1Department of Biological Sciences and Program in Neuroscience, Biomedical Research Facility, Florida State University, Tallahassee, Florida 32306; and 2Zoology and Wildlife Sciences, Auburn University, Auburn, Alabama 36849-5414
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ABSTRACT |
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Fadool, D. A., K. Tucker, J. J. Phillips, and J. A. Simmen. Brain Insulin Receptor Causes Activity-Dependent Current Suppression in the Olfactory Bulb Through Multiple Phosphorylation of Kv1.3. J. Neurophysiol. 83: 2332-2348, 2000. Insulin and insulin receptor (IR) kinase are found in abundance in discrete brain regions yet insulin signaling in the CNS is not understood. Because it is known that the highest brain insulin-binding affinities, insulin-receptor density, and IR kinase activity are localized to the olfactory bulb, we sought to explore the downstream substrates for IR kinase in this region of the brain to better elucidate the function of insulin signaling in the CNS. First, we demonstrate that IR is postnatally and developmentally expressed in specific lamina of the highly plastic olfactory bulb (OB). ELISA testing confirms that insulin is present in the developing and adult OB. Plasma insulin levels are elevated above that found in the OB, which perhaps suggests a differential insulin pool. Olfactory bulb insulin levels appear not to be static, however, but are elevated as much as 15-fold after a 72-h fasting period. Bath application of insulin to cultured OB neurons acutely induces outward current suppression as studied by the use of traditional whole-cell and single-channel patch-clamp recording techniques. Modulation of OB neurons is restricted to current magnitude; IR kinase activation does not modulate current kinetics of inactivation or deactivation. Transient transfection of human embryonic kidney cells with cloned Kv1.3 ion channel, which carries a large proportion of the outward current in these neurons, revealed that current suppression was the result of multiple tyrosine phosphorylation of Kv1.3 channel. Y to F single-point mutations in the channel or deletion of the kinase domain in IR blocks insulin-induced modulation and phosphorylation of Kv1.3. Neuromodulation of Kv1.3 current in OB neurons is activity dependent and is eliminated after 20 days of odor/sensory deprivation induced by unilateral naris occlusion at postnatal day 1. IR kinase but not Kv1.3 expression is downregulated in the OB ipsilateral to the occlusion, as demonstrated in cryosections of right (control) and left (sensory-deprived) OB immunolabeled with antibodies directed against these proteins, respectively. Collectively, these data support the hypothesis that the hormone insulin acts as a multiply functioning molecule in the brain: IR signaling in the CNS could act as a traditional growth factor during development, be altered during energy metabolism, and simultaneously function to modulate electrical activity via phosphorylation of voltage-gated ion channels.
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INTRODUCTION |
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One-third of all mammalian proteins are thought to
contain covalently bound phosphate (Hubbard and Cohen
1993). In the cell, phosphorylation is a reversible, dynamic
process involving integrated networks and coordinated actions of
protein kinases and protein phosphatases (reviewed in Hunter 1998
; Sun
and Tonks 1994
). The insulin receptor belongs to a family of related
receptor-linked protein tyrosine kinases that includes insulin growth
factor (IGF) I and II, relaxins, the invertebrate bombyxins, and
molluscan insulin-like peptides (De Meyts et al. 1995
).
The function of insulin signaling in the brain is unclear but has been
widely sought. Insulin and IGF are synthesized by neurons in the
olfactory bulb, hippocampus, and cerebellum (Werther et al.
1990
); are temporally related to local neuronal proliferation
(Bondy et al. 1992
; Giacobini et al.
1995); and are released on depolarization (Boyd et al. 1985
).
Putative functions for insulin in the CNS immediately evoke comparisons
with the large body of research on Diabetes mellitus. This
research has elucidated the insulin signaling system as a flexible
network of interacting proteins (reviewed in White 1997) whereby a
major downstream target for IR and IGF receptor kinases is insulin
receptor substrate (IRS) (Adamo et al. 1989
;
Myers and White 1996
; Skolnik et al.
1993
). IRS has been shown to link the IR kinase with other
proteins by acting as a multisite "docking protein" to bind
signal-transducing molecules containing Src-homology-2 (SH2) and
Src-homology-3 (SH3) domains (Myers and White 1993
; Sun et al. 1991
). In certain areas of the brain such as
the olfactory bulb, however, IRS is weak or absent (Folli et al.
1994
). It is not clear whether there is a different downstream
transduction cascade for insulin signaling in the brain versus that of
the periphery.
Another protein substrate that could serve the role of IRS in the
olfactory bulb is a voltage-gated ion channel, Kv1.3, a mammalian
homologue of the Shaker family that is highly localized to
the olfactory bulb and cortex (Kues and Wunder 1992) and
has been shown to carry a large proportion of the outward current in
olfactory bulb neurons (Fadool and Levitan 1998
). The
Kv1.X family of ion channels contains several tyrosine residues that, when phosphorylated, could serve as recognition sites for
SH2-containing proteins. These channels also contain proline-rich
sequences for protein-protein interactions with SH3-containing protein
kinases (Holmes et al. 1996
); we therefore suggest
another multiply phosphorylated protein substrate, an ion channel,
could serve as the interacting downstream target for brain insulin
signaling, similar to IRS.
Insulin signaling in the adult brain may also be involved in sculpting
and maintaining synaptic circuitry. The synaptic connections in the
olfactory system have long been explored because of the system's
well-known capacity for continual neurogenesis (Graziadei and
Monti-Graziadei 1978). Olfactory receptor neurons that contain specific G-protein-coupled olfactory receptors to encode a given odorant molecule must be regenerated from a basal cell population and
reestablish a proper topographical map within the olfactory bulb to
ensure odor quality coding (Bozza and Kauer 1998
;
Ressler et al. 1994
). Given that receptor-linked
tyrosine kinases have been demonstrated to produce short-term
modulatory changes in neuronal excitability (Bowlby et al.
1997
; Fadool and Levitan 1998
; Huang et
al. 1993
; Jonas et al. 1996
; Tricarico et
al. 1997
; Wang and Salter 1994
; Wilson
and Kaczmarek 1993
) and the finding that the highest brain
insulin-binding affinities, IR density, and IR kinase activity are
localized to the olfactory bulb (Baskin et al. 1983
;
Gupta et al. 1992
; Hill et al. 1986
), we
found this area of the brain to be fortuitous as a model to study
neuromodulation by insulin signaling. This study provides evidence that
the downstream substrate of insulin signaling in the olfactory bulb is
a voltage-gated ion channel, Kv1.3. We show that insulin stimulation of
IR kinase causes multiple phosphorylation of Kv1.3 at discreet tyrosine residues to induce current suppression of the ion channel. The source
of insulin in the olfactory bulb is unknown but the hormone is retained
or elevated in the olfactory bulb after a period of fasting. IR kinase
expression and neuromodulation of Kv1.3 is significantly reduced after
unilateral naris occlusion, which ties insulin signaling to
sensory/odor experience. These findings demonstrate that insulin
signaling in the brain produces modulatory changes at the level of the
ion channel and can be altered by external sensory experience and feeding.
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METHODS |
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Solutions and reagents
Human embryonic kidney (HEK 293) cell patch pipette solution
contained (in mM): 30 KCl, 120 NaCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 2 CaCl2 (pH 7.4). Olfactory
bulb neuron (OBN) patch pipette solution contained (in mM): 145 KCl, 10 HEPES, 10 ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 MgCl2, 0.20 sodium adenosine 5'-triphosphate
(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(hydroxymethyl)aminomethane (Tris), 250 NaCl, 5 ethylenediaminetetraacetic acid (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).
Homogenization buffer (HB) contained (in mM): 320 sucrose, 10 Tris, 50 KCl, 1 EDTA (pH 7.8). All salts were purchased from Sigma Chemical Co.
or Fisher Scientific. Tissue culture and transfection reagents were
purchased from Gibco/BRL. TTX was purchased from Calbiochem. Human
recombinant insulin was purchased from Roche (Boehringer Mannheim).
cDNA constructs and antibodies
Kv1.3 channels were expressed transiently in human embryonic
kidney (HEK 293) cells using the Invitrogen vector pcDNA3.
Kv1.3 was ligated into pcDNA3 at the unique Hind
III site of the multiple cloning region, placing the channel-coding
region downstream from a CMV 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 Sal I and Xba I, and
ligated into pcDNA3 between the Xho I and
Xba I sites in the multiple cloning region. A
kinase-deficient insulin receptor (IRtrunc) was constructed by
truncating 417 bases of the 3' IR-coding region. This mutant has an
unstable subunit, has no kinase activity in vitro or in vivo and
does not mediate insulin-stimulated uptake of 2-deoxyglucose (Ellis et al. 1986
). The IRtrunc was constructed by the
use of a unique Mst I site (bp 3944) in the IR cDNA. IR pECE
was double-digested with Sal I and Mst I, and the
~4.4-kb fragment was gel-purified. pcDNA3 was
linearized with Xho I and Xba I. Both the
fragment and vector were filled in with Klenow (Sambrook et al.
1989
) and blunt-end ligated. Junctions were sequenced by the
use of a cycle-sequencing reaction (PRISM) and an automated sequencer
(Applied Biosystems Inc., Princeton, NJ) to verify the mutation and
detect PCR errors.
A rabbit polyclonal antiserum, raised against a MalE fusion protein
(New England BioLabs) 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 the olfactory bulb, 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, Inc.) that recognizes
phosphotyrosine. Monoclonal antiserum against
-tubulin III was
purchased from Sigma and used as a neural marker. Polyclonal antibody
directed against the
subunit of the human insulin receptor was
purchased from Upstate Biotechnology.
Naris occlusions
Neonatal unilateral anosmia was established by naris occlusions
by the use of a modification of the techniques described by Meisami
(1976) and Philpot et al. (1997)
as
approved by Auburn University Animal Care Facility and AVMA-approved
methods. Postnatal day (P)1 Sprague-Dawley rats were removed from the
mother and anesthetized with hypothermia for 7 min. The left naris was
cauterized by insertion of a heated 2-mm metal probe, twice for 3 s. A similar protocol applying the probe to the shank of the nose
served as the matched sham treatment. Occluded and sham litter mates
were warmed to 37°C after the protocol and returned to the mother. Litters were culled to 12 animals to ensure equal nursing of sham versus occluded animals during the several-day period of tissue healing. Gloves and minimal contact ensured zero rejection rate by the mother.
Primary cell culture
Olfactory bulbs were harvested from 24-h-old Sprague-Dawley rats
and neuronal primary cultures were prepared using the procedure of
Huettner and Baughman (1986) as modified by Egan et al.
(1992a)
. Animals were killed by decapitation according
to AVMA-approved methods. Olfactory bulbs were removed quickly from the
cranium and placed into 10 ml serum-free Dulbecco's modified Eagle
medium (DMEM; Gibco/BRL) 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 Biochemicals)
for 1 h at 37°C in the 5% CO2 incubator.
The bulbs were then washed in DMEM containing 5% fetal bovine serum
(FBS; Gibco/BRL) and 5 mg/ml trypsin inhibitor (Boehringer Mannheim)
for 10 min to stop the enzymatic activity of the papain. Cells were
dissociated by trituration by the use of a graded-size series of
fire-polished siliconized Pasteur pipettes; the resulting neuron and
glia suspension was plated onto poly-D-lysine hydrobromide
(MW 49,300-53,000; Sigma) coated 12-mm glass coverslips, and incubated
in DMEM supplemented with 2% penicillin/streptomycin and 5% FBS
(Gibco/BRL). Cytosine arabinoside (10 µM; Sigma) was added to the
medium for 36 h between days 3 and 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 mo. Neurons were used for patch recording or immunocytochemistry 2 to
32 days after plating. Although the effect of insulin stimulation was
performed as a paired measure within a single neuron and was observed
independent of days in vitro (DIV), voltage-activated currents
increased in magnitude as the neurons developed over DIV. To control
for this variable, entire data sets were collected within 24- to 36-h
intervals for proper statistical comparisons.
Maintenance of HEK 293 cell cultures and transfection
HEK 293 cells were maintained in minimal essential medium (MEM), 2% penicillin/streptomycin, and 10% FBS (Gibco/BRL). Before transfection, cells were grown to confluency (7 days), dissociated with trypsin-EDTA (Sigma) and mechanical trituration, diluted in MEM to a concentration of 600 cells/µl, and replated on Corning dishes (Catalog number 25000; Fisher Scientific). cDNA was introduced into the HEK 293 cells with a lipofectamine reagent (Gibco/BRL) 3 to 4 days after cell passage. At the time of transfection, the cells were approximately 20-30% confluent. Lipofectamine and cDNA were allowed to complex for 15 min. The DNA/lipofectamine complex was diluted in 1 ml of serum-reduced OptiMEM (Gibco/BRL), and cells were transfected for 4.5 to 5 h 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 by cotransfecting with pHook (Invitrogen) as a means of rapidly selecting transfected cells. pHook encodes a transmembrane domain from the platelet derived growth factor receptor (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 allowed recognition of transfected cells. More than 85% of bead-labeled cells expressed Kv1.3 current. Double, sequential labeling with antibodies directed against the Kv1.3 channel and insulin receptor, respectively, was used to confirm the uptake and colocalization of more than one cDNA construct per cell. On the basis of confocal microscopic visualization, we observed that if an HEK 293 cell incorporated the cDNA construct for the ion channel, it also incorporated the cDNA for the kinase with a >95% efficiency. Typically, single-channel events could be detected as early as 9 h posttransfection and macroscopic currents were observed in the range of 24 to 60 h.
Site-directed mutagenesis
The parent Kv1.3 clone was propagated in Escherichia
coli DH-1. Plasmid DNA preparation was according to standard
methods by the use of a Qiagen Plasmid Kit (Qiagen Inc.) followed by
phenol-chloroform extraction and ethanol precipitation (Sambrook
et al. 1989). All Kv1.3 channel mutants were constructed with
the use of two sequential polymerase chain reactions (PCRs) in an
Eri-Comp thermocycler (Twin Block System, San Diego, CA), with the use
of Taq polymerase (Promega). The circularized plasmid
containing the channel gene served as the DNA template. For each
tyrosine mutation, three oligonucleotides, each 15-24 bases 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 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 with the use of T4 DNA ligase
(Promega). All resulting mutant constructs were sequenced as described previously.
Electrophysiology of HEK 293 cells
Macroscopic currents in cell-attached membrane patches were
recorded 24-60 h after transfection with the use of an Axopatch-200B amplifier (Axon Instruments). Cells were visualized at ×40
magnification with the use of an inverted microscope equipped with
Hoffman modulation contrast optics (Axiovert 135, Carl Zeiss).
Electrodes were fabricated from Jencons glass (Catalog number M15/10,
Jencons Limited, Bedfordshire, UK), fire-polished to approximately 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 with the use of 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.
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 90 mV, and the voltage was stepped to depolarizing potentials for a pulse duration of 1,000 ms. Stimuli were delivered at
60-s or longer intervals to prevent cumulative inactivation of the
Kv1.3 channel (Marom et al. 1993
). Peak current
amplitudes, channel inactivation kinetics, deactivation kinetics,
voltage at one-half maximum activation, and channel conductance were
measured before and 20-25 min after insulin application to permit a
paired statistical comparison.
Electrophysiology of OBNs
OBNs were voltage-clamped in the whole-cell or cell-attached
recording configuration. Patches were held at 90 mV and stepped to
+40 mV for a pulse duration of 1 or 5 s, at a stimulating interval of 1 min. The effect of insulin on OBN whole-cell currents was tested
as a paired measurement of peak current amplitude, channel inactivation
kinetics, and deactivation kinetics, before and after a 20-min bath
application of insulin. The effect of insulin on OBN unitary currents
was tested by tip-filling the electrode with control patch solution and
backfilling the electrode with insulin to activate the kinase. The
initial currents were taken as the control condition and the modulatory
effect of the kinase could be observed within a 5-min period. This is
in agreement with the average time course for tip diffusion on the
basis of previous experimentation (Fadool and Ache 1992
;
Fadool et al. 1995
; Fadool and Levitan
1998
).
All electrophysiological data were analyzed using software written in
the Levitan Laboratory (Dr. Irwin Levitan, University of Pennsylvania,
Philadelphia, PA), in combination with the analysis packages Origin
(MicroCal Software) and Quattro Pro (Borland International). 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. The
V1/2, the voltage at which half of the
channels were activated, was calculated by fitting normalized peak tail
currents at different holding potentials to a Boltzmann function. The
slope of this function, or the value for the steepness of the voltage
dependence, is reported as k. Differences between control
and treatment groups within single cells were analyzed by paired
t-test. Statistical significance in all tests was defined at
the 0.95 confidence level.
Immunoprecipitation, membrane preparation, and ELISA assay
HEK 293 cells were transfected as described earlier, but at
80-90% confluency and with a total of 7.0 µg cDNA per 60-mm dish. Equal amounts of channel and IR cDNA were mixed. Cells were harvested 2 days posttransfection 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 h with 3 mg/ml Protein A-sepharose (Amersham-Pharmacia), 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 3 µg/ml 4G10 antibody, followed by a 2-h incubation with
Protein A-sepharose and centrifugation as before. The
immunoprecipitates were washed four times with ice-cold PPI solution
(modified to contain 0.1% Triton X-100). Lysates and washed
immunoprecipitates were diluted in sodium dodecyl sulfate (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 4%
nonfat milk and incubated overnight at 4°C in primary antibody
against Kv1.3, then with horseradish peroxidase-conjugated donkey
anti-rabbit secondary antibody (Amersham-Pharmacia) for 90 min at room
temperature. ECL (Amersham-Pharmacia) exposure on Fuji RX film (Fisher)
was used to visualize labeled protein. Films were scanned with a
Hewlett-Packard Photosmart Scanner (model 106-816, Hewlett-Packard) and
analyzed with Quantiscan software (Biosoft, Cambridge, UK).
For immunoprecipitation of tyrosine-phosphorylated proteins from native olfactory bulb neurons, the olfactory bulbs (OBs) from left naris-occluded animals were exposed, but not removed, from the cranium. In this in situ state, brains with intact OBs were stimulated with either optiMEM or 50 µg/ml insulin in optiMEM for 20 min in a 37°C incubator. Right versus left OBs were then removed and homogenized 50 strokes by Kontes tissue grinder (size 20) in ice-cold PPI solution. The lysate clarification, immunoprecipitation, and SDS-PAGE analysis were as described previously for HEK 293 cells.
OB membranes were prepared by harvesting tissue from animals at various
postnatal stages and homogenizing the OBs 50 strokes by Kontes tissue
grinder (size 20) in HB solution on ice. The mixture was centrifuged
twice at ~2,400 g (3,800 rpm) for 30 min at 4°C in an
Eppendorf model 5416 to remove cellular debris. The combined
supernatant was centrifuged in a Beckman ultracentrifuge (model L8-M,
Beckman) at 110,000 g (40,000 rpm) for 2.5 h at 4°C. The resulting pellet was resuspended in HB solution and tip-sonicated on ice three times for 20 s with a Tekmar Sonicator (setting 50). Protein concentration was determined by Bradford assay and samples were
stored at 80°C until use.
Plasma and olfactory bulb insulin levels were determined using a solid-phase two-site enzyme immunoassay (APLCO, Wilham, NH) with slight modification of the manufacturer's defined protocol. Briefly, trunk or left atria blood was collected into heparinized tubes and the plasma fraction was collected by centrifugation at 4°C for 10 min at 14,000 g. OB were homogenized as before but the provided zero-standard solution was used instead of HB solution. Insulin in the plasma or OB samples was reacted for 2 h at 300 rpm at room temperature with peroxidase-conjugated anti-insulin antibodies and anti-insulin antibodies bound to microplate walls. After thorough washing, the bound conjugate was detected by reaction with 3,3',5,5'-tetramethybenzidine (TMB) and reincubated for an additional 30 min at 300 rpm at room temperature. The reaction was quenched with perchloric acid and the colorimetric endpoint was read at 450 nM in a spectrophotometer (model Ultraspec 2000, Pharmacia Biotech).
Immunocytochemistry
For cryosections, whole OBs were fixed in 4% paraformaldehyde
for 3 h followed by overnight infiltration with 10% sucrose, then
4 h with 30% sucrose. OBs were cut to 9- to 12-µm thickness on
a Microm Laborgeräte GmgH microtome-cryostat (Carl Zeiss). Sections were transferred to 1% gelatin-coated glass slides (Sigma) and stored at 20°C until use. Before immunolabeling, slides were briefly fixed in 1% paraformaldehyde for 5 min to promote adhesion of
the section to the gelatin slide. Cultured OBNs and transfected HEK 293 cells were rinsed once in PBS and then lightly fixed in St. Marie
Fixative (95% EtOH/5% acetic acid) for 10 min at
20°C. The
fixative for cultured cells or cryosections was removed by rinsing with
two changes of PBS for 10 min each, two changes of PBS with 0.1%
Triton X (PBST), and nonspecific binding was blocked by incubation for
30 min in PBST containing 1-2% Albumin Fraction V (fatty acid free;
Sigma) (Block). Cryosections or cultured cells were incubated with
primary antiserum diluted in Block for 90 min at room temperature,
washed with three changes of PBST, and then were reincubated for 90 min
at room temperature with a fluorescein-conjugated goat anti-rabbit
secondary antibody (Boehringer-Mannheim) in Block. Sections or cultured
cells were washed with two changes of PBST for 10 min, two changes of
PBS for 10 min, rinsed in millipore water, and mounted in 60/40
glycerol/PBS with 0.02% p-phenylene-diamine added to
prevent photobleaching. Photomicroscopy was performed at ×40 with the
use of a CH-2 Olympus microscope equipped with epifluorescence. Laser
confocal microscopy was performed on a BioRad MRC1000 fitted with a
Zeiss Axioskop using a ×40 Plan neofluor objective. Changes in
fluorescence intensity were compared with the use of a quantitative
densitometric analysis of pixel density computed by Quantiscan software (Biosoft).
Counting of immunopositive cells was performed by examining five fields
of view in each of two to four experiments. The examiner was blind to
the experimental condition. The mean ± SE was calculated for the
number of immunolabeled cells across approximately 40 neurons in the
field of view. Cells were confirmed as neurons by double-labeling with
-tubulin III as a neural marker.
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RESULTS |
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Insulin-induced current suppression is kinase activity dependent
Kv1.3 plus IR kinase cotransfected HEK 293 cells were
voltage-clamped in the cell-attached configuration and stepped to
various depolarizing potentials from rest (90 mV) under control and
insulin-stimulated conditions. Stimulation with 0.1 µg/ml insulin
caused a significant decrease in peak current magnitude of the Kv1.3
ion channel (Fig. 1, paired
t-test; control = 1,124 ± 146 pA; plus insulin
832 ± 148 pA, n = 7). Modulation of the channel
was acute, occurring in the window of 20 min. Only the peak current
magnitude was significantly modulated by activation of the kinase;
channel inactivation kinetics, deactivation kinetics, conductance, and
voltage dependence were not affected (Fig. 1B, Table
1). Interestingly we found that the
modulation of Kv1.3 was dependent on the concentration of insulin
activating the IR kinase: 0.1-1.0 µg/ml insulin evoked current
suppression, whereas 50-100 µg/ml insulin induced only a slowing of
the deactivation kinetics and no change in other Kv1.3 current
properties (paired t-test, see Table 1). Because higher
concentrations of insulin can potentially cross-react with the IGF-I or
-II receptor expressed endogenously in the HEK 293 cells or in native
OB neurons (Rotwein et al. 1988
; Smith et al. 1988
; Werther et al. 1990
), we elected to
perform all subsequent experiments at the lower concentration range.
Reported brain insulin contained within the OB and its half-maximum
binding affinity is within this physiological range (Baskin et
al. 1983
; Gupta et al. 1992
).
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The Kv1.3 ion channel and the IR kinase could not be
coimmunoprecipitated, whether with the use of our heterologous
expression system (HEK 293 cells) or immunoprecipitating the native
proteins from the OB (data not shown), thereby indicating that the two proteins did not directly or strongly interact. We thus tested whether
neuromodulation of Kv1.3 by insulin activation of IR kinase involved
phosphorylation as opposed to a direct coupling. A kinase-deficient insulin receptor (IRtrunc) was constructed by truncating 417 bases of
the IR-coding region. This change to the IR kinase is known to cause
instability of the subunit and deletion of its subsequent kinase
activity via blocking autophosphorylation (Ellis et al. 1986
). HEK 293 cells cotransfected with Kv1.3 + IRtrunc
displayed a peak current magnitude that was not significantly different after insulin stimulation (paired t-test; control = 1,304 ± 246 pA; plus insulin = 1,171 ± 192 pA,
n = 10).
To ensure that the expressed channel and kinase were colocalized to
the same HEK 293 cell, we examined the efficiency of cotransfecting the
respective cDNA constructs with the use of confocal microscopy combined
with double-labeling for the channel and the kinase (Fig. 1C). These studies consistently confirmed that if a cell
took up the cDNA for one construct, it took up both cDNAs (or more) nonselectively. Although we could not quantify the absolute equivalence of the expression of each protein within a single cell,
immunofluorescent signal was observed for both the channel and the
kinase in over 98% of observed cells (n = 20 fields of
view), assurance that expression of the two proteins was colocalized.
General transfection efficiency using lipofectamine reagent was high
(60-80%) as confirmed by -galactosidase staining of Lac Z + Kv1.3
cotransfected HEK 293 cells. Efficiency was highly dependent on cell
density (percentage confluence), cDNA concentration and purity, and
attachment substrate, conditions that were optimized as stated in the
METHODS section.
Multiple sites for tyrosine phosphorylation revealed by mutagenesis
Because kinase activity was necessary for insulin-induced current
suppression of Kv1.3 ion channel by IR (Fig. 1) and activation of the
IR has been shown to phosphorylate the ion channel (Bowlby et
al. 1997), we explored whether removal of the tyrosine
phosphorylation motif in the ion channel could reverse both the
tyrosine phosphorylation of Kv1.3 and its physiological modulation by
insulin. Single-point Y to F mutations were made at six sites in the
Kv1.3 channel sequence (Fig.
2A) that contained strong
consensus sequences (flanking acidic residues and downstream
hydrophobic residues) for tyrosine-specific phosphorylation based on
combinatorial peptide libraries (Sun and Tonks 1994
).
The triple-Y site at 111-113 was treated as a unit and mutated as a
single cassette.
|
Wild-type Kv1.3 or one of the four Kv1.3 mutant channels was
cotransfected with IR kinase and tested for insulin-induced modulation of current magnitude (Fig. 2B). Cotransfected HEK 293 cells
were held at rest (90 mV) and stepped to a single depolarizing
potential at an interpulse interval of 1 min to prevent cumulative
inactivation of Kv1.3 current. Most patch recordings could be held for
0.5 h under this repetitive stimulation protocol. After allowing
the cell to stabilize for 5 min, insulin was applied to the bath of the
recording chamber. The value for peak current magnitude under control
conditions was taken just before insulin application and the paired
insulin treatment value was taken at the end of the 0.5-h period. Y449F
Kv1.3 + IR kinase-transfected HEK 293 cells exhibited a significant
decrease in mean current magnitude of 195 ± 72 pA (paired
t-test, n = 8), which correlates to a
percentage decrease of 22%. This was not unlike the WT Kv1.3 + IR
kinase-transfected cells that responded with a 26% decrease in mean
current magnitude (n = 12) in response to insulin
stimulation (Fig. 2B). All other Kv1.3 mutant channels
(YYY111-113FFF Kv1.3, Y137F Kv1.3, and Y479F Kv1.3) coexpressed with
IR kinase exhibited no significant change in mean current magnitude in
response to insulin stimulation (paired t-test of mean
current values) that correlated to a 7% decrease for Y137F Kv1.3
(n = 9), a 4% decrease for Y479F Kv1.3
(n = 9), and a 6% decrease for YYY111-113 Kv1.3
(n = 6); compared with WT Kv1.3 no insulin stimulation
responded with a 6% decrease (n = 9) over the 0.5-h
recording period.
Identical cotransfections as in the preceding patch-clamp experiments were established for the WT or Kv1.3 ion channel mutants and IR kinase to test for tyrosine-specific phosphorylation by immunoprecipitation of tyrosine-phosphorylated proteins. Immunoprecipitates were separated by SDS-PAGE, followed by Western analysis that probed for Kv1.3 ion channel (Fig. 2C). WT Kv1.3 or Y449F Kv1.3 plus IR kinase-cotransfected HEK 293 cells demonstrated increased tyrosine phosphorylation of the Kv1.3 ion channel after 20-min stimulation with the hormone insulin. Quantitative densitometry indicated that WT Kv1.3 and Y449F Kv1.3 showed a 1.9 ± 0.2-fold (n = 11) and a 3.9 ± 1.3-fold (n = 6) increase, respectively, in the presence of insulin stimulation over that of basal phosphorylation (Fig. 2D). Three of the channel mutants, YYY111-113FFF, Y137F, and Y479F, however, showed no increase in phosphorylation in the presence of insulin (Fig. 2D) [fold increase for YYY111-113FFF Kv1.3 = 1.0 ± 0.1 (n = 5), for Y137F Kv1.3 = 1.1 ± 0.1 (n = 5), and Y479F Kv1.3 = 1.5 ± 0.3 (n = 5)]. These data indicate that three tyrosine residues (tyr111-113, tyr137, and tyr479) are targets for insulin-induced current suppression of Kv1.3 ion channel by tyrosine phosphorylation; with the removal of these tyrosine targets, insulin-induced increase in phosphorylation is not observed. Combined analysis of data in Figs. 1 and 2 strongly suggests that tyrosine phosphorylation at multiple sites is coupled to current suppression of the Kv1.3 ion channel.
Whole-cell and unitary currents are suppressed by insulin stimulation of native olfactory bulb neurons
We next pursued whether insulin neuromodulation of native currents
occurred in the olfactory bulb. Our previous work has shown that a
large component of the outward voltage-activated current in
voltage-clamped whole-cell recordings of olfactory bulb neurons is
carried by a Kv1.3-like current that is based on several properties including the blockability of the current by a selective scorpion toxin, margatoxin (Fadool and Levitan 1998).
Bath-applied insulin significantly suppressed outward current in native
olfactory bulb neurons (Fig. 3, paired
t-test; control = 1,541 ± 141 pA; plus insulin = 1,170 ± 106 pA, n = 7). Similarly
to what was found in Kv1.3 + IR-cotransfected HEK 293 cells, modulation
of the neurons affected the peak current magnitude and not the kinetics
of inactivation or deactivation (Table
2). There is similarity between the
insulin-like growth factor I (IGF I) and insulin receptors; each
receptor binds to its specific ligand but may also cross-react with the
other receptor at lower affinity. Because of this fact, we questioned whether insulin-like growth factor, that binds with only 10- to 100-fold less affinity, could cross-react with IR kinase in these neurons. To test this possibility we applied 50 ng/ml IGF I to OBNs and
found there was no acute modulation of Kv1.3 current properties (Fig.
3, paired t-test; control = 783 ± 82 pA; plus IGFI = 684 ± 66 pA, n = 8).
|
|
With the use of patch-recording solutions that minimize the activation of calcium- or sodium-activated potassium currents (see METHODS), Kv1.3 unitary currents can be recorded in the cell-attached configuration and readily identified by the characteristic C-type inactivation observed under a long depolarized step of 5 s. The patch electrode was first tip-filled with control patch solution (Fig. 4A, Control) and then back-filled with 0.1 µg/ml insulin (Fig. 4A, Insulin). This allowed a baseline recording under control conditions before gravity perfusion of the hormone (see METHODS). Because of the low probability of recording one channel in the patch as opposed to several, combined with the property of cumulative inactivation for these channels, this approach did not yield a large number of single-channel transitions for extensive nonstationary kinetic analysis. Nonetheless, with the use of this recording paradigm, patches maintained with a back-filled insulin electrode displayed channel activity that moved into a closed or inactivated state within four to five voltage-stimulating sweeps and failed to reopen (n = 3). Patches that were back-filled with control patch solution (n = 4) maintained channel activity for the duration of the recording (up to 1 h).
|
Because Kv1.3 exhibits nonstationary behavior, ensemble averaging was
performed on the single-channel data by averaging idealized records
under control and insulin-exposed conditions. A time-dependent open
probability (Propen) was calculated as:
Propen (at t = i seconds) = (number of records with channel open at
t = i)/(total number of records in the
ensemble) (Aldrich and Yellen 1983). Because
Propen changes with time, the probability that
the channel was open was computed by finding all the detectable opening
and closing transitions and constructing a schematic representation that had a value of 0 when the channel was closed, and 1 when it was
open. By performing this calculation every 500 µs for eight uniform
voltage steps of 5-s duration, a representation of the channel open
probability as a function of time and treatment was created (Fig.
4B). The ensemble mean number of channels open at time
t = 2 s was calculated as:
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Plasma and brain insulin measurements under feeding and fasting conditions
To probe the capacity of native Kv1.3 neuromodulation by insulin, we chose to measure insulin levels in both the plasma and the olfactory bulb of feeding adult rats (Fig. 5). Adult rats allowed to freely feed for 72 h had a plasma insulin level of 2.6 ± 1.0 ng/ml (n = 6), whereas rats that fasted for the same time interval had a significantly reduced plasma insulin level of 0.06 ± 0.04 ng/ml (n = 3; Student's t-test). This represents a mean 43-fold increase in plasma insulin levels on feeding. Interestingly, the opposite response occurred in the olfactory bulb in response to feeding/fasting. Insulin levels thereby decreased in the olfactory bulb on feeding and significantly increased 15-fold on a 72-h fast [control = 0.25 ± 0.1 ng/g wet tissue homogenate (n = 5) and fasted = 3.7 ± 1.3 ng/g wet tissue homogenate (n = 4), Student's t-test]. Insulin levels were not as high in P20 animals as compared with adults; however, the same response to fasting was measured. P20 rats demonstrated a fourfold increase in OB insulin (control = 0.18 ng/g wet tissue homogenate and fasted = 0.74 ng/g wet tissue homogenate, n = 6-8 each, significantly different Student's t-test).
|
Localization and developmental expression of Kv1.3 and IR kinase in the olfactory bulb
Once we confirmed the presence of significant levels of insulin in
the olfactory bulb, we explored the localization and developmental expression of the receptors for the ligand. Purified membranes from
adult rat olfactory bulbs were separated by SDS-PAGE followed by
Western analysis. The nitrocellulose was probed with antiserum against
the subunit of the human IR kinase. A band of approximately 120 kDa
was observed in three brain regions (olfactory bulb, cerebral hemisphere, and cerebellum) that was absent in nonneural tissue (Fig.
6A). IR kinase was
developmentally expressed in olfactory bulb membrane harvested from
animals at various postnatal stages (P1 to P16) and the kinase
persisted in the adult animal, which was defined at P60 (Fig.
6B).
|
The developmental expression of Kv1.3 and IR kinase was followed in
cryosections of rat olfactory bulb harvested at various postnatal
stages over a 1-mo time period (P1 to P29) (Fig.
7). If the Kv1.3 ion
channel is modulated in native olfactory bulb, it would be beneficial
to demonstrate that the two proteins were localized to the same cell
types and during the same developmental time point. Unfortunately, the
best commercially available IR kinase antibody that we tested in
cryosections was a rabbit polyclonal antiserum, so we were unable to
design traditional double-immunolabeling experiments with the Kv1.3
channel (also a rabbit polyclonal). Moreover, we were not successful in
sequential double-labeling experiments for the channel and the kinase,
which always demonstrated less intense immunolabeling with the antibody
applied first in the series, most likely the result of washing and
reincubation with the second primary antiserum (data not shown).
Instead we double-labeled cryosections with the mouse monoclonal neural
marker tubulin III and compared kinase and channel labeling in
sequentially cut sections (Fig. 7B). As also demonstrated by
Western blot analysis (Fig. 6B), IR kinase was not strongly
expressed in the first few days of postnatal development but Kv1.3 was
ubiquitously expressed nonselectively throughout the bulb. During the
first 10 days to 1 mo (P10-P29), both proteins became strongly
expressed in the outer nerve layer (ONL). After 3 wk (P20-P29)
distinct labeling of the dendrites of the mitral cells leading from the
glomerular layer (GML) and strong expression in the external plexiform
layer (EPL) was additionally visible. Both Kv1.3 and IR kinase labeled the inner granule cell layer (GCL, see Fig. 7A) but the
expression of these proteins in this layer was independent of postnatal
development (data not shown). In primary cell culture, olfactory bulb
neurons increased labeling for Kv1.3 and IR kinase respectively, with days in vitro (DIV), as imaged by confocal microscopy through day 10 (Fig. 8A). Neurons with
bipolar and tripolar morphology, indicative of granule and mitral cell
types, respectively, both contained Kv1.3 and IR kinase as evidenced by
immunolabeling and similar electrophysiological modulation by insulin.
A total of 136 cells across 40 micrographs were examined for IR or
Kv1.3 fluorescent signal intensity on DIV 2, 5, and 7. A total of three measurements were taken per neuron and each measurement was
background-subtracted before calculation of a mean signal intensity for
that cell. Three to five neurons were sampled per micrograph (Fig.
8B). The line graphs in Fig. 8B imply a
time-dependent increase in protein expression over DIV for both of
these proteins.
|
|
Odor/sensory deprivation alters phosphorylation, IR kinase expression, and neuromodulation of Kv1.3
Because IR kinase expression was highly localized to the outer
nerve, glomerular, and external plexiform layers and this pattern of
expression was developmentally regulated, we hypothesized that IR
kinase and its subsequent neuromodulation of Kv1.3 ion channel may be
involved in odor sensation or coding. We tested this hypothesis by
performing left-naris occlusions by cauterization 24 h after birth
and compared IR kinase expression, Kv1.3 expression, and total tyrosine
phosphorylation in the olfactory bulb ipsilateral to the occlusion and
its contralateral control 20 to 30 days after sensory/odor deprivation.
We observed what previous studies have shown (reviewed in Brunjes
1994), that there is a differential in size of the olfactory bulbs
after naris occlusion. The left olfactory bulb was significantly
smaller (approximately 19%) in size (0.023 ± 0.0009 vs.
0.029 ± 0.001 g, paired t-test, n = 11) than the contralateral control olfactory bulb as can be visualized in the photograph in Fig. 9A.
Naris occlusion reduced the thickness of the ONL, disrupted the ordered
array of the glomeruli, reduced the size of the external plexiform
layer, and the demarcation between the mitral and granule cell layer
was lost (Fig. 9B). Even though the total size of the
olfactory bulb was reduced and the ordered array of the neural lamina
was clearly disrupted by naris occlusion, anti-
tubulin III strongly
labeled the ONL, GML, and EPL in both the control and naris-occluded
olfactory bulb at P30 (Fig. 9C, left).
Double-labeling this same section with anti-IR kinase demonstrated a
marked reduction in IR kinase signal in all three regions (Fig.
9C, right). IR kinase immunolabeling in the GCL
did not appear affected by 30 days of naris occlusion (data not shown).
Mean decrease in IR fluorescence signal as measured as a percentage
drop in pixel density from the right (control) to the left
(naris-occluded) olfactory bulb was the following: 54 ± 3% in
the ONL, 27 ± 4% in the GML, 13 ± 5% in the EPL, and 3 ± 5% in the GCL. These data were collected by densitometric analysis of 10 individual sections generated from three different litters of naris-occluded animals.
|
Left (ipsilateral to naris occlusion) and right (contralateral control)
olfactory bulb tissue were independently placed into primary cell
culture 20 days after odor/sensory deprivation. Cultures derived from
naris-occluded animals contained a higher proportion of fibroblasts and
a lesser proportion of total neurons than that of control cultures.
Cultures derived from naris-occluded versus control animals were plated
at densities so as to contain approximately equal neurons as indexed by
anti- tubulin III immunoreactivity. In double-blind experiments, 10 to 20 fields of view were counted for Kv1.3 and IR kinase
immunoreactive neurons across the two culture subtypes. Cultures
derived from naris-occluded animals had significantly fewer neurons
immunoreactive for IR kinase but a similar number of neurons
immunoreactive for Kv1.3 (Student's t-test, Fig.
10A). Voltage-clamp
recordings taken from these two sets of cultures demonstrated similar
mean outward current magnitude and kinetics, implying that the
expressed Kv1.3 ion channel protein in these P20 cultured neurons was
not functionally altered (Table 2); however, neurons cultured from
naris-occluded animals lacked insulin-induced current suppression of
the outward Kv1.3 current (Fig. 10B). These data suggest
that kinase activity in the olfactory bulb is dependent on sensory
experience in the naris.
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DISCUSSION |
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The discovery of the hormone insulin and IR kinase in the CNS has
prompted many investigators to reconsider the physiological role for
insulin signaling in the brain, once considered an insulin-independent organ (Zaia and Piantanelli 1997). We show that insulin
induces current suppression of a predominant voltage-gated ion channel (Kv1.3) in the olfactory bulb. Modulation is the result of tyrosine phosphorylation of Kv1.3 channel protein at multiple discreet sites
similar to that of IRS, the traditional downstream target for IR kinase
in the periphery. IR kinase is developmentally expressed in the neural
lamina of the OB and the pattern of expression is disrupted after 20 to
30 days of odor/sensory deprivation induced by unilateral naris
occlusion. Modulation of OB neuronal outward current by insulin is
activity dependent and is not observed after naris closure. The
continual regulation of neuronal excitability by brain IR kinase could
be one of many physiological roles for insulin signaling in the
CNS
short-term modulation of ongoing electrical activity to fine-tune
developing and ongoing neuronal circuits via phosphorylation.
Our data indicate that insulin activation of IR kinase in a
heterologous expression system induces current suppression and concomitant tyrosine phosphorylation of Kv1.3 ion channel. Modulation of the channel is dependent on the kinase activity of IR, as
demonstrated by the lack of Kv1.3 current suppression induced by the IR
mutant (IRtrunc). We do not know, however, whether the signaling
cascade is direct or whether there is an intermediate step between IR kinase activation and Kv1.3 channel tyrosine phosphorylation. nShc is
an example of an adaptor protein that is readily phosphorylated by IR
kinase (Nakamura et al. 1998; Paez-Espinosa et
al. 1998
), is highly expressed in the OB (Yip et al.
1980
), and could recruit a variety of protein modules to affect
Kv1.3 channel properties or phosphorylation state via SH2 or SH3
recognition sites in the ion channel. The cellular tyrosine kinase src
is also highly expressed in the OB independent of postnatal development
(data not shown) and we have previously demonstrated that
Srcpp60 perfused OBNs undergo a time-dependent
decrease in current magnitude (Fadool and Levitan 1998
).
Hilborn and colleagues (1998)
have demonstrated that
members of the receptor-linked tyrosine kinases modulate sodium channel
properties indirectly via src signaling pathways. That insulin induces
current suppression of outward currents in native olfactory bulb
neurons as well as in Kv1.3 + IR cotransfected HEK 293 cells implies
that insulin activation of IR kinase triggers the same event in both
systems, regardless of whether it is a direct or indirect mechanism.
In the olfactory bulb, soon after the action potential discharge begins
in the mitral cell (Kauer 1991), the pattern and
intensity of the discharge is shaped by the action of local circuits
(Hamilton and Kauer 1989
) and the inherent ionic
conductances of the neurons (Mori 1987
). K channels make
major contributions to these conductances (Egan et al.
1992b
). To understand the capacity of electrical signaling in
the OB, one must ultimately elucidate the mechanisms by which channel
proteins respond to biochemical changes at specific modulatory sites
(Levitan 1994
). Site-directed mutagenesis of tyrosine phosphorylation recognition motifs in Kv1.3 demonstrates that multiple sites in a single protein are modulated by IR kinase activation. Although our investigation was restricted to Kv1.3 channel
protein, there are other types of potassium voltage-gated ion channels
that underlie currents in mitral and granule cells (e.g., Kv1.4; A-type
currents) (Bardoni et al. 1996
; Chen and Shepherd
1997
) and which contain similar consensus sequences for tyrosine phosphorylation (Jonas and Kaczmarek
1996
). GABAminergic and glutaminergic synaptic pathways have
been well defined in specific olfactory connections involved in odor
processing (reviewed in Issacson and Strowbridge 1998
; Shipley and
Ennis 1996
; Trombley and Shepherd 1993
) receptors that could be
modulated by IR kinase phosphorylation, as has been demonstrated in
other cell systems for
-adrenergic,
-aminobutyric acid (GABA),
and N-methyl-D-aspartate (NMDA) receptors
(Chen and Leonard 1996
; Karoor et al.
1998
; Wang and Salter 1994
).
The brain insulin receptor and
subunits have different
molecular weights because of differences in carbohydrate moieties of
the peripheral IR kinase (Heidenreich et al. 1983
;
Hendricks et al. 1984
; Yip et al. 1980
).
In the olfactory bulb and two other brain regions we found that the IR
kinase
subunit was slightly larger (~120 kDa) than that reported
for the peripheral IR kinase (97 kDa) (Waldbillig and LeRoith
1987
). It has been suggested that the brain IR may be a hybrid
consisting of an
insulin receptor subunit and an
IGF
I receptor subunit (Moxham et al. 1989
;
Waldbillig and LeRoith 1987
). The hybrid receptors would have dual specificity for insulin and IGF I. IGF I IR has been reported
in the GML, EPL, and MCL of the olfactory bulb (Bondy et al.
1992
; Russo et al. 1994
; Werther et al.
1990
), which is not unlike our present observed distribution
for IR kinase in these neural lamina (Fig. 7). The potential for a
hybrid receptor therefore exists; however, the dual specificity of
ligand activation is unclear given that IGF I appears not to modulate
Kv1.3 channel properties in these neurons (Table 2).
IR knock-out mice have been shown to display normal features at birth,
but on feeding die of diabetic ketoacidosis within a few days
(Accili 1997). By using naris occlusion, we were able to
restrict the IR kinase decrease in expression, presumably, to only the
OB to discern whether IR kinase or tyrosine phosphorylation was tied
with sensory experience. Our data indicate that there is a decrease in
tyrosine phosphorylation after naris occlusion and a reduction of IR
kinase in the ONL, EPL, and MCL. Based on autoradiography studies
(Baskin et al. 1983
; Young et al. 1980
) and insulin binding sites in subcellular fractions (dendrodendritic synaptosomes) (Matsumoto and Rhoads 1990
), insulin has
been proposed as a modulator of mitral-granule cell synaptic
transmission as well as final output of the mitral cells to the
olfactory cortex (Baskin et al. 1983
). Sensory
experience or a developmental critical period appears to be essential
in expression of insulin receptors in the external plexiform and mitral
cell layers that contain these synaptic connections (Fig. 9). The most
marked change after naris occlusion, 30-day lack of sensory input, was
the decrease of IR kinase in the ONL. The failure of axonal maintenance
from the periphery was disrupted as was the robust expression of IR kinase in these axons. Activation of IR kinase may be necessary in this
area of the brain that supports continual synaptogenesis and neuronal
remodeling of peripheral axons throughout adult life to fine-tune the
proper topographical map for odor coding within the OB. Future
experimentation in this area is needed to explore this potential
function of brain insulin signaling.
In diabetic-induced rats, levels of plasma insulin appreciably fall,
whereas levels of brain insulin receptor binding and total IR protein
remain unaltered (Gupta et al. 1992). Diabetes also
causes an increase in brain IR kinase activity that is markedly localized to the olfactory bulb (Folli et al. 1994
;
Gupta et al. 1992
). This is not unlike what we observed
in the olfactory bulb, ipsilateral to naris occlusion. Whereas the
total IR protein was not altered across the control and naris-occluded
treatments (data not shown; SDS-PAGE), IR immunoreactivity was reduced
in discreet cell types (Figs. 8 and 9). The kinase activity, however,
was altered in these neurons, as evidenced by changes in total tyrosine phosphorylation (data not shown) and lack of insulin-induced current suppression (Fig. 10). Clearly alteration of IR kinase activity induced
by a metabolic or other disease state (reviewed in Wickelgren 1998
)
would alter the current properties of Kv1.3 as did changes in IR kinase
activities produced by sensory/odor deprivation.
It is possible that insulin in the brain could also be used as a
satiety factor. After a meal, blood insulin levels would rise in
response to increased serum levels of glucose. This peripheral insulin
is conjectured to cross the blood-brain barrier and chaperon glucose
into brain neurons. Our data suggest that there is a clear differential
between the levels of insulin in the plasma and that found in the OB.
OB insulin levels are low after a meal. Eating has been shown to alter
the response of the olfactory system to sensory input (Cain
1975). Insulin-induced modulation of electrical signaling in
the OB could afford a potential mechanism. In alignment with this
hypothesis is the fact that brain insulin is reported to be highly
retained during periods of starvation (Cashion et al.
1996
). Our data indicate that insulin is elevated in the
olfactory bulb after a 72-h fast. This elevation in insulin could
modulate total outward current in OBNs to change the shape and duration of action potentials produced in these neurons. Further experiments are
required to delineate whether insulin is produced locally by the OBNs
in response to fasting or whether it is retained or even selectively
pumped across the blood-brain barrier.
Insulin receptor kinase modulates the activity of a predominant
voltage-gated ion channel by tyrosine phosphorylation at multiple sites
in the amino and carboxyl terminus of Kv1.3 ion channel protein. In a
region of the brain where IRS expression is reported weak or totally
absent (Folli et al. 1994), the downstream docking protein for insulin receptor activation may be the ion channel itself.
Kv1.3 ion channel and IRS are similar in that they both lack
Asp-Phe-Gly and Ala-Pro-Glu motifs diagnostic of a protein kinase;
neither has endogenous kinase activity or undergoes autophosphorylation (Sun et al. 1991
). As the putative roles for the brain
IR are explored, it will be important not to limit its potential role to a single function, especially given the regenerative nature of the
olfactory system, where a molecule acting as a combined neuromodulator,
mediator of energy metabolism, and a growth factor would be well served.
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ACKNOWLEDGMENTS |
---|
The authors thank Drs. Ray Henry, Steve Kempf, Mary Mendonca, Sadik Tuzan, Marie Wooten, and Michael Wooten for access to technical equipment necessary to perform our study. We thank T. Tabb for technical assistance; D. Person, B. Rimel, and S. Wazeerud-din for assistance with the occlusions and ELISA protocols; and Drs. Jim Fadool, Frank Simmen, and Vitaly Vodanyoy for constructive reading of the manuscript.
This research was supported by National Institute on Deafness and Other Communication Disorders Grant R29DC-03387 to D. A. Fadool, a Howard Hughes Precollege Life Scholars Award at Auburn University to J. A. Simmen, and an undergraduate scholarship from the Department of Microbiology/Botany of Auburn University to J. J. Phillips.
Present addresses: K. Tucker, Biomedical Research Facility, Dept. of Biological Sciences, Florida State University, Tallahassee, FL 32306; J. J. Phillips, University of Alabama at Birmingham, School of Medicine, Birmingham, AL 35216-6903; J. A. Simmen, Yale University, Undergraduate Studies, New Haven, CT 06520.
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FOOTNOTES |
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Present address and address for reprint requests: D. A. Fadool, Biomedical Research Facility, Dept. of Biological Sciences, Florida State University, Tallahassee, FL 32306.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 October 1999; accepted in final form 5 January 2000.
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REFERENCES |
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