures were prepared using the procedure of Huettner and Baughman (1986) as modified by Egan et al. (1992). Briefly, animals were killed by decapitation according to American Veterinary Medical
Association-approved methods. Olfactory bulbs were removed
quickly from the cranium and placed into 10 ml serum-free DMEM
(Life Technologies) equilibrated previously at 37°C in a 5%
CO2 incubator. Olfactory bulbs from four to five animals
were incubated whole in a physiological saline solution containing
cysteine-activated papain (200 U; Worthington, Freehold, NJ) for 1 hr
at 37°C in the 5% CO2 incubator. The bulbs were then
washed in DMEM containing 5% fetal bovine serum (Life Technologies)
and 5 mg/ml trypsin inhibitor (Boehringer Mannheim) for 10 min to stop
the enzymatic activity of the papain. Cells were dissociated by
trituration using a graded-size series of fire-polished siliconized
Pasteur pipettes; the resulting neuron and glia suspension was plated
onto poly-D-lysine hydrobromide-coated (molecular
weight, 49,300-53,000; Sigma) 12 mm glass coverslips, and
incubated in DMEM supplemented with 2% penicillin-streptomycin and
5% fetal bovine serum (Life Technologies). Cytosine arabinoside (10 µM; Sigma) was added to the medium for 36 hr between days 3-5 to stop the overgrowth of dividing cells and to promote better survival of the neurons. Growth medium was changed twice a week, allowing viable neurons for at least 2 months. Neurons were used for
patch recording or immunocytochemistry 3-32 d after plating.
Maintenance of HEK 293 cell cultures and transfection. HEK
293 cells were maintained in minimal essential medium (MEM), 2% penicillin-streptomycin, and 10% fetal bovine serum (Life
Technologies). Before transfection, cells were grown to confluency (7 d), dissociated with trypsin-EDTA (Sigma) and mechanical trituration,
diluted in MEM to a concentration of 600 cells/µl, and replated on
Corning dishes (catalog #25000; Fisher Scientific, Houston, TX). cDNA was introduced into the HEK 293 cells with a lipofectamine reagent (Life Technologies) 3-4 d after cell passage. At the time of
transfection, the cells were ~20-30% confluent. Lipofectamine and
cDNA were allowed to complex for 30 min. The DNA-lipofectamine complex
was diluted in 1 ml of serum-reduced OptiMEM (Life Technologies), and
cells were transfected for 4.5-5 hr with 1 µg of Kv1.3 cDNA or with
0.75 µg each of Kv1.3 and IR cDNA per 35 mm dish. Plasmid DNA with no
coding insert served as the control.
Transfection efficiency was monitored in parallel plates by
transfecting with a Lac Z expression plasmid and subsequently staining
the  -galactosidase reaction product. Staining efficiency was
routinely 70-90%, and physiological expression of Kv1.3 was observed
in 40-60% of the cells. In later experiments, constructs were
cotransfected with pHook (Invitrogen) as a means of rapidly selecting
transfected cells. pHook encodes a transmembrane domain from the
PDGF-R, which is then anchored on the extracellular side of the plasma
membrane. Before patch recording, a brief incubation with an
appropriate antibody linked to a 5 µM polystyrene bead allows recognition of transfected cells. More than 85% of bead-labeled cells expressed Kv1.3 current. Typically, single channel events could
be detected as early as 9 hr post-transfection, and macroscopic currents were observed in the range of 24-72 hr.
Tyrosine phosphorylation of Kv1.3. Cells were transfected as
above, but at 80-90% confluency and with a total of 7 µg cDNA per
60 mm dish. Equal amounts of channel and IR cDNA were mixed. HEK 293 cells were harvested 2 d post-transfection by lysis in ice-cold
PPI solution. The lysates were clarified by centrifugation at 14,000 × g for 10 min at 4°C and incubation for 1 hr with 3 mg/ml Protein A-Sepharose (Pharmacia, Piscataway, NJ), followed by
another centrifugation step to remove the Protein A-Sepharose. Tyrosine-phosphorylated proteins were immunoprecipitated from the
clarified lysate by overnight incubation at 4°C with 5 µg/ml 4G10
antibody, followed by a 2 hr incubation with Protein A-Sepharose and
centrifugation as above. The immunoprecipitates were washed 4 times
with ice-cold PPI solution (modified to contain 0.1% Triton X-100).
Lysates and washed immunoprecipitates were diluted in SDS gel loading
buffer (Sambrook et al., 1989) containing 1 mM Na3VO4, and proteins were separated on
10% acrylamide SDS gels and transferred to nitrocellulose for Western
blot analysis. Blots were blocked with 5% nonfat milk and incubated
overnight at 4°C in primary antibody against Kv1.3, then with
horseradish peroxidase-conjugated goat anti-rabbit secondary antibody
(Amersham, Arlington Heights, IL) for 90 min at room temperature. ECL
(Amersham) exposure on Fuji RX film (Fisher Scientific) was used to
visualize labeled protein.
Electrophysiology of OBNs. OBNs were voltage-clamped in the
whole-cell recording configuration using an Axopatch-1B amplifier (Axon
Instruments, Foster City, CA). Cells were visualized at 40×
magnification using a phase contrast water immersion lens (Carl Zeiss,
Thornwood, NJ). Electrodes were fabricated from Jencons glass (catalog
#M15/10; Jencons Limited, Bedfordshire, England), fire-polished to ~1
µm, and coated near the tip with beeswax to reduce the electrode
capacitance. Pipette resistances were between 9 and 14 M . All
voltage signals were generated, and data were acquired using a
microstar DAP 800/2 board (Microstar Lab, Bellevue, WA). The amplifier
output was filtered at 2 kHz, digitized at 2-5 kHz, and stored for
later analysis.
Typically cells were held at  90 mV and stepped to depolarizing
potentials for a pulse duration of 400 msec at a stimulating interval
of 30-60 sec. In the studies involving MgTx block of OBN outward
current, picomolar concentrations of MgTx were applied to the bath.
Peak current amplitudes from OBNs in the whole-cell configuration
before and 10 min after MgTx application were measured to permit a
paired statistical comparison. Parallel experiments with Kv1.3
expressed in HEK 293 cells demonstrated that MgTx block was complete in
<10 min.
The effect on OBN outward current of 0.5 U of
c-Srcpp60 (Upstate Biotechnology) plus 200 µM MgATP (Sigma), applied via the recording electrode to
allow access to the cell cytosol, was tested by the same stimulation
protocol as above. Immediately on breakthrough to the whole-cell
configuration, the initial peak current magnitude was recorded as the 0 time condition. The amount of current remaining at 24 min, in the
absence or presence of Src, was normalized to the 0 time value. Boiled
c-Srcpp60, ATP alone, and
c-Srcpp60 ATP served as negative controls.
The effect of bath-applied pervanadate, insulin, or NGF on whole-cell
OBN current was determined by measuring peak current amplitude,
inactivation rate, and deactivation rate before and 5-20 min after
application to carry out a within cell or paired statistical
comparison. In the case of pervanadate, cells were pretreated for 5-10
min with the carrier 0.003% H2O2 + 54 µM catalase to confirm the stability of the patch.
Electrophysiology of HEK 293 cells. Macroscopic currents in
cell-attached membrane patches were recorded 24-72 hr after
transfection. The Kv1.3 channel expression was so robust that it was
not possible to record whole-cell currents without saturating the
amplifier. The diameter of the patch electrode, and hence number of ion
channels sampled, was held uniform by checking the bubble number of the pipette immediately after electrode fabrication and polishing (Mittman
et al., 1987). Patches were held routinely at a holding potential of
80 or 90 mV, and the voltage was stepped to depolarizing potentials
for a pulse duration of 1000 msec. Stimuli were delivered at 45 sec or
longer intervals to prevent cumulative inactivation of the Kv1.3
channel (Marom et al., 1993). Testing for MgTx block was identical to
that described above for the OBNs, with the exception that the toxin
was applied in the patch electrode. Pervanadate and insulin treatment
and analysis were also the same as described for OBNs above.
All electrophysiological data were analyzed using software written in
our laboratory, in combination with the analysis packages Origin
(MicroCal Software, Northampton, MA) and Quattro Pro (Borland International, Scotts Valley, CA). Data traces were subtracted linearly
for leakage conductance. Functional expression of Kv1.3 current was
defined as the presence of a nonohmic current at depolarizing voltages.
The inactivation of the macroscopic current was fit to the sum of two
exponentials by minimizing the sums of squares. The two inactivation
time constants were combined by multiplying each by its weight and
summing as described previously (Kupper et al., 1995). The deactivation
of the macroscopic current was fit similarly, but to a single
exponential. Differences between control and treatment groups within
single cells were analyzed by paired t test with statistical
significance defined at the 0.95 confidence level.
Immunocytochemistry. Cultured OBNs and transfected HEK 293 cells were rinsed once in PBS and then lightly fixed in St. Marie fixative (95% EtOH and 5% acetic acid) for 10 min at 20°C. The fixative was removed by rinsing with three changes of PBS for 10 min
each, and nonspecific binding was blocked by incubation for 25 min in
PBS containing 1% albumin Fraction V (fatty acid free; Sigma) and
2-5% goat serum (PBS-Block). Neurons or HEK 293 cells were incubated
with primary antiserum diluted in PBS-Block for 2 hr at room
temperature, washed with three changes of PBS, and then were
reincubated for 2 hr at room temperature with a fluorescein-conjugated
goat anti-rabbit secondary antibody (Boehringer Mannheim or Amersham)
in PBS-Block. Neurons or HEK 293 cells were washed with three changes
of PBS for 10 min, rinsed in millipore water, and mounted in 60:40
glycerol-PBS with 0.1% p-phenylene diamine added to
prevent photobleaching. Photomicroscopy was performed at 40× with a
Ph3 objective using a Microphot-fxa Nikon (Columbia, MD) microscope
equipped with fluorescence. Microscope use was generously provided by
Dr. John Dowling (Biological Laboratories, Harvard University, Boston,
MA).
Site-directed mutagenesis. The parent Kv1.3 clone was
propagated in Escherichia coli DH-1. Plasmid DNA preparation
was by standard methods using a Qiagen (Chatsworth, PA) plasmid kit
followed by phenol-chloroform extraction and ethanol precipitation
(Sambrook et al., 1989). All Kv1.3 channel mutants were constructed
using two sequential PCRs (Landt et al., 1990) in an Eri-Comp
(Twin Block System, San Diego, CA) thermocycler, using Taq
polymerase (Promega, Madison, WI). The circularized plasmid containing
the channel gene served as the DNA template. For each tyrosine
mutation, three oligonucleotides, each 15-24 b in length, were
synthesized. Two of the oligonucleotides were complementary to
sequences on opposite sides of the tyrosine residue to be mutated, and
the third was a mutant primer with a single base change to convert the
tyrosine to phenylalanine. In the case of YYY111-113, the three
adjacent tyrosines were treated as a unit and mutated together to
phenylalanines. The first PCR used the mutagenic primer and the
upstream primer. The second PCR used the amplified, gel-purified product of the first reaction (Gene Clean II; Bio Labs 101, Vista, CA)
and the downstream oligonucleotide as primers. In this way a
stretch of mutant DNA flanked by two unique restriction sites was
obtained; the product was double digested and ligated into the parent
channel backbone using T4 DNA ligase (Promega). The resulting mutant
construct was sequenced using a cycle-sequencing reaction (Prism) and
an automated sequencer (Applied Biosystems Inc., Princeton, NJ) to
verify the mutation and detect PCR errors.
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RESULTS |
Characteristics of cultured OBNs
OBNs from neonatal rats can be maintained in culture for as long
as 2 months and are readily amenable to patch recording (Huettner and
Baughman, 1986; Egan et al., 1992). The cultures contain both glial-like and neuronal cell types that we characterized
immunocytochemically with a neural cell-typing kit (Boehringer
Mannheim). Astrocytes (GFAP-positive) constitute the majority of a
confluent mitotic cell layer that also contains scattered fibroblasts
(fibronectin positive) and oligodendrocyte-like cells (Gal C-positive).
On top of this layer are two types of neurons that can be distinguished by their morphology. The first type is pyramidal shaped and has a
single large neurite extending from each of its corners. The second
type is bipolar, which in later stages of cell culture, is seen also in
clusters independent of the supporting glial-like cell layer (Frosch
and Dichter, 1984). As described previously for olfactory bulb neurons
(Trombley and Westbrook, 1990; Bufler et al., 1992), the pyramidal
cells are probably mitral-tufted cells, and the bipolar cells are
probably granule cells. The cultured neurons could be divided into two
distinct subsets based on the rate of inactivation of the
voltage-dependent outward current (data not shown); however all cells
responded to insulin and Src in the same way, and there was no obvious
correlation between inactivation rate and cell morphology, and
hence the data were not divided by cell type.
Modulation of voltage-dependent outward current in OBNs
by insulin
Several recent studies have demonstrated that the activation of
receptor tyrosine kinases by appropriate ligands can modulate neuronal
ion channels (Jonas et al., 1996; Hilborn et al., 1998). Because
insulin receptors are prominent in the olfactory bulb (Gupta et al.,
1992; Folli et al., 1994), we tested the effects of insulin on outward
currents in cultured olfactory bulb neurons. As shown in Figure
1A, insulin treatment
decreases the amplitude of the outward current evoked by a depolarizing
voltage pulse. Although TrkA receptors that bind NGF are also present
in the olfactory bulb (Sobreviela et al., 1994), prolonged NGF
treatment does not affect the amplitude of the outward current (Fig.
1B). This result demonstrates that there is selective
coupling of some, but not all, receptor tyrosine kinases to potassium
channels in OBNs.
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Figure 1.
Effects of insulin and nerve growth factor on OBN
outward current. Whole-cell outward current responses were elicited by
voltage steps to +40 mV from a holding potential of 90 mV. Cells were
allowed to stabilize for 10 min after achieving the whole-cell
recording configuration, and the control outward current was recorded
(t = 0). Insulin (10 µg/ml)
(A) or nerve growth factor (100 nM)
(B) was added to the bath at
t = 0, and outward current was measured at 1 min
intervals thereafter. The traces shown were elicited 10 (A) or 50 min (B) after
addition of the ligand.
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As shown in Figure 2, A
and B, the suppression of outward current by insulin can be
seen at all depolarizing voltages at which outward current can be
evoked. The effect of insulin takes several minutes to develop (Fig.
2C), consistent with the idea that modulation of current
involves the activation of an intracellular metabolic cascade
downstream from the insulin receptor tyrosine kinase. Generally a
steady state suppression of ~50% is reached 10-20 min after
application of insulin.
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Figure 2.
Characteristics of the OBN response to insulin.
A, Outward current was elicited by voltage steps every
30 sec from a holding potential of 90 mV to depolarizing potentials
between 70 and 0 mV (5 mV increments). Shown are the currents
elicited before (Control) and 10 min after
(Insulin) application of 10 µg/ml insulin to the bath.
B, Plot of the peak current amplitude as a function of
the voltage during the depolarizing pulse. C, Change in
peak outward current as a function of time after addition
(arrow) of 10 µg/ml insulin ( ) or control OBN bath
solution ( ). The current at each time point was normalized to the
current at t = 0 for that cell.
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Modulation of voltage-dependent outward current in OBNs
by Src
Among the downstream targets of receptor tyrosine kinases are
members of the Src family of nonreceptor tyrosine kinases (Hilborn et
al., 1998). Because it is known that Src can interact with and suppress
the activity of several cloned potassium channels (Holmes et al.,
1996b; Fadool et al., 1997), we investigated whether Src might mimic
the actions of insulin on outward current in cultured OBNs. As
shown in Figure 3A, when
active recombinant Src kinase together with MgATP is included in the
patch electrode, the outward current evoked by a depolarizing step to
+40 mV decreases with time after achieving the whole-cell patch
recording configuration. The time course of suppression by Src (Fig.
3B) is comparable to that after insulin treatment (Fig.
2C), although it is not clear whether this prolonged time
course reflects the involvement of a downstream cascade or
simply the time required for Src to diffuse from the electrode into
the cell. Less than half of the outward current remains after 24 min in
the whole-cell recording configuration when Src is included in the
patch electrode, whereas there is no significant decrease in the
current during this time in the absence of Src (Fig.
3B,C). Heat-inactivated Src does
not suppress the outward current (Fig. 3C), and Src is
ineffective in the absence of ATP (data not shown), suggesting that the
tyrosine kinase activity of Src is required to produce the modulation.
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Figure 3.
Effect of Src on OBN outward current.
A, Outward currents were elicited by depolarizing from
90 to +40 mV at 1 min intervals. Whole-cell patch electrodes were
back-filled with either control patch solution
(Control) or 0.5 U of recombinant c-Src
(c-Srcpp60). Every second
pulse during a 24 min recording period is shown for each cell.
B, Change in peak outward current as a function of time
in a control () and an Src-treated () cell. Normalization as in
Figure 2. C, Mean normalized peak current amplitudes
after 24 min of recording for cells treated with control patch solution
(Control) or active c-Src
(c-Srcpp60). *Significantly
different, Student's t test. Shown also for comparison
is the normalized peak current remaining after the same time period in
cells treated with heat-inactivated c-Src
(Heat-inactivated).
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Modulation of voltage-dependent outward current in OBNs
by pervanadate
To determine whether the outward current in OBNs can be modulated
by endogenous tyrosine kinases, we treated cells with pervanadate while
recording current in the whole-cell patch configuration. At
concentrations in the micromolar range, pervanadate specifically inhibits tyrosine phosphatases and has little effect on
serine-threonine phosphatases (Bourgoin and Grinstein, 1992). As shown
in Figure 4A, the
outward current amplitude in response to a depolarizing pulse to +40 mV
is substantially decreased after 10 min treatment with 100 µM pervanadate. Suppression of current was seen with as
little as 10 µM pervanadate (data not shown). The time
course (Fig. 4B) and extent (Fig. 4C) of
the suppression by pervanadate are comparable to those produced by
insulin or Src treatment. These results demonstrate that there is a
constitutive phosphorylation and dephosphorylation cycle in OBNs,
resulting from the activities of endogenous tyrosine kinases and
phosphatases, that can influence the amplitude of the voltage-dependent
outward current.
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Figure 4.
Effect of pervanadate on OBN outward current.
A, Outward current elicited by a depolarization from
90 to +40 mV before (Control) and 5 min after
(Pervanadate) addition of 100 µM sodium
pervanadate to the bath. B, Change in peak outward
current as a function of time after addition (arrow) of
either carrier solution () or pervanadate (). Normalization as in
Figure 2. C, Mean normalized peak current amplitude
after 5 min of recording for cells treated with carrier solution
(Control) or 100 µM pervanadate
(Pervanadate). *Significantly different, paired
t test.
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Characterization of the voltage-dependent outward current
in OBNs
OBNs express a variety of potassium channels that normally
contribute to the total outward current (Wang et al., 1996; Chen and
Shepherd, 1997). Because the concentrations of sodium and calcium in
our whole-cell patch electrodes were kept very low, the prominent
sodium-dependent (Egan et al., 1992) and calcium-dependent (Egan et
al., 1993) potassium channels in these neurons are not activated under
the conditions of our experiments. Thus Kv channels, several of which
are expressed in the olfactory bulb, must be responsible for most of
the outward current seen in Figures 1-4. Several lines of evidence
suggest that a major portion of the Kv current is carried by channels
that resemble the cloned Kv1.3 potassium channel. First, it is known
that the rate of C-type inactivation of cloned Kv1.3 expressed in
Xenopus oocytes increases as a function of time when
membrane patches are detached from the cell in the inside-out recording
configuration (Marom et al., 1993). Similarly, the inactivation rate of
native Kv1.3 in T lymphocytes increases with time in the whole-cell
recording configuration (Oleson et al., 1993). As shown in Figure
5A, the voltage-dependent outward current in OBNs also exhibits faster C-type inactivation with
time in the whole-cell recording configuration, comparable to the
change in inactivation kinetics of Kv1.3 in detached inside-out patches
from HEK 293 cells (Fig. 5B).
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Figure 5.
Comparison of outward current in OBNs and HEK 293 cells. A, Inactivation kinetics of OBN outward current
immediately (t = 0 min) and 10 min after
(t = 10 min) achieving the whole-cell recording
configuration. B, Inactivation kinetics of Kv1.3 current
in a cell-attached patch on a HEK 293 cell and 10 min after detaching
in the inside-out patch configuration. For both A and
B, the peak currents are normalized to better visualize
the inactivation kinetics. C, Plot of the normalized
peak outward current amplitude in an OBN 10 min after application of
the indicated concentration of MgTx to the bath. Each point represents
the mean for three to four cells; currents were normalized to the
initial current for each cell. D, Kv1.3 currents in a
cell-attached patch on a HEK 293 cell evoked by a depolarizing voltage
step from 90 to +40 mV at the indicated times after achieving a
gigaohm seal. 100 pM MgTx was present in the patch
electrode.
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Pharmacological experiments also suggest that much of the
voltage-dependent outward current in OBNs is Kv1.3-like. The scorpion toxin MgTx selectively binds to and blocks Kv1.3 with very high affinity in the picomolar range; in contrast, MgTx blocks other Kv
channels, only at much higher (micromolar) concentrations (Knaus et
al., 1995). We find that 100-250 pM MgTx blocks 50-83%
of the outward current in OBNs (Fig. 5C) and all of the
Kv1.3 current in HEK 293 cells (Fig. 5D) within 10 min.
Finally, we used immunocytochemical analysis to determine whether Kv1.3
is present in the cultured OBNs. Previous experiments using in
situ hybridization demonstrated that the highest concentration of
Kv1.3 messenger RNA is in the olfactory bulb and olfactory cortex (Kues
and Wunder, 1992). As shown in Figure 6,
an antibody directed against a putative extracellular loop of Kv1.3
stained HEK 293 cells that had been transfected with Kv1.3 cDNA. Note that not all the cells present in the field of view (Fig.
6A) were stained with the antibody (Fig.
6B), reflecting the fact that transfection efficiency
was <100%. In contrast, all of the OBNs in the field (Fig.
6C) stained with the anti-Kv1.3 antibody (Fig.
6D), whereas the mitotic glial-like cell layer on
which the neurons were growing did not stain. Both the mitral and
granule neurons appeared to stain equally well for Kv1.3.
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Figure 6.
Expression of Kv1.3 in HEK 293 cells and OBNs.
Photomicrographs of Kv1.3-transfected HEK 293 cells (A,
B) and OBNs (C, D), under bright-field
illumination (A, C) and epifluorescence (B,
D). Cells were immunolabeled with an antibody directed
against a putative extracellular domain in Kv1.3 and visualized with a
fluorescent-conjugated secondary antibody.
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|
Mutational analysis of pervanadate-induced modulation of Kv1.3
These findings suggested that mutational analysis of cloned Kv1.3
expressed in HEK 293 cells might provide insight into the molecular
details of modulation of outward current in OBNs. As shown in Figure
7B and Table
1, pervanadate treatment decreases the
amplitude of wild-type Kv1.3 current without influencing channel kinetics, confirming our previous report (Holmes et al., 1996a). Because there are at least six tyrosine residues in the channel protein
that might serve as substrates for tyrosine kinases (Fig. 7A), we performed an extensive mutational analysis. The
three adjacent tyrosine residues at positions 111-113 were treated as a unit and mutated together to phenylalanines, and the three tyrosines at positions 137, 449, and 479 were mutated individually to
phenylalanines; the properties of each of the four Kv1.3 mutant
channels were then examined. All of the mutant channels expressed well
in HEK 293 cells and could be activated by depolarization, and none of the mutations altered the basal inactivation or deactivation kinetics of Kv1.3 (Table 1).
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Figure 7.
Mutational analysis of the effect of pervanadate
on Kv1.3 expressed in HEK 293 cells. A, Schematic
representation of the positions of the tyrosine residues in Kv1.3 that
were mutated to phenylalanines. B-F,
Kv1.3 outward currents evoked in cell-attached membrane patches by
depolarizations from 80 to +40 mV, 20 min after addition of carrier
(Control) or 100 µM sodium
pervanadate (+Pervanadate) to the bath. The particular
mutant channel analyzed is indicated in each panel (WT,
wild-type).
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|
Neither Y137 nor Y479 appears to be necessary for the suppression of
current by pervanadate, because the modulation persists in the Y137F
(Fig. 7D) and Y479F (Fig. 7F) mutant
channels (see also Table 1). In contrast, both Y449 and at least one of
the three tyrosines in the YYY111-113 triplet are necessary for the modulation because no effect of pervanadate is seen in either the
YYY111-113FFF (Fig. 7C) or the Y449F (Fig. 7E)
mutant channel. As shown in Figure 8,
these two mutant channels do not respond even to prolonged treatment
with pervanadate.
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Figure 8.
Time course of current suppression by pervanadate
for wild-type (WT) and mutant Kv1.3 channels.
Outward currents in cell-attached patches on channel-expressing HEK 293 cells elicited by depolarizations from 80 to +40 mV at 1 min
intervals. 100 µM sodium pervanadate was added at the
arrow. Control () indicates WT Kv1.3 treated with
carrier solution instead of pervanadate.
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|
Mutational analysis of insulin-induced modulation of Kv1.3
Because insulin can participate in a variety of signaling pathways
and may have effects that are independent of tyrosine phosphorylation, we used an immunoprecipitation-Western blot strategy (Holmes et al.,
1996a) to measure the effect of insulin on the tyrosine phosphorylation of Kv1.3 in HEK 293 cells. As shown in Figure
9B, insulin treatment increases the tyrosine phosphorylation of Kv1.3 without affecting the
level of channel protein (Fig. 9A). Hence we examined the effects of insulin on the functional properties of wild-type and mutant
Kv1.3 channels as described above for pervanadate. Insulin suppresses
wild-type Kv1.3 current in HEK 293 cells cotransfected with the IR
(Fig. 10B, Table
2). Although Y449 does not appear to be
necessary for the current suppression (Fig. 10E),
mutation of the YYY111-113 triplet (Fig. 10C), Y137 (Fig.
10D), or Y479 (Fig. 10F) to
phenylalanine eliminates this response to insulin (Table 2).
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Figure 9.
Insulin increases the tyrosine phosphorylation of
Kv1.3. HEK 293 cells were transfected with vector alone or cDNA
encoding Kv1.3 and the IR. Two days later, the serum-containing medium
was replaced with Opti-MEM (Life Technologies) for 20 min, and cells
were then treated with either Opti-MEM vehicle () or 20 µg/ml
insulin (+) for 20 min. A, Western blot to measure the
amount of Kv1.3 protein expressed. B,
Immunoprecipitation-Western blot to measure tyrosine-phosphorylated
Kv1.3. The prominent protein band below the Kv1.3 band is the heavy
chain of IgG.
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View larger version (16K):
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[in a new window]
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Figure 10.
Mutational analysis of the effect of insulin on
Kv1.3 expressed together with the IR in HEK 293 cells.
A, Schematic representation of the positions of the
tyrosine residues in Kv1.3 that were mutated to phenylalanines.
B-F, Kv1.3 outward currents evoked in
cell-attached membrane patches by depolarizations from 80 to +40 mV,
20 min after addition of vehicle (Control) or 10 µg/ml insulin (+ Insulin) to the bath. The particular
mutant channel analyzed is indicated in each panel (WT,
wild-type).
|
|
| |
DISCUSSION |
The olfactory bulb receives odor information from the primary
olfactory receptor neurons and processes it in a way that serves as the
basis for olfactory perception (Chen and Shepherd, 1997). At least two
peripheral processes in olfaction, pheromone transduction (Zufall and
Hatt, 1991) and termination of the odorant response (Boekhoff et al.,
1992), involve intracellular messenger systems and protein
phosphorylation. In contrast, little is known about the role of
phosphorylation in information processing in the olfactory bulb.
Insulin receptors and insulin-dependent tyrosine kinase activity are
present at high levels in the rat olfactory bulb (Gupta et al., 1992;
Folli et al., 1994), and the Trk receptors for several different
neurotrophins have also been detected (Sobreviela et al., 1994; Roskams
et al., 1996; Yan et al., 1997).
We show here that insulin treatment suppresses the voltage-dependent
outward current in cultured OBNs. Insulin has also been shown to
influence ionic currents in Aplysia neurons (Jonas et al.,
1996). Both mitral cells and granule cells, the two major morphologically distinguishable neuron types in the olfactory bulb,
appear to be affected in the same way by insulin. Because the
nonreceptor tyrosine kinase Src plays a key role in the pathways activated by many receptor tyrosine kinases, we asked whether Src might
participate in this action of insulin on OBN outward current. Direct
application of active Src kinase to the cytoplasm of OBNs mimics the
suppression produced by insulin treatment. It has been shown recently
that several different growth factor receptor tyrosine kinases inhibit
sodium currents in PC12 cells via the Src-signaling pathway (Hilborn et
al., 1998). However, we cannot rule out the possibility that, in OBNs,
insulin and Src operate via parallel but independent pathways to
produce similar suppression of outward current. It is interesting that
inhibition of endogenous tyrosine phosphatase activity in the OBNs with
pervanadate also leads to suppression of the outward current,
presumably via the actions of endogenous tyrosine kinase(s). Again,
this may be a parallel phenomenon, and we cannot be certain that the
endogenous tyrosine kinase activity is important for the action of
insulin. Future experiments with inhibitors that interfere with the
coupling of the insulin receptor tyrosine kinase to specific downstream signaling pathways will be required to address this question.
It is difficult to identify specific amino acid residues that
contribute to the modulation of native ion channels in neurons. However, the Kv1.3 potassium channel is expressed prominently in
olfactory bulb and in the cultured OBNs. Although clearly there are
other potassium channels in these neurons, MgTx sensitivity and other
criteria suggest that as much as 80% of the voltage-dependent outward
current may be attributable to Kv1.3 channels. In addition, we have
shown previously that cloned Kv1.3 expressed in HEK 293 cells, like the
voltage-dependent outward current in OBNs, is suppressed by insulin
(Bowlby et al., 1997), Src (Fadool et al., 1997), and pervanadate
(Holmes et al., 1996a) treatment. Accordingly, we decided to carry out
detailed mutational analysis of Kv1.3 to determine whether specific
tyrosine residues are required for its modulation by these treatments.
We showed previously that at least one tyrosine residue, at position
449 of Kv1.3, is involved in the modulation by pervanadate (Holmes et
al., 1996a) and that Y449 together with another tyrosine residue at
position 137 is required for current suppression by Src (Fadool et al.,
1997). The fact that several tyrosines are involved in the modulation of Kv1.3 by Src prompted us to re-examine the effect of pervanadate. The more extensive mutational analysis of the pervanadate response presented here demonstrates that at least one additional tyrosine residue in the YYY111-113 triplet is required together with Y449 for
pervanadate modulation. In the case of insulin, the modulation is even
more complex, because the YYY111-113 triplet, Y137, and Y479 are all
required for the suppression of current. Adding to the complexity is
our finding that activation of another growth factor receptor, the
EGFr, also suppresses Kv1.3, but only Y479 is necessary for this
response (Bowlby et al., 1997). It is striking that different tyrosines
or combinations of tyrosines are involved in the responses of Kv1.3 to
these different agents, and yet in each case the current amplitude is
suppressed. Although the mutational analysis demonstrates that the
tyrosines are necessary for the responses, it has not yet been shown
unequivocally that it is their phosphorylation that is required. If
this turns out to be the case, it seems likely that the specific kinase
or set of kinases that phosphorylates the channel is different for each
of the modulatory phenomena.
Both serine-threonine and tyrosine phosphorylation pathways can
modulate potassium currents in OBNs, and such modulation probably plays
an important role in the response of the olfactory bulb to sensory
input. The present data add to our growing awareness of the complexity
of the signal transduction pathways that impinge on ion channels.
Unraveling the molecular details of the responses of particular ion
channels to activation of these pathways will be essential for
understanding information processing in the olfactory system as well as
in other parts of the brain.
| |
FOOTNOTES |
Received Jan. 26, 1998; revised May 29, 1998; accepted June 2, 1998.
This work was supported by grants from the National Institutes of
Health to I.B.L. and a National Institutes of Health national research
service award and a FIRST award to D.A.F. We are grateful to Richard
Huganir and Richard Roth for cDNA constructs, James Douglass for
antibodies, Jing Wang and James Fadool for technical hints, and Jeremy
Scarpate, Deanne Tabb, and especially Kristal Tucker for technical
assistance.
Correspondence should be addressed to Dr. Debra A. Fadool, Department
of Zoology and Wildlife Sciences, 331 Funchess Hall, Auburn University,
Auburn, AL 36849-5415.
| |
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Top
Abstract
Introduction
