1Whitney Laboratory, University of Florida, St. Augustine 32086; and 2Department of Zoology, 3Department of Neuroscience, and 4Center for Smell and Taste, University of Florida, Gainesville, Florida 32610
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Zhainazarov, Asylbek B.,
Richard Doolin,
John-David Herlihy, and
Barry W. Ache.
Odor-Stimulated Phosphatidylinositol 3-Kinase in Lobster
Olfactory Receptor Cells.
J. Neurophysiol. 85: 2537-2544, 2001.
Two antagonists of phosphoinositide
3-OH kinases (PI3Ks), LY294002 and Wortmannin, reduced the magnitude of
the receptor potential in lobster olfactory receptor neurons (ORNs)
recorded by patch clamping the cells in vivo. An antibody directed
against the c-terminus of human PI3K-P110 detected a molecule of
predicted size in the outer dendrites of the ORNs. Two
3-phosphoinositides, PI(3,4)P2 (1-4 µM) and
PI(3,4,5)P3 (1-4 µM) applied to the
cytoplasmic side of inside-out patches taken from cultured lobster
ORNs, reversibly activated a Na+-gated channel
previously implicated in the transduction cascade in these cells.
3-Phosphoinositides were the most effective phosphoinositide (1 µM)
in enhancing the open probability of the channel. Collectively, these
results implicate 3-phosphoinositides in lobster olfactory transduction and raise the need to consider the 3-phosphoinositide pathway in olfactory transduction.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphoinositides are
integral membrane constituents in eukaryotic cells that also function
in transmembrane signaling (review: Zhang and Majerus
1998). The discovery of D-3 phosphorylated inositol lipids and
their synthesis by a family of phosphoinositide 3-OH kinases (PI3Ks)
has fostered important new insight into cell signal transduction
(Fruman et al. 1998
; Toker and Cantley
1997
). With different specificity, these enzymes phosphorylate
phosphatidylinositol (PI) itself, phosphatidylinositol 4-phosphate
[PI(4)P], and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] in
the D-3 position of the inositol ring to generate phosphatidylinositol
3-phosphate [PI(3)P], phosphatidylinositol 3,4-bisphosphate
[PI(3,4)P2], and phosphatidylinositol
3,4,5-trisphosphate [PI(3,4,5)P3], respectively.
In higher animals, at least, there is a relatively large, constitutive
pool of PI(3)P present in resting cells, in contrast to endogenously
low levels of PI(3,4)P2 and
PI(3,4,5)P3 that are transiently and rapidly
elevated in response to a wide range of external stimuli and are
thought to have signaling function (review: Kapeller and Cantley
1994). The latter 3-phosphoinositides are generated by a
family of heterodimeric PI3Ks, isoforms of p110 PI3Ks, consisting of a
110-kDa catalytic subunit and an associated 50- to 100-kDa
noncatalytic, regulatory subunit. While they are capable of
phosphorylating PI and PI(4)P in vitro, they exhibit a preference for
PI(4,5)P2 as a substrate within cells
(Hawkins et al. 1992
). These enzymes can be activated by
a wide array of ligands such as hormones, neurotransmitters, growth
factors, and cytokines acting through both tyrosine kinases and
G-protein-coupled receptors, depending on the particular isoform
(Tang and Downes 1997
). The G-protein-activated PI3K
(p110gamma) appears to be a distinct form of the enzyme. It associates
with a noncatalytic p101 subunit that is responsible for the substrate
selectivity of the enzyme by sensitizing the catalytic subunit toward
G
in the presence of
PI(4,5)P2 (Maier et al. 1999
). In
general, the downstream components of PI3K-dependent signaling pathways are still being identified.
3-Phosphoinositides have yet to be implicated in sensory transduction,
although other phosphoinositides can modulate ion channels implicated
in olfactory transduction in lobsters (Zhainazarov and Ache
1999) and in vertebrate phototransduction (Womack et al.
2000
). In olfaction, the role of phosphatidylinositol (PI) signaling in general is unclear (reviews: Schild and Restrepo 1998
; Zhainazarov and Ache 1995
), and even
controversial (Brunet et al. 1996
; Gold
1999
). Interest in PI signaling in olfaction, however, has
focused on the canonical phosphoinositide turnover pathway in which
PI(4,5)P2 is cleaved into
inositol(1,4,5)trisphosphate (IP3) and
diacylglycerol by odor-stimulated phospholipase C. It remains to be
determined whether PI signaling in olfactory transduction could be
mediated at least in part through activation of one or more PI3Ks.
The involvement of PI signaling in olfaction is perhaps best
established in lobster olfactory receptor neurons (ORNs), where it has
been possible to both functionally and molecularly implicate a plasma
membrane-associated IP3 receptor in activation
of the cells (Fadool and Ache 1992; Munger et al.
2000
). We therefore attempted to implicate the
3-phosphoinositide pathway in these cells. We report that two
antagonists of PI3Ks, LY294002 and Wortmannin, reduce the magnitude of
the receptor potential in lobster ORNs. We show that an antibody
directed against the c-terminus of human PI3K-P110
detected a
molecule of appropriate molecular weight in the outer dendrite
(transduction zone) of the lobster ORNs. We also show that two
3-phosphoinositides, PI(3,4)P2 (1-4 µM) and
PI(3,4,5)P3 (1-4 µM), applied to the
cytoplasmic side of inside-out patches taken from cultured lobster ORNs
reversibly activate a Na+-gated channel
previously implicated in the transduction cascade in these cells, and
that 3-phosphoinositides are the most effective phosphoinositides
(1 µM) in enhancing the open probability of the channel. These
results collectively implicate 3-phosphoinositides in lobster olfactory
transduction and raise the possibility of a general role for the
3-phosphoinositide pathway in olfactory transduction.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and preparations
Adult specimens of the Caribbean spiny lobster, Panulirus
argus, were collected in the Florida Keys and maintained in the laboratory in running seawater on a diet of fish, squid, and shrimp. Data were obtained from two different preparations. Whole cell, current-clamp recordings were obtained from the ORNs in vivo as described in detail elsewhere (Michel et al. 1991), with
several modifications. In the present study, the olfactory organ
(lateral antennular filament) was hemisected in a dorsal/ventral axis, and the sections were mounted flat on the bottom of a recording chamber, allowing odorants and/or drugs to be "spritzed" directly on to the olfactory sensilla (aesthetascs) from a multibarrel pipette.
To digest away the sheath covering the clusters of ORNs, the recording
chamber was filled with L-cysteine-activated papain [Sigma Type IV, 0.17 mg/ml in Panulirus saline (PS); see
Solutions] for 1 min. The cells were then rinsed with
Ca-free PS (see Solutions), and the bath was replaced with
trypsin (1 mg/ml in Ca-free PS) for 1 min, before returning to PS for
recording. Unitary currents were obtained from the ORNs in vitro.
Primary cultures of the ORNs were prepared as described earlier
(Fadool et al. 1991
). Cells were given fresh medium
every third day and used within 1-7 days of plating.
Whole cell recording
The receptor potential was recorded from the soma of the cells
using conventional whole cell patch-clamp recording. Patch pipettes
were fabricated from borosilicate filament glass (1.50 mm OD, 0.86 mm
ID; Sutter Instrument) and fire-polished to a tip diameter about 1 µm. The pipette resistance was 5-9 M when filled with normal
patch pipette solution and formed seals with resistances of 4-10 G
.
Signals were recorded with a Dagan 3900 amplifier, low-pass filtered at
2 kHz (4-pole low-pass Bessel filter), directly digitized at 2-5 kHz,
and analyzed using pClamp 8 software (Axon Instruments). A reference
electrode was connected to the bath solution through a 3 M KCl/agar
bridge. The series resistance was <10 M
. All potentials were
corrected for the junction potentials at the pipette tip and at the
indifferent electrode as described by Neher
(1995). The magnitude of the receptor potential was
determined as the maximum amplitude of the "plateau" since membrane
voltage was not clamped and a few cells crossed the threshold for
discharging, giving the appearance of a phaso-tonic receptor potential.
Experiments were carried out at room temperature (20-22°C).
Single-channel recording
Membrane patches were pulled from the soma of the cultured ORNs,
and voltage-clamp recordings were performed using the inside-out configuration of the patch-clamp technique, as described earlier (Zhainazarov and Ache 1995). The pipettes were filled
with PS. Single-channel currents were measured with an Axopatch 200A
patch-clamp amplifier (Axon Instruments, Foster City, CA), low-pass
filtered at 1 kHz (
3 dB; 4-pole Bessel filter), digitized at 10 kHz
(A/D, D/A interface, DigiData-1200A; software, pClamp 7.0; Axon
Instruments), and stored on a computer hard disk for later analysis. A
rotary perfusion system (RSC-100, Bio-Logic, Claix, France) was used to
apply different solutions to the isolated membrane patches from one of
up to nine different reservoirs (Zhainazarov and Ache 1999
). Unless stated otherwise, recordings were performed at a holding potential of
60 mV. The recordings were referenced to a
Ag-AgCl wire electrode connected to the bath solution through a 3 M KCl
agar bridge. All recordings were made at room temperature (20-22°).
Single-channel current was analyzed using pClamp 7.0 software as
described earlier (Zhainazarov and Ache 1999). Briefly,
membrane patches typically contained more than one channel, so the open probability of a channel was calculated using the equation
Po =
I
/(Ni), where
I
is the
mean current over the interval of interest, N is the number
of channels in the patch, and i is the single-channel
current amplitude. Current amplitude histograms were used to measure
single-channel current amplitudes. Ns given in this context
refer to the number of single-channel current traces, each of 1-min
minimum duration, analyzed for a given membrane potential. When the
number of channels in a patch was difficult to determine reliably,
NPo was used as a measure of the
channel activity. In estimating the relative efficacy of a
phosphoinoside in activating a Na+-activated
channel, the first minute of channel activity after application of
phosphoinoside was excluded from calculation of the channel open
probability. The baseline of the single-channel current traces are
depicted by dashed lines. The data points are presented as means ± SE of n observations.
Western blotting and immunocytochemistry
Protein preparation was carried out as described by Xu et
al. (1999). Briefly, olfactory sensilla were shaved from 20-30
frozen lobster olfactory organs and the sensilla transferred to a
1.5-ml microcentrifuge tube containing 300 µl of homogenization
buffer consisting of 120 mM NaCl, 5 mM KCl, 1.6 mM
KH2PO4, 1.2 mM
MgSO4, 25 mM NaHCO3, 7.5 mM
glucose, 2 mM ethyleneglycoltetraacetic acid, 3 µg/ml
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 10 µg/ml benzamidine, pH 7.4. Hairs were homogenized with a plastic Teflon pestle, sonicated for 10 s, and centrifuged (1,000 × g) to remove the cuticle. ORN somata were
dissected from 10 shaved olfactory organs and homogenized and prepared
as above. Protein concentrations were determined by Bio-Rad Protein
Assay. Samples were kept frozen at
80°C.
Protein samples were boiled for 5 min in 1 × sodium dodecyl
sulfate (SDS) loading buffer then run on 4-12% NuPAGE Bis-Tris Gels
with a MES SDS Running Buffer. Ten micrograms of sample were loaded per
well. Electrophoresis was carried out with a Hoefer miniVE Vertical
Electrophoresis System. Separated proteins were transferred onto
nitrocellulose using semidry electrophoresis apparatus. Once
transferred, the blots were blocked overnight in 5% dried milk powder
in Tris-buffered saline plus Tween 20 (TBST). Blots were probed for
2 h with a commercially available goat anti-PI3K antibody raised
against the carboxy terminus of PI3-kinase p110, but cross reactive
with all isoforms of p110 PI 3-kinase (Santa Cruz Biotechnology). As a
control, the primary antibody was preabsorbed for 2 h in a 10-fold
excess of the blocking peptide (Santa Cruz Biotechnology) prior to
probing the blot. Both the primary and the preabsorbed control were
used at a final dilution of 1:1,000 in blocking solution. The blots
were washed three times in TBST for 5 min and then probed with a donkey
anti-goat horseradish peroxidase-conjugated secondary antibody at
1:10,000 (Jackson ImmunoResearch Laboratories) in blocking solution and washed three times in TBST for 5 min. The blots were developed using
enhanced chemoluminescence (Amersham Pharmacia Biotech) exposed to
Hyperfilm (Amersham Pharmacia Biotech).
Solutions
PS contained (in mM) 458 NaCl, 13.4 KCl, 13.4 Na2SO4, 13.6 CaCl2, 9.8 MgCl2, 2 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), at a pH of 7.4 adjusted with 1 M NaOH. Patch pipettes were filled with either PS (cell-free recording) or normal patch pipette solution consisting of (in mM) 180 K-acetate, 30 NaCl, 11 ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 CaCl2, 10 HEPES, and 522 glucose, pH 7.2 adjusted with Tris base (whole cell recording). Sodium-free solution consisted of (in mM) 210 KCl, 11 EGTA, 1 CaCl2, 696 glucose, and 10 HEPES, at a pH of 7.4 adjusted with 1 M
tris[hydroxymethyl]amino-methane (Tris base). The calculated free
calcium concentration in the sodium-free solution was 10 nM (Chelator)
(Schoenmakers et al. 1992
). In some experiments, part of
the KCl in the sodium-free solution was substituted by an equivalent
concentration of NaCl as described in the text and figure legends.
PI(3)P, PI(3,4)P2, and
PI(3,4,5)P3 stock solutions (1 mM) were prepared
by dispersing the inositol phosphates in distilled water with 30 min
sonication on ice, aliquoted, and stored at
20°C for use within 3 days. Stock solutions were diluted to working solutions of the stated concentration and sonicated for an additional 30 min on ice before use.
PI(3)P, PI(3,4)P2, and
PI(3,4,5)P3 were purchased from Matreya (Pleasant
Gap, PA).
The odorant consisted of an extract of a commercial marine aquarium fish food, TetraMarin (TET; Tetra Werke, Melle, Germany), made by mixing 2 g of dry flakes in 60 ml PS, centrifuging the resulting suspension at 1,400g, and filtering through no. 3 Whatmann paper. The resulting stock solution was adjusted to pH 7.4 with 1 M NaOH and stored frozen in 5-ml aliquots until needed, at which point aliquots were thawed and diluted the stated number of times with PS.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blockers of PI3K activity block the receptor potential in lobster ORNs in vivo
LY294002 (50 µM), a synthetic membrane-permeant inhibitor of
PI3K (Vlahos et al. 1994), markedly and reversibly
reduced the amplitude of the receptor potential (Fig.
1A). It was not necessary to
preincubate the cell with the drug to see this effect. There was no
obvious effect of the drug on the membrane potential in the absence of
the odor. The drug was effective on all cells tested, and reduced the
receptor potential on average to 24 ± 10% of its initial
magnitude (means ± SE in this and all subsequent whole cell data,
n = 12, Fig. 1B). The effect of the drug was
fully reversible on wash out. The effect of the drug was concentration dependent; lowering the concentration to 10 µM reduced the receptor potential to 64 ± 11% of its initial amplitude
(n = 5, data not shown).
|
Wortmannin (100 nM), a naturally occurring membrane-permeant inhibitor
of PI3K (e.g., Okada et al. 1994), also reduced the amplitude of the receptor potential in a manner similar to that seen
with the synthetic inhibitor in 8 of 14 cells tested (Fig. 2A). In these cells, the drug
reduced the receptor potential on average to 64 ± 10% of its
initial amplitude, with no obvious effect on the membrane potential in
the absence of the odor (Fig. 2B). The effect of the drug
was fully reversible on wash out, with the receptor potential
recovering on average 96 ± 9% of its initial amplitude. Ten
micromolar LY294002 and 0.1 µM Wortmannin reduced the amplitude of
the receptor potential to essentially the same level, suggesting that
Wortmannin was the more potent of the two inhibitors in this system.
The effect of Wortmannin on the receptor potential in the remaining six
cells was confounded by the fact that the drug alone depolarized the
cells and increased the amplitude of the receptor potential to 138 ± 2% of its pretreatment magnitude (data not shown). Testing 200 nM
LY294002 on two of these cells, however, inhibited the receptor
potential in the same manner as it did in all other cells (data not
shown), suggesting that Wortmannin acted nonspecifically on these six
cells, perhaps as an odorant. Unlike the synthetic drug LY294002,
Wortmannin is naturally derived and more likely to contain
water-soluble compounds capable of activating these chemosensitive
cells.
|
PI3K is enriched in the transduction compartment lobster ORNs in vivo
Scraping the olfactory sensilla from the lobster olfactory organ
produces a membrane preparation that is highly enriched in the outer
dendrites of the ORNs. An antibody raised against the c-terminus of
human PI3K-P110 recognized a protein of predicted size,
approximately 110 kDa, in a Western blot of membranes obtained from the
olfactory sensilla (Fig. 3). The staining
in this tissue was enriched compared with that in the remainder of the
organ, which consists primarily of the soma and inner dendrites of the ORNs. The immunostaining could be eliminated by preabsorbing the antibody with the antigenic peptide (Fig. 3).
|
Exogenous PI(3,4,5)P3 and PI(3,4)P2 target a component of the transduction cascade, the Na+-activated nonselective cation channel, in cultured lobster ORNs
Applying 1 µM PI(3,4,5)P3 to inside-out
patches containing Na+-activated channel activity
evoked single-channel openings, even in the absence of
Na+, one instance of which is shown in Fig.
4. The effect of
PI(3,4,5)P3 was immediate, reversed within 10-30
s of wash out, and was seen in all of 12 patches. In none of 10 attempts did 1 µM PI(3,4,5)P3 elicit
single-channel openings in patches that failed to display Na+-activated channel activity (data not shown).
The effect was concentration dependent. In a typical experiment,
increasing PI(3,4,5)P3 from 1 to 4 µM in the
absence of Na+ increased channel activity (Fig.
5A). The
NPo in this instance increased from
0.24 to 0.52. Similar concentration-dependent effects of
PI(3,4,5)P3 were observed in all of five patches
tested. Co-application of 30 mM Na+ with 1 µM
PI(3,4,5)P3 induced a greater level of channel
activity in the same patch than did application of 30 mM
Na+ alone (Fig. 4; n = 5). In a
typical experiment, the channel activity (NPo) increased from 0.02 at 30 mM
Na+ to 0.52 at 30 mM Na+ + 1 µM PI(3,4,5)P3. At a membrane potential of
60 mV, the channel opened to a single level with an amplitude of
1.5 ± 0.1 pA (n = 3) for 30 mM
Na+,
1.6 ± 0.1 pA (n = 3)
for 1 µM PI(3,4,5)P3, and
1.5 ± 0.1 pA (n = 3) for 30 mM Na+ + 1 µM
PI(3,4,5)P3. The slope conductance was not
affected by PI(3,4,5)P3 (Fig. 5B).
Between
100 and
50 mV, the slope conductance was 42.2 ± 2.6 pS for 30 mM Na+, 43.7 ± 2.7 pS for 1 µM
PI(3,4,5)P3, and 48.2 ± 5.7 pS for 30 mM
Na+ + 1 µM PI(3,4,5)P3.
|
|
Applying 1-4 µM PI(3,4)P2 also activated the channel in the absence of Na+ and increased the channel open probability in the presence of 30 mM Na+, as shown for one patch exposed to 1 µM PI(3,4)P2 in Fig. 6, A and B. The same effect was seen in 13 other patches, each exposed to a single concentration PI(3,4)P2 ranging from 1 to 4 µM. As with PI(3,4,5)P3, the effect of PI(3,4)P2 was also rapid and concentration dependent. In contrast to PI(3,4,5)P3, however, PI(3,4)P2 typically required about 1 min for channel activity to reach a stable level. On wash out, the effect of PI(3,4)P2 reversed slowly over 20-30 min. Applying 1-4 µM PI(3)P failed to activate the channel in the absence of Na+, or increase the open probability of the channel when activated by 30 mM Na+, as shown for one patch exposed to 1 µM PI(3)P in Fig. 6, C and D. The same effect was seen in four other patches, each exposed to a single concentration PI(3)P2 ranging from 1 to 4 µM.
|
PI(3,4,5)P3 is the most effective phosphoinositide on the Na+-activated nonselective cation channel
We estimated relative efficacy of phosphoinositide activation
(PR) using the following equation
![]() |
(1) |
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We pharmacologically implicated PI3K in the odor activation of lobster ORNs, immunocytochemically localized a PI3K-like molecule to the transduction zone, identified an ion channel in the transduction cascade as a potential target for the action of 3-phosphoinositides, and showed that 3-phosphoinositides are the most effective phosphoinositide activating the channel. These results collectively suggest that 3-phosphoinositides play an important role in lobster olfactory transduction.
Four lines of evidence support the involvement of PI3K in the
transduction sequence. 1) LY294002, a synthetic antagonist
of PI3K, reduced the magnitude of the receptor potential in the cells. The drug has a very selective structure-activity relationship for PI3K
since analogues of LY294002 with only slight changes in structure cause
marked decreases in inhibition in binding assays (Vlahos et al.
1994). 2) Wortmannin, a naturally occurring
antagonist of PI3K, mimicked the action of LY294002 in those cells in
which it could be tested effectively. 3) An antibody
directed against the c-terminus of human PI3K-P110
stained a
molecule of appropriate molecular weight in the olfactory sensilla,
suggesting that an appropriate molecular target for the two
pharmacological probes occurs in the transduction zone of the cells.
Finally, 4) the substrate for PI3K, phosphatidylinositol
4,5-bisphosphate (PIP2), is constitutively
present in the ORNs. Depleting membrane patches of cultured lobster
ORNs of endogenous PIP2 by applying recombinant PLC
alters ion channel activity in the patch, which can be restored by subsequent application of exogenous PIP2
(Zhainazarov and Ache 1999
).
We assume that the antibody directed against the c-terminus of human
PI3K-p110 stained a lobster homologue, as it recognized an antigen
of the correct molecular weight, and this immunoreactivity was lost
when the antibody is preabsorbed with antigenic peptide. Although
raised against human p110
, the anti-PI3K-p110
, antibody recognizes all the mammalian p110 isoforms (
,
,
, and
),
allowing that the protein recognized in the lobster ORN could be any of these four isoforms. The most likely candidate, however, is likely to
be a lobster p110 gamma, because p110 gamma can be activated by
G
/
, as well as
G
(Stoyanov et al. 1995
), and
olfactory signaling in lobsters utilizes G-protein-coupled receptors
(Fadool et al. 1995
).
The action of the 3-phosphoinositides on the lobster olfactory
Na+-gated cation channel reveals a potential
target for the products of PI3K activity in the transduction cascade.
This channel has been proposed to secondarily amplify the primary
transduction current in lobster ORNs (Zhainazarov et al.
1998). Here, we show that PI(3,4,5)P3 and
PI(3,4)P2 directly activate the
Na+-gated cation channel and enhance the
Na+ sensitivity of the channel when co-applied
with Na+. We assume that this action is selective
since 1) neither phosphoinositide induced channel openings
in patches that fail to exhibit Na+-gated channel
activity, 2) the channel openings they evoked had a
single-channel conductance similar to that of the
Na+-gated channel (Zhainazarov and Ache
1995
), and 3) PI(3)P had no effect on the channel.
Membrane phosphoinositides also have such dual trigger/regulatory
action on ATP-sensitive K+ channels
(Baukrowitz et al. 1998
; Shyng and Nichols
1998
), suggesting that this may be a common mechanism by which
phosphoinositides control ion channel function, even if the specific
effects (e.g., increased or decreased open probability) vary for
different channel types.
The action of PI(3,4,5)P3 and PI(3,4)P2 on the channel in isolated membrane patches is consistent with the pharmacological effect of LY294002 and Wortmannin on the cells in vivo, suggesting that the olfactory Na+-gated cation channel is indeed a potential target for one or more products of PI3K metabolism. Blocking the production of 3-phosphoinositides would be expected to reduce the amplifying function of the Na+-gated cation channel on the receptor current. This in turn would reduce the magnitude of a depolarizing receptor potential, which was the pharmacological effect we obtained from treating the cells in vivo with LY294002 or Wortmannin.
PI(3,4,5)P3 was the most potent
phosphoinositide, as measured by the ability of the lipid to activate
and regulate the channel, although PI(3,4)P2,
PI(4)P, and PI(4,5)P2 also had measurable activity. The sensitivity to multiple phosphoinositides does not reflect a generalized effect of charge per se, with more highly charged
molecules being more active, since InsP3, for
example, with six negative charges per molecule, had no significant
effect on the Na+-gated channel either in the
presence or absence of Na+ (Zhainazarov
and Ache 1995). Presumably, both the hydrophobic tail and the
negatively charged hydrophilic head are required for phosphoinositides
to regulate the channel. It is not clear, however, that the channel
would be activated/modulated by multiple phosphoinositides in vivo.
PI(4,5)P2 is the substrate for PI3K, so it is
possible that application of exogeneous
PI(4,5)P2, and perhaps PI(4)P as a precursor for
PI(4,5)P2, shifts the equilibrium of PI3K toward
production of 3-phosphoinositides, although the constitutive activity
of the enzyme is thought to be low (Zhang and Majerus
1998
). P110 PI3Ks exhibit a preference for
PI(4,5)P2 as a substrate within cells
(Hawkins et al. 1992
), suggesting that
PI(3,4,5)P3 would be the primary product of
odor-stimulated PI3K in the lobster cells.
This study provides the first evidence that
3-phosphoinositides can have signaling function in an olfactory
receptor cell. Still to be resolved is the exact role of these lipids
in the activation sequence and whether, as in NT3-induced synaptic
potentiation (Yang et al. 2001), the 3-phosphoinositide
pathway works in concert with the canonical phosphoinositide turnover
pathway shown earlier to be involved in activation of these cells
(Fadool and Ache 1992
). These results open a new avenue
of inquiry that may help resolve the controversy presently surrounding
the role of PI signaling in olfactory transduction in other animals.
![]() |
ACKNOWLEDGMENTS |
---|
We thank A. Hastings for preparation of the cultured cells, D. Migliaro for assistance with Western blotting, and M. Milstead for assistance with the illustrations.
This work was supported by National Institute on Deafness and Other Communication Disorders Grant DC-01655.
Present address of A. B. Zhainazarov: Dept. of Cardiology, Children's Hospital and Harvard Medical School, John F. Enders Building, Rm. 1316, 320 Longwood Ave., Boston, MA 02115.
![]() |
FOOTNOTES |
---|
Address for reprint requests: B. W. Ache, Whitney Laboratory, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32086.
Received 27 November 2000; accepted in final form 14 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|