From the
Light stimulates phosphatidylinositol bisphosphate
phospholipase C (PLC) activity in Drosophila photoreceptors.
We have investigated the mechanism of this reaction by assaying PLC
activity in Drosophila head membranes using exogenous
phospholipid substrates. PLC activation depends on the photoconversion
of rhodopsin to metarhodopsin and is reduced in
norpA
Excitation of invertebrate photoreceptors is initiated by
photoconversion of rhodopsin into metarhodopsin. This stimulates a
cascade of events in photoreceptor cell rhabdomeres which leads to
opening of plasma membrane cation channels. It has been demonstrated
that photoactivated rhodopsin catalyzes nucleotide exchange on a G
protein and that light stimulates hydrolysis of
PIP
The
Drosophila norpA gene encodes a photoreceptor PLC that is
obligatory for photoexcitation. Strong norpA alleles are blind
and exhibit a loss of light-induced electrical
responses
(2, 3) . About 90% of the PLC activity in
Drosophila head homogenates is associated with membranes from
the compound eyes. This activity is strongly reduced in norpA mutants
(4) . The major PLC from Drosophila heads
which is absent in norpA mutants has been purified
(5) .
This protein, the product of the norpA gene, is a homolog of
mammalian PLC-
Several invertebrate G protein
subunits that may participate in phototransduction have recently been
identified.
These findings suggest that NorpA PLC is
coupled to light activated rhodopsin (metarhodopsin) via a G protein
with G
All fly stocks were white-eyed to eliminate the
light blocking effects of colored pigments.
w
Final reaction
mixtures contained 50 mM HEPES (pH 7.1), 100 mM NaCl,
50 mM sucrose, 24 mM KCl, 2 mM MOPSO, 0.5
mg/ml bovine serum albumin, 3.5 mM MgCl
PLC activity in Drosophila head membranes was assayed in the presence of 10 µM
GTP
We
examined the Ca
To examine the role of G protein in NorpA PLC
activation, we measured the effects of GTP, GTP
G proteins couple photoexcitation of rhodopsin to activation
of PLC in invertebrate photoreceptors
(1, 30) . Although
there have been no previously published measurements of light and G
protein-dependent PLC activity in Drosophila heads, Devary
et al.(19) have reported evidence that light and a G
protein stimulates inositol phospholipid hydrolysis in Musca eye membranes. Our findings with Drosophila are similar to that
report, but there are some notable differences. Whereas Devary et
al.
(19) reported that blue light stimulates inositol
phosphate production in the absence of added nucleotide and in the
presence of 100 µM GTP, we found no stimulation under
these conditions. In their study, GTP
Although both we and Devary et al.(19) have interpreted the guanyl nucleotide effects to indicate
involvement of a G protein, these results do not preclude the
possibility that GTP
We have
demonstrated that a G protein is absolutely required for blue light
stimulation by analyzing PLC activation in the G protein-deficient
mutant G
Recent
evidence suggests that the phototransduction G protein
The
mechanism by which the photoreceptor G protein activates NorpA PLC is
not known. NorpA is most similar in amino acid sequence to vertebrate
PLC-
The
Ca
The predominant source of
light-mobilized Ca
The decrease in PLC
activity that we observed at 5 µM free Ca
We thank Drs. Patrick Dolph and Charles Zuker for
G
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
PLC and
ninaE
rhodopsin mutants. NorpA PLC is
stimulated by light at free Ca
concentrations between
10 nM and 1 µM. This finding is consistent with a
Ca
-mediated positive feedback mechanism that
contributes to the rapid temporal response of invertebrate
photoreceptor cells. The guanyl nucleotide dependence of
light-stimulated PLC activity indicates that a G protein regulates
NorpA. This was confirmed by the observation that light stimulation of
PLC activity is deficient in mutants that lack the eye-specific G
protein
subunit G
e. These results indicate that G
e
functions as the
subunit of the G protein coupling rhodopsin to
NorpA PLC.
(
)
into IP
and
diacylglycerol
(1) . Light causes an increase in cytoplasmic
Ca
, opening of plasma membrane cation channels, and
influx of Na
and Ca
.
(6). Immunological analysis indicates that NorpA is
present in all adult photoreceptor cell types and is specifically
localized to rhabdomeres
(7) .
,
, and
subunits of light-stimulated G
protein have been isolated from squid
photoreceptors
(8, 9, 10, 11) . Two
photoreceptor-specific G protein
isoforms, DGq1 and
DGq2
(12) , and a photoreceptor-specific G
, G
e (13),
have been cloned from Drosophila. Recent studies suggest that
DGq1 is the
subunit of a G protein that couples light-stimulated
rhodopsin to PLC
(14) and that G
e is the
subunit of
this G protein. Immunoreactive G
e is present in photoreceptor cell
rhabdomeres
(15) , and G
e mutants are deficient in
light-stimulated GTP
S binding in compound
eyes
(15, 16) . G
e mutants also exhibit
reduced light sensitivity and slowed excitation and deactivation
kinetics
(16) .
e as its
subunit. To confirm this, we determined the
nucleotide dependence of PLC activity in Drosophila head
membranes and compared light stimulation of PLC activity in
G
e
and G
e
mutant flies. We found that light-sensitive NorpA PLC activity in
Drosophila head membranes is guanyl nucleotide-sensitive and
dependent on normal G
e function.
Materials
Bovine brain PIP and PE
were purchased from Sigma, PS from Avanti, and
[
H]PIP
(74-370 GBq/mmol) from
DuPont NEN. PS and PE were stored in CHCl
, PIP
,
and [
H]PIP
in
CHCl
:MeOH:HCl (75:25:1) under argon at -20 °C.
Nucleotides, creatine phosphate, creatine phosphokinase, and protease
inhibitors were purchased from Sigma. Nucleotides were made up as
described
(17) and stored at -70 °C in Tris buffer.
The buffer for the guanine nucleotides also contained 1 mM
dithiothreitol.
,
w
;G
e
,
w
;G
e
;P[w
,Rh1-G
e
],
and w;ninaE
flies were maintained as
described previously
(15) . w,norpA
were raised in the dark to prevent light-dependent retinal
degeneration
(18) . Heads were collected from
ninaE
flies that were less than 3 days
old to avoid time-dependent retinal degeneration
(18) .
Head Membrane Preparation
Flies were dark-adapted
for 2-24 h, frozen in liquid nitrogen, and stored at -70
°C. Heads were collected as described
(13) and crude
membranes prepared by a procedure similar to that of Devary et
al.(19) . Heads were homogenized in 250 mM
sucrose, 120 mM KCl, 5 mM MgCl, 1
mM EGTA, 10 mM MOPSO (pH 7.0), 1 mM
dithiothreitol, 10 µg/ml leupeptin, 1 µg/ml pepstatin A) using
a glass/Teflon homogenizer. The suspension was allowed to settle for 5
min to remove large debris, decanted, and crude membranes prepared by
centrifugation at 14,000
g, followed by resuspension
in 0.5 volume of the same buffer. Membrane preparations were aliquoted
and stored in foil-wrapped tubes in liquid nitrogen. Protein
concentration was assayed by Bradford (Bio-Rad) using bovine serum
albumin standards. All procedures were performed in a darkroom under
infrared illumination using night vision goggles.
Substrate Preparation
Total head membrane PLC
activity was assayed using OGM (100 mM NaCl, 50 mM
HEPES (pH 7.1), 2% octyl--glycopyranoside, 100 µM
PIP
, 35,000 dpm
[
H]PIP
/reaction). Light-inducible PLC
activity was measured on PLV (100 mM NaCl, 50 mM
HEPES (pH 7.1), 500 µM mixed phospholipids (1:2:2 molar
ratio of PIP
:PE:PS), 35,000 dpm
[
H]PIP
/reaction)
(20) .
Phospholipids were mixed, dried under a stream of argon in a
siliconized polypropylene tube, suspended in argon-saturated substrate
buffer (100 mM NaCl, 50 mM HEPES (pH 7.1), plus 2%
octyl-
-glycopyranoside for OGM), vortexed 5 min (PLV only), then
sonicated at room temperature for 2 min in a Branson water bath
sonicator. The substrates were stable for several hours at room
temperature and were used within 1 h of preparation.
PLC Assay
Membrane preparations were thawed and
illuminated at room temperature using a 100-watt tungsten quartz
halogen light source with red (Schott RG-610) or blue (Schott BG-28)
filters. In the standard procedure, assays were initiated by addition
of 10 µl of illuminated membranes (8-10 µg of protein) to
40 µl of reaction mixture composed of 20 µl of phospholipid
substrate, 10 µl of reaction buffer (150 mM HEPES, pH 7.1,
300 mM NaCl, 2.5 mg/ml bovine serum albumin, 12.5 mM
MgCl, 9 mM EGTA), 5 µl of ATP regenerating
system (10 mM ATP, 50 mM creatine phosphate, 500
units/ml creatine phosphokinase), 2.5 µl of 200 µM
GTP
S, and 2.5 µl of either 6.6 mM (for PLV substrate)
or 38.2 mM CaCl
(for OGM substrate) and incubated
for 4 min at 30 °C. The reaction was terminated by addition of 50
µl of 10% (w/v) trichloroacetic acid and 25 µl of 10 mg/ml
bovine serum albumin. After 15 min on ice, samples were centrifuged at
4 °C for 4 min at 9000
g, and 100-µl
supernatants containing soluble inositol phosphates scintillation
counted for [
H]IP
. Substrate
blanks obtained by adding trichloroacetic acid to the reaction mix
before the membranes were subtracted from each data point.
Ca
concentrations were varied by adding equal volumes
of CaCl
stocks of differing concentrations. Free
Ca
concentration was calculated using MaxC (Shareware
from C. Patton, Hopkins Marine Station, Stanford University), and the
estimated concentrations of free Ca
produced by the
CaCl
, EGTA, MgCl
, and HEPES stocks used in
these experiments verified using Rhod-2
(21) .
, 2
mM EGTA, 1 mM ATP, 5 mM creatine phosphate,
50 units/ml phosphocreatine kinase, 10 µM GTP
S, 0.2
mM dithiothreitol, 2 µg/ml leupeptin, 0.2 µg/ml
pepstatin A, 35,000 dpm [
H]PIP
],
plus 20 µM PIP
, 40 µM PE, 40
µM PS, 50 nM free Ca
(PLV) or
0.4% octyl-
-glycopyranoside, 20 µM PIP
, 5
µM free Ca
(OGM).
Assay of Light-stimulated NorpA PLC Activity in
Drosophila Head Membranes
Blue and red illuminations were used
to regulate PLC activity in Drosophila head membrane
preparations. Rh1 rhodopsin, the predominant photopigment in
Drosophila compound eyes, absorbs maximally at 480 nm and is
converted to its active metarhodopsin form by blue light. Rh1
metarhodopsin absorbs maximally at 560 nm and is photoconverted back to
inactive rhodopsin by red light.
S using exogenous [
H]PIP
incorporated into either PLV or OGM. With the PLV substrate, blue
illumination of head membranes from dark-adapted control
(w
) flies stimulated PLC activity
2-fold above dark levels (Fig. 1). There was no stimulation with
red light. High light-independent levels of PLC activity were found
using the OGM substrate (Fig. 1), suggesting that the detergent
uncouples PLC from receptor activation.
Figure 1:
Blue light stimulation
of phospholipase C activity in Drosophila head membranes.
Phospholipase C activity of w head membranes on
[H]PIP
-labeled phospholipid vesicles
(PLV) or octyl-
-glycopyranoside micelles (OGM).
The flash times were 1 min for red (Schott RG-610) and 10 s for blue
(Schott BG-28) illumination. IP is mixed inositol phosphates. Values
are means ± sem from multiple experiments (PLV, n = 18) (OGM, n =
4).
The relationship between
metarhodopsin formation and PLC stimulation with PLV substrate was
examined by titrating Drosophila head membranes with blue
light (Fig. 2). With the light intensity attenuated by a 3 OD
neutral density filter the level of PLC activity increased with
exposure time. Maximal PLC activity was stimulated by a 10-s flash. A
1000-fold increase in intensity of the 10-s flash had little additional
effect on PLC activity. However, illumination times of 30 s or 1 min
reduced activity (data not shown). A possible explanation for this
decrease in activity might be decay caused by extended incubation of
the membranes at room temperature. To test for this decay, membranes
were flashed for 10 s at maximal intensity and held for varying lengths
of time before addition to the substrate mix. We found that PLC
activity remained at maximal levels for at least 2 min following the
flash of blue light. The activity then slowly decreased to background
levels by 10 min (data not shown). Since the membranes maintained full
potential for PLC stimulation for 2-min post-flash, the reduced PLC
activity at 30-s and 1-min flash times is likely to be the result of
specific inactivation mechanisms rather than nonspecific decay.
Figure 2:
Changes
in light-stimulated phospholipase C activity with intensity and time of
illumination. w head membranes were flashed with blue light
for different times and assayed for PLC activity on PLV substrate.
Neutral density filters of optical density (OD) 1, 2, and 3 were
employed to vary light intensity. Values are means ± S.E. (n = 3 or 4) from multiple
experiments.
Light stimulation of PLC depends on the state of the
phototransduction apparatus prior to preparation of the membranes.
Exposure to red illumination did not reduce PLC activity in membranes
prepared from dark-adapted flies. However, when membranes were prepared
from non-dark-adapted flies, blue light did not stimulate PLC, and red
illumination reduced PLC activity to the level found in dark-adapted
flies (data not shown). Dark adaptation of previously frozen heads was
ineffective in lowering basal activity. We unexpectedly found that when
membranes from dark-adapted flies were first flashed with red light,
they became refractory to subsequent stimulation by blue light. This
suggests that red light induces an insufficiency of some factor
required for PLC activation. Byk et al.(22) reported
that illumination of Musca eye membranes with orange light,
which photoconverts metarhodopsin to rhodopsin, caused release of
arrestin from the membranes. It is possible that other components of
the phototransduction cascade are also released from rhabdomeric
membranes under these conditions.
PLC Activity in norpA and ninaE Mutants
It has
been reported that most of the PLC activity in Drosophila head
membranes corresponds to NorpA PLC
(4, 5, 23) .
Consistent with these reports, we found that PLC activity in
norpA mutants was only 5% of blue
light-stimulated activity of control white-eyed flies when assayed with
PLV substrate (Fig. 3). norpA
is
a strong allele which exhibits severe deficiencies in electroretinogram
light sensitivity
(24) and NorpA protein levels
(23) .
When assayed with the OGM substrate, PLC activity in
norpA
head membranes was approximately
25% of wild type activity (Fig. 3). The levels of Rh1, DGq, and
G
e proteins in dark raised norpA
head membranes were normal when analyzed by immunoblotting (data
not shown).
Figure 3:
Phospholipase C activity in norpA and ninaE mutants. Head membranes from control w and white-eyed norpA and ninaE flies were
flashed with red (1 min) or blue (10 s) light and assayed on either PLV
or OGM substrates. Values are means ± S.D. (n =
3) from a representative experiment.
To confirm that light stimulation of PLC activity
requires rhodopsin, ninaE head
membranes were assayed. ninaE encodes the opsin component of
Rh1 rhodopsin
(25, 26) . ninaE
mutants exhibit electroretinogram abnormalities and express less
than 2% of wild type Rh1 rhodopsin levels
(24) .
ninaE
PLC activity assayed with PLV
substrate was insensitive to blue light and was only 25% of
unstimulated wild type levels (Fig. 3). The low basal activity of
ninaE
suggests that most of the basal
activity of wild type membranes on PLV substrate is
metarhodopsin-dependent. Nearly wild type levels of PLC activity were
observed with OGM substrate, indicating that normal levels of norpA PLC
are present in the ninaE
membranes.
Immunoblot analyses demonstrated that the levels of DGq and G
e in
ninaE
head membranes were normal (data
not shown).
Effect of Free Ca
Caon PLC
Activity
is an important mediator of
both excitation and inactivation processes in invertebrate
photoreceptors (1). Cytoplasmic Ca
increases as a
consequence of IP
-induced mobilization from internal
Ca
stores and uptake of extracellular Ca
through light-sensitive plasma membrane cation channels.
dependence of PLC activation. With
PLV substrate, PLC activity increased when free Ca
was raised from 10 nM to 1 µM
(Fig. 4, upper panel). Blue light stimulated PLC
activity over this range of free Ca
concentration.
The largest blue light effect was between 10 and 200 nM free
Ca
. PLC activity was maximal at 1 µM
free Ca
, but was nearly insensitive to light at this
Ca
level. PLC activity assayed with OGM substrate was
also dependent on Ca
. Maximal activity occurred at 5
µM and was independent of light over the entire range of
Ca
concentration (Fig. 4, lower
panel). This is similar to the previously reported Ca
sensitivity of PIP
hydrolysis using crude and
partially purified norpA PLC
(5) .
Figure 4:
Ca titration of
phospholipase C. w head membranes were maintained in the dark
or flashed with red (1 min) or blue light (10 s) and assayed for PLC
activity at different free Ca
concentrations. PLV
substrate (upper panel). Values are means ± S.E. (n = 3 or 4) from multiple experiments. OGM substrate
(lower panel). Values are means ± S.D. (n = 3) from a representative
experiment.
G Protein Requirement for Light-stimulated PLC
Activity
Stimulatory or inhibitory effects of guanine nucleotide
analogs on enzymatic activities are classic indicators of G protein
regulation. Guanine nucleotide analogs affect PLC activity in various
vertebrate cells
(20) as well as in the photoreceptors of
Musca(19) , Limulus(27) , and
squid
(28) .
S, and GDP
S on
PLC activity in Drosophila head membranes using the PLV
substrate. Our standard PLC assay included 10 µM
GTP
S, a nonhydrolyzable GTP analog which irreversibly activates G
protein
subunits following nucleotide exchange. Varying GTP
S
concentration from 1 µM to 1 mM did not affect
either basal or light-stimulated PLC activity levels (data not shown).
When assayed in the absence of added nucleotide, or in the presence of
10 µM to 1 mM GTP, PLC activity was insensitive
to light and about 50% lower than basal activity in the presence of
GTP
S (Fig. 5). The reduced basal activity and loss of light
sensitivity in the absence of GTP
S is consistent with G protein
regulation of norpA PLC. GDP
S, an analog of GDP resistant to
phosphorylation by nucleoside diphosphate kinase
(29) , inhibits
G protein-mediated signaling pathways by maintaining G
in the
inactive, GDP bound state. The presence of 250 µM
GDP
S in our assays inhibited PLC activity on PLV substrate and
eliminated blue light stimulation. This provides further evidence that
NorpA PLC is regulated by a G protein (Fig. 5).
Figure 5:
Effects of guanine nucleotides on light
stimulation of phospholipase C. w head membranes were
maintained in the dark or flashed with red (1 min) or blue (10 s) light
and assayed for PLC activity on PLV substrate in the presence or
absence of different guanine nucleotides. Black filled
columns, no guanine nucleotide; black with white diagonal
stripe columns, 10 µM GTPS; gray filled
columns, 1 mM GTP; white with black diagonal stripe
columns, 250 µM GDP
S. Values are means ±
S.E. (n = 3 to 9) from multiple
experiments.
We used
another Drosophila mutant to determine whether light
activation of NorpA PLC requires Ge, a photoreceptor-specific G
protein subunit. G
e
flies have less
than 1% of wild type levels of immunoreactive G
e and are defective
in light-stimulated GTP
S
binding
(15, 16) . The photoreceptors of
G
e
flies also exhibit abnormal
excitation and recovery as measured in whole cell patch clamp
recordings
(16) . We found that the basal PLC activity in
G
e
head membranes using the PLV
substrate was about 50% of control levels, whereas the activity
following blue illumination was 38% of control. No significant (p = 0.19 by analysis of variance) blue light stimulation in
the G
e
membranes was evident
(Fig. 6, left panel). Analysis of a P
element-transformed rescue strain
G
e
;P[w
,G
e
] (16) confirmed that the G
e
mutation
was responsible for the elimination of metarhodopsin-dependent PLC
activation. These transgenic flies have about 10% of normal levels of
G
e, but exhibit 75% of wild type light induced
GTP
S binding activity
(15) and nearly normal
activation and deactivation kinetics
(16) . The rescue gene
restored blue light-stimulated PLC activity on mixed PLV substrate to
73% of control (Fig. 6, left panel). This blue light
stimulation was highly significant (p = 0.00025 by
analysis of variance). The PLC activities of the
G
e
and
G
e
;P[w
,G
e
] head membranes on the OGM substrate were equivalent to wild type,
indicating normal levels of functional NorpA PLC (Fig. 6,
right panel).
Figure 6:
Phospholipase C activity in ge mutants. Head membranes from w, G
e, and
G
eP[g
e
] flies were maintained in the dark or flashed with red (1 min) or
blue (10 s) light and assayed for PLC activity. PLV substrate (left
panel). Values are means ± S.E. (n = 5 or
7) from multiple experiments. OGM substrate (right panel).
Values are means ± S.E. (n = 3) from multiple
experiments.
S stimulated PLC under both
blue and red illumination, but in our study GTP
S was only
effective under blue illumination. The differences in guanine
nucleotide effects probably reflect differences in the substrates and
membranes used in the PLC assays. Devary et al.
(19) used endogenous
H-labeled inositol phospholipid
substrate, whereas we used exogenous [
H]PIP
in either phospholipid vesicles or detergent micelles. We did not
assay PLC using the Devary et al.(19) method, because
we were unable to label endogenous PIP
with
[
H]myo-inositol using their conditions.
It is clear from our results using the two different
[
H]PIP
substrates, PLV and OGM, that
PLC regulation by G protein depends on the substrate environment. With
the PLV substrate, we observed metarhodopsin-dependent, G
protein-mediated PLC stimulation. However, with OGM, we observed high
levels of light and G protein-independent PLC activity. Other
differences in our preparations may also account for these
discrepencies. Devary et al.(19) prepared membranes
from Musca eyes, whereas we used whole Drosophila heads, which contain additional G
proteins
(31, 32) . The eye and brain G proteins in our
preparations may hydrolyze the added GTP to GDP, and the resulting high
levels of GDP may be inhibitory. The absence of a GTP effect on other G
protein-coupled signaling systems has been reported by
others
(20) .
S or GDP
S may also have direct effects on
NorpA PLC. Potential GTP binding motifs have been identified in the
NorpA protein sequence
(33) , but the ability of NorpA PLC to
bind or hydrolyze GTP has not been demonstrated.
e
. The loss of
light-stimulated PLC activity in the
G
e
mutant and its restoration in the
G
e
;P[w
,G
e
] rescue flies suggests that G
e functions as the
subunit
of the G protein that couples NorpA to metarhodopsin. G
e is
required for light-stimulated G protein function
(15, 16) and it is necessary both for photoexcitation and for
termination of the phototransduction cascade
(16) . Additionally,
immunoprecipitation of G
e is enhanced by GTP
S, suggesting
interaction between G
e and a G
subunit
(15) .
subunit is
DGq1
(14) . DGq is present in rhabdomeres, and affinity-purified
DGq antibody blocks light-stimulated GTP hydrolysis (14). Dominantly
active DGq1 mutants exhibit light-independent GTPase activity
and abnormal electrophysiological light responses. Furthermore,
interaction between dominant DGq1 and light-dependent
degeneration mutants indicate that the DGq1 mutations cause
constitutive activation of PLC
(14) . Interaction of DGq with PLC
is consistent with its structure. DGq is most similar in deduced amino
acid sequence to the vertebrate Gq
class
(12, 34) ,
which activate PLC
(35, 36, 37) .
4
(33, 38) . Reconstitution studies have shown
that different PLC-
isoforms can be directly activated by either
or both Gq
or G
subunits
(39, 40, 41) . Vertebrate PLC-
4 was
stimulated by Gq
and not by G
in transiently transfected
cells
(42) . However, in common with other PLC
isoforms
(43) , NorpA PLC has a pleckstrin homology domain in its
amino-terminal region. This motif may be a site of G
interaction in the
-adrenergic receptor kinase (44, 45). A recent
study reports binding between G
and pleckstrin homology
domains from nine different proteins
(46) . Our results do not
resolve the issue of whether NorpA PLC is directly regulated by DGq or
by a
complex containing G
e. This will require the
identification of a Drosophila phototransduction G
subunit and expression and reconstitution studies.
dependence of PLC activity in Drosophila heads is consistent with a positive feedback model of PLC
activation in which light-induced Ca
mobilization
accelerates the response to light. PLC enzymes require Ca
for catalytic activity, and activation of PLC by micromolar
Ca
has been reported for many tissues and cell
types
(47, 48) . Payne and Fein
(49) reported a
nonlinear decrease in time to peak of the light response of Limulus ventral photoreceptors with increasing flash intensity. They
suggested that a messenger produced as part of the signaling cascade
accelerates the production of new messenger. They proposed that this
messenger is Ca
, since injection of the
Ca
buffer EGTA suppressed nonlinearity and slowed the
response to bright flashes.
in Drosophila photoreceptors reportedly is from entry of extracellular
Ca
through light-activated
channels
(50, 51) . The light-dependent Ca
influx generates highly localized transients adjacent to
rhabdomeres
(51) . Experiments with Ca
chelators of various affinities indicate that local
Ca
concentrations extend into the micromolar
Ca
range
(51) . Positive feedback of NorpA PLC
during the Ca
transients may contribute to the rapid
temporal responses of photoreceptor cells.
indicates that Ca
-regulated inactivation
mechanisms act at or before NorpA in the phototransduction cascade.
This may reflect Ca
and diacylglycerol-dependent
stimulation of inaC protein kinase C
(52, 53) or arrestin phosphorylation and activation by calmodulin
kinase
(54) .
, phosphatidylinositol 4,5-bisphosphate; IP
,
inositol trisphosphate; PLC, phosphatidylinositol-specific
phospholipase C; IP, mixed inositol phosphates; PLV, mixed phospholipid
vesicles; PE, phosphatidylethanolamine; PS, phosphatidylserine; OGM,
octyl-
-glycopyranoside micelles; MOPSO,
3-(N-morpholino)-2-hydroxypropanesulfonic acid; GTP
S,
guanosine 5`-3-O-(thio)triphosphate; GDP
S, guanyl-5`-yl
thiophosphate.
e
and
G
e
;P[w
,G
e
] and Dr. William Pak for norpA
and
w;ninaE
flies. We thank Julietta Schoo
for maintaining fly stocks, Dr. David Teng for comments on the
manuscript, and Dr. David Hyde for communicating results prior to
publication.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.