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The Journal of Neuroscience, September 15, 2000, 20(18):6797-6803
Phenotypes of trpl Mutants and Interactions between
the Transient Receptor Potential (TRP) and TRP-Like Channels in
Drosophila
Hung-Tat
Leung,
Chaoxian
Geng, and
William L.
Pak
Department of Biological Sciences, Purdue University, West
Lafayette, Indiana 47907-1392
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ABSTRACT |
The trp and trpl genes are thought to
encode two classes of light-activated ion channels in
Drosophila. A previous report indicated that a null
trpl mutant does not display any mutant phenotype. This
lack of detectable mutant phenotypes made it difficult to suggest
functions for the transient receptor potential-like (TRPL) channel in
photoreceptor responses. Here, the properties of trpl
photoreceptor responses were studied by using electroretinogram (ERG)
and intracellular recording techniques in combination with light
stimuli of relatively long durations. Distinct mutant phenotypes were
detectable under these conditions. These consisted of a reduced sustained component, oscillations superimposed on the response, a
poststimulus hyperpolarization, and altered adaptation properties to
dim background light. Comparison of photoreceptor responses obtained
from wild type, trp, and trpl showed that
the responses obtained from the trp and
trpl null mutants did not sum up to that of the
wild-type response. To explain the nonlinear summation at the peak of
the response, Reuss et al. (1997) proposed that Ca2+
ions entering through the TRP channel modulate TRP and TRPL channel activities differentially. However, nonlinear summation was present not
only at the peak but throughout the duration of response. Two lines of
evidence are presented to suggest that, in addition to the interaction
proposed by Reuss et al. (1997), there are other forms of interactions
between TRP and TRPL channels, probably involving the channel proteins themselves.
Key words:
Drosophila; phototransduction; trpl
phenotypes; TRP channel; TRPL channel; channel interactions
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INTRODUCTION |
Drosophila photoreceptors
respond to light with a depolarization mediated by a phospholipase C
(PLC)-dependent signaling pathway (Bloomquist et al., 1988), resulting
in the opening of two types of cation channels, transient receptor
potential (TRP) and TRP-like (TRPL). The mechanism of activation of
either channel is not known. The TRP and TRPL channel subunits are
encoded by the transient receptor potential (trp) and
trp-like (trpl) genes, respectively (Montell and Rubin, 1989; Wong et al., 1989; Phillips et al., 1992).
The protein products of trp and trpl share 39%
amino acid identity and some homology to neuronal voltage-gated channel
subunits, although neither TRP nor TRPL is voltage-gated (Stühmer
et al., 1989; Phillips et al., 1992). The TRP channel is highly
calcium-permeable, whereas the TRPL channel is nonspecifically
cation-selective (Hardie and Minke, 1992; Niemeyer et al., 1996; Reuss
et al., 1997).
Previous workers have demonstrated that trp mutants exhibit
severely impaired photoreceptor responses (Cosens and Manning, 1969;
Minke et al., 1975; Pak, 1979; Minke, 1982). However, a null
trpl mutant,
trpl302, has been reported not
to show any mutant phenotype (Niemeyer et al., 1996), raising questions
about the role of the TRPL channel in phototransduction. It was
suggested that TRP and TRPL might play overlapping roles. Subsequently,
Reuss et al. (1997) showed differences in ionic permeabilities between
wild type and trpl302.
However, the differences were subtle, and the question still remained
whether trpl302 has a
clear-cut phenotype. Previous studies of photoreceptor responses of
trpl302 were performed on
dissociated photoreceptors (Hardie, 1991), using patch-clamp techniques
(Hamill et al., 1981) and short-duration light stimuli. In an attempt
to keep the preparations as physiologically intact as possible, we used
living flies to perform extracellular (electroretinograms, ERG) and
intracellular recordings of photoreceptor responses. Moreover, we used
light stimuli of relatively long durations to explore properties of the
sustained component of the receptor potential. We show that, under
these recording conditions, trpl302 photoreceptor
responses do display distinct mutant phenotypes.
TRP and TRPL channel activities appear to influence each other. For
example, the similarity in the current amplitudes obtained from
trpl302 and wild type was
explained in terms of differential regulation of the two channels by
Ca2+ entering through the TRP channel
(Reuss et al., 1997). These authors found that an increase in the
internal Ca2+ concentration first
facilitated and then suppressed the TRP channel while suppressing the
TRPL channel. Thus, during the initial part of the photoreceptor
response in wild-type flies the calcium ions coming in through the TRP
channels would facilitate the activities of TRP channels but suppress
those of TRPL channels. Because the TRP channels are the only major
contributors to the early part of the response in both wild type and
trpl302, their peak response
amplitudes would be similar. Such interactions are important because
they modulate and shape the responses of photoreceptors. We, therefore,
sought to determine what other forms of interactions might be present
between TRP and TRPL channels.
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MATERIALS AND METHODS |
Materials. The wild-type strain used was Oregon R. All flies were marked with the mutations w
(white) or cn bw (cinnabar brown) to
remove the screening pigments in the eye. The
trpl302 mutant was generated
by Niemeyer et al. (1996). All other mutants were generated on an
Oregon R background by ethylmethane sulfonate mutagenesis in this
laboratory. The light stimuli, originating from a tungsten halogen lamp
(Bausch & Lomb, Rochester, NY), were delivered to the preparation with
a fiber optics light guide. The unattenuated intensity at the level of
the fly was ~800 µW/cm2.
Electroretinograms (ERGs). The ERGs were obtained as
described in Larrivee et al. (1981), using glass microelectrodes filled with Hoyle's saline. White light stimuli attenuated by two log units
were used. Signals were filtered below 100 Hz and sampled at 5 kHz with
an analog-to-digital converter (Digidata 1200A), and the data were
acquired and analyzed in a computer with Axoscope (Axon Instruments,
Foster City, CA).
Intracellular recordings. Intracellular recordings were
performed as described in Johnson and Pak (1986) on 1-d-old flies. The
electrodes were introduced through a cut that covered small parts of
both the cornea and the head. Vacuum grease was applied on the cut to
prevent fluid loss. The recording electrodes had 30-50 M resistance
when filled with 2 M KCl. Signals were filtered out below
50 Hz and sampled at 2 kHz with an analog-to-digital converter
(Digidata 1200A), and the data were acquired and analyzed in a computer
with Axoscope (Axon Instruments).
Western blot analysis. Flies were raised at 25°C in a 12 hr light/dark illumination cycle. Heads were collected from six female and six male flies at 1 d after eclosion and homogenized in 60 µl of SDS-PAGE sample buffer containing 50 mM
dithiothreitol (DTT). The homogenate was boiled for 5 min and
centrifuged (12,000 × g for 3 min), and 10 µl of the
supernatant was loaded onto SDS/8% acrylamide gels. To confirm the
uniformity of total protein loading in each lane, we stained one
of the several identically loaded gels with Coomassie blue. Western
blots were generated by a standard protocol. The anti-TRP monoclonal
antibody (Pollock et al., 1995) was used at 1:3000 dilution.
Confocal microscopy. After dissection, fly eyes were fixed
in 4% formaldehyde (in PBS with 0.3% Triton X-100) for 1 hr and incubated in PBS containing 4% normal goat serum for 2 hr. Filamentous actin of the rhabdomeres was stained with
phalloidin-tetramethylrhodamine B isothiocyanate (Sigma, St. Louis,
MO). Optical sections of ~1 µm thickness were viewed by confocal microscopy.
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RESULTS |
Phenotypes of trpl302
Among the parameters of photoreceptor responses that were examined
by using ERG and intracellular recording techniques were (1) the time
course of decay of the response during stimulus; (2)
V-log I relations, which relate response
amplitudes with the log of stimulus intensity, determined at two time
points: the peak of the response and 2 sec after the onset of response;
(3) oscillations superimposed on the response; (4) poststimulus
hyperpolarizations; (5) adaptation to background illuminations of
different intensities; (6) speed of recovery from a previous
stimulation (refractory period); and (7) response latency.
As reported by others (Niemeyer et al., 1996; Reuss et al., 1997), the
peak response amplitudes obtained from
trpl302 and wild type were
similar (Fig. 1A,B).
However, the sustained components of the two responses had very
different amplitudes and time courses of decay. During a bright 20 sec
light stimulus the responses of both wild type and
trpl302 declined from the peak
amplitude to a lower sustained level (Fig. 1A).
However, the trpl302 response
declined to a much lower level so that the response remaining 20 sec
after the onset was significantly smaller in trpl302 than in wild type. The
smaller sustained amplitudes of the trpl responses could be
detected even 2 sec after the stimulus onset. As before, the peak
amplitudes of the wild-type and
trpl302 responses were
similar, but the amplitudes measured at 2 sec after the onset of
response were significantly smaller in
trpl302 than in wild type
(Fig. 1B). Tables 1 and
2 summarize the results of amplitude
measurements at the response peak and 2 sec after the response onset
obtained at several stimulus intensities. Although the peak amplitudes
were similar between wild type and
trpl302 (Table 1), the
normalized amplitudes measured at 2 sec after the response onset were
all significantly smaller in
trpl302 than in wild type
(Table 2).
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Figure 1.
Electrophysiologically detectable phenotypes of
trpl302. A,
Comparison of representative receptor potentials elicited from wild
type, trpl302, and
trpP343 by using prolonged
stimuli. The trpl302
receptor potential had a peak amplitude similar to that of wild type
but a substantially smaller sustained component, although not as small
as that of trpP343. White
light stimuli of 20 sec duration were used without any attenuation (log
I/I0 = 0, where
I = stimulus intensity used and
I0 = maximum stimulus intensity
available). B, Comparison of representative receptor
potentials elicited from wild type (a) and
trpl302
(b) by using shorter stimuli viewed at faster
sweep speed than in A. The stimuli were 2 sec white
lights attenuated by two log units (log
I/I0 =  2). The
trpl302 receptor potential
showed oscillations superimposed on the response, a reduced sustained
amplitude, and a poststimulus hyperpolarization.
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In addition to the smaller sustained component, the
trpl302 response often
was accompanied by oscillations superimposed on the response during
stimulus and a hyperpolarization immediately after the stimulus
termination (Fig. 1B-b). To determine the frequency of occurrence of these two response phenotypes, we obtained ERGs from
30 trpl302 and 30 wild-type
flies. As summarized in Table 3, 80% (24 of 30) and 37% (11 of 30) of
trpl302 flies exhibited
oscillations and poststimulus hyperpolarizations, respectively, whereas
none of the wild-type flies showed either property. Response
oscillations and poststimulus hyperpolarizations also were examined in
intracellularly recorded responses, allowing the average amplitude of
hyperpolarizations to be determined. Of the 12 cells that were
analyzed, 10 showed oscillations (83%) and seven (58%) showed
hyperpolarizations, yielding similar frequencies of occurrence of these
properties as in ERG recordings. The average amplitude of
hyperpolarization in the seven cells was 2.2 ± 0.6 mV when light
intensities were attenuated by two log units, i.e., log
I/I0 = 2, where
I and I0 are as defined in
the Figure 1 legend.
Still another characteristic of
trpl302 was its inability to
adapt to dim background illuminations (Fig.
2). Light adaptation refers to the
ability of photoreceptors to adjust their sensitivity in response to
changing background illuminations, allowing them to operate over a wide
range of illuminations. Figure 2A compares the
V-log I curves obtained from wild type and
trpl302 normalized to the
maximal response obtained from each cell, using the brightest stimulus
available in the absence of background illumination.
V-log I curves obtained from wild-type
photoreceptors shifted to increasingly higher intensities and saturated
at progressively lower amplitudes as background illuminations increased
in intensity (Fig. 2A). For a test stimulus of given
intensity the largest response was obtained with no background
illumination, and progressively smaller responses were obtained with
background illuminations of increasing intensity (Fig.
2A-a,B-a). The
trpl302 photoreceptors
responded similarly at higher background illuminations, but the
V-log I curve obtained at the lowest background that
was tested was indistinguishable from that generated in the absence of
background illumination [compare open diamonds (5
BG) with open squares (Dark BG) in Figure
2A-b,B-b], suggesting that trpl photoreceptors failed to adapted to the dim background light.
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Figure 2.
Inability of
trpl302 to adapt to a dim
background illumination. A, V-log
I curves for wild type (a) and
trpl302
(b) determined at four different intensities of
background illumination. V-log I curves
relate the response amplitudes to relative stimulus intensities, which
are given in log units. Both the test and background stimuli were white
lights. Before each test stimulus a background light of 1 min duration
was turned on first, and the 2 sec test stimulus was presented at the
very end of the 1 min background. For each cell that was examined, all
responses were normalized with respect to the maximal peak amplitude
obtained in that cell by using the brightest stimulus (log
I/I0= 0) in the absence of
background illumination (n = 5). The average
maximal peak amplitudes obtained from wild type and
trpl302 were 28 ± 4.2 mV (n = 5) and 26.1 ± 3.3 mV
(n = 5), respectively. Dark BG, No
background illumination (open squares); 5
BG, background illumination attenuated by five log units
(open diamonds); 4 BG, attenuated by
four log units (open circles); 3 BG,
attenuated by three log units (open triangles). Unlike
in wild type, the V-log I curve of
trpl302 obtained at 5 BG
is indistinguishable from that obtained in Dark BG. B,
Receptor potentials obtained from wild type (a)
and trpl302
(b) by using maximum intensity white test stimuli
at different background intensities and by using the protocol described
in A. In
trpl302 the receptor
potentials recorded in dark and 5 log backgrounds are very similar in
amplitude and waveform.
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Two other response properties that were tested were unaltered in
trpl302, as was the case of
peak amplitudes that were described previously. They were the
refractory period and response latency. The refractory period was
defined, in terms of the two-stimulus protocol illustrated in Figure
3, as the time required for the response
to the second stimulus, R2, to attain nearly the same amplitude as the
response to the first stimulus, R1. The refractory periods of wild-type and trpl302 responses were
indistinguishable in this protocol. In both cases R2 had an amplitude
similar to that of R1 when two strong 2 sec stimuli, S1 and S2, were
presented only 2 sec apart. The response latency was defined as the
time interval between the beginning of a stimulus and the onset of
response. The latency of R2 was shorter than that of R1 in both
trpl302 and wild-type flies. Moreover,
the magnitudes of latency were also very similar (Fig.
3C).
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Figure 3.
Comparison of the refractory periods
and response latencies of the receptor potentials obtained from wild
type, trpl302, and
trpP343. The
stimulus protocol is shown at the top. After a 2 min
dark adaptation two 2 sec stimuli (S1 and
S2) were presented 20 sec apart, and the corresponding
responses (R1 and R2) were recorded.
A, The responses, R1 and R2, obtained in the above
protocol are shown superimposed to allow for a comparison of
amplitudes: a, wild type; b,
trpl302; c,
trpP343. The term
"refractory period" refers to the time required for the second
response (R2) to attain a response amplitude similar to
that of the first (R1). R2s of both wild type and
trpl302 have amplitudes
similar to those of R1s. R2 of
trpP343, however, is
much smaller than that of R1. The stimulus intensity was attenuated by
one log unit (log I/I0 = 1). B, The initial 120 msec of the responses shown in
A are presented at a higher sweep speed than in
A to allow for a comparison of latencies:
a, wild type; b,
trpl302; c,
trpP343.
Arrows indicate the beginning of light stimuli. The
response latency is defined as the time between the beginning of
stimulus and the onset of the response. C, Histogram
showing the response latencies of R1 and R2 obtained from wild type,
trpl302, and
trpP343
(n = 10). In both wild type and
trpl302 the response
latencies of R1 and R2 are similar in magnitude, and in both the R2
latency is significantly shorter than that of R1. In
trpP343, on the
other hand, both R1 and R2 latencies are much longer than
those of the other genotypes; moreover, the R2 latency is significantly
longer than that of R1. D, Summing the
trpl302 and
trpP343
responses does not reproduce the wild-type response. The
shaded area represents the summation of
the trpl302 and
trpP343 responses.
The summed response has a larger peak amplitude but a smaller sustained
component than the wild-type response.
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Phenotypes of trp
The phenotypes of trp have been described extensively
by previous workers (Cosens and Manning, 1969; Minke et al., 1975; Pak, 1979; Minke, 1982). The present study of trp was performed
(1) to serve as controls for some of the experiments to be described and (2) to assess the relative contributions of TRP and TRPL activities to the photoreceptor response. As has been shown by others, a functionally null trp mutant,
trpP343, had no sustained
response during a strong prolonged light stimulation (Fig.
3D), a reduced peak amplitude (Fig. 3A-c,D), a
longer-than-normal refractory period (Fig. 3A-c), and a
longer-than-normal response latency (Fig. 3B-c,C). A
near-null trp mutant,
trpP301, gave similar results
(see Fig. 5A, B-b,C-b,D). As shown in Figure 3D,
the peak amplitude of
trpP343, elicited by a
bright stimulus, was ~ that of the wild type, whereas that
of trpl302 was approximately
the same as wild type. There was no sustained component in a
trpP343 response, and
there was a significantly reduced sustained component in
trpl302. The summed response
of trpP343 and
trpl302 had a peak amplitude
larger than that of wild type and a sustained component smaller than
that of wild type (Fig. 3D). This observation suggested that
there might be functional interactions between TRP and TRPL channel
activities. We therefore performed the following experiments to see
whether there is any other evidence of possible interactions between
TRP and TRPL and to obtain clues to the nature of the interactions, if
any are present.
TRPL helps to maintain a higher amount of TRP in
InaDP215
Western blot analyses performed on 1-d-old
trpl302 showed that the
trpl302 mutation did not
affect the amount of the TRP protein (Fig.
4A). The
InaD gene encodes a PDZ protein to which several proteins involved in phototransduction, including TRP, bind to form a signaling complex (for review, see Montell, 1999; Tsunoda et al., 1999). In the
InaDP215 mutant the TRP
protein reportedly cannot bind to the signaling complex because of a
defect in the TRP-binding site on the INAD protein (Shieh and Zhu,
1996; Chevesich et al., 1997; Tsunoda et al., 1997). Tsunoda et al.
(1997) have reported that the amount of TRP was normal in newly eclosed
InaDP215 flies but
decreased to an undetectable level by 10 d after eclosion. Consistent with this report, the amount of TRP in 1-d-old
InaDP215 fly heads was
~70% of the wild-type amount (Fig. 4A). If the TRPL protein were removed from
InaDP215 (i.e., in the
trpl302InaDP215
double mutant), however, the amount of TRP was reduced to <10% of the
wild-type amount. To see whether degeneration of the rhabdomeres could
be the cause of this large reduction in the amount of TRP in the
trpl302InaDP215
double mutant, we performed confocal microscopy on the 1-d-old double
mutant. The results (Fig. 4B) showed that the
rhabdomeres of the double mutant were intact at this age, excluding
degeneration of the rhabdomeres as a primary cause of the reduction in
TRP in very young
trpl302InaDP215.
The above data, thus, suggested that the amount of TRP in 1-d-old InaDP215 fly heads
depended strongly on the presence of TRPL.
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Figure 4.
A, Western blot analysis showing
the relative quantity of the TRP protein in wild type,
trpl302,
trpl302InaDP215,
and InaDP215. The
amount of TRP in
trpl302 is
indistinguishable from that in wild type. Although the amount of TRP in
InaDP215 is reduced
only slightly, that of
trpl302InaDP215
is <10% of the wild-type amount. B, Confocal
micrograph showing normal-looking rhabdomeres of the
trpl302InaDP215
double mutant. The rhabdomeres were visualized by staining F-actin with
phalloidin.
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TRP that is not bound to INAD contributes significantly to
photoreceptor response in the presence, but not in the absence, of
TRPL
To explore how the binding of TRP to the signaling complex through
INAD might affect the photoreceptor responses, we compared the
responses of InaDP215
and trpP301.
trpP301 is a near-null
mutant that has no immunodetectable TRP protein but has small yet
detectable physiological activities of TRP when examined in the double
mutant
trpl302;trpP301.
Thus, 1-d-old InaDP215
heads contained ~70% of the wild-type amount of TRP (Fig.
4A), which presumably was not bound to the signaling
complex, whereas trpP301
heads contained a small, undetectable amount of TRP that, however, was
bound properly to the complex. If the TRP protein that was not bound to
the signaling complex were nonfunctional, then one might expect the
phenotype of InaDP215 to
be similar to that of
trpP301. The comparison
between the photoreceptor responses of
InaDP215 and
trpP301 showed
otherwise. The responses of
InaDP215 were more
wild-type-like (less severe) in the peak amplitude (Fig.
5A), amount of decay during
light illumination (Fig. 5A), refractory period (Fig.
5B), and response latency (Fig. 5C,D) than those
of trpP301. The results
suggested that either some TRP remained bound to the signaling complex
in InaDP215 mutants or
the TRP channels that were not bound to the signaling complex
contributed significantly to photoreceptor responses.
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Figure 5.
Comparison of the properties of the receptor
potentials obtained from
InaDP215 and
trpP301.
A, Representative receptor potentials recorded from wild
type, InaDP215, and
trpP301 by using
prolonged, unattenuated white light stimuli. Although both
InaDP215 and
trpP301 receptor
potentials decay toward the baseline, the
InaDP215 receptor
potential decays more slowly than that of
trpP301.
B-D, The differences in the properties of
InaDP215 and
trpP301 responses
revealed in the two-stimuli protocol described in Figure 3. As in
Figure 3, A and B, the R1 and R2
responses are shown superimposed in B and
C. In B, the R2 amplitude is a
significantly larger fraction of the R1 amplitude in
InaDP215 than in
trpP301 at any point
in the response time course so that the time integral of R2 is a
significantly larger fraction of that of R1. C, The
initial 130 msec portions of the responses of
InaDP215 and
trpP301 in
B are presented at a higher sweep speed to allow for a
comparison of latencies. D, Histogram comparing the
response latencies of R1 and R2 from analysis of records similar to
those in C (n = 10) obtained from
wild type, InaDP215,
and trpP301. The
response latency of R2 is shorter than that of R1 in wild type,
approximately the same as that of R1 in
InaDP215, and longer
than that of R1 in
trpP301.
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A very different picture emerged if the TRPL protein were removed from
the above two mutants via the construction of the double mutants
trpl302InaDP215
and
trpl302;trpP301.
As may be seen in Figure
6A, the responses of
both
trpl302InaDP215
and
trpl302;trpP301
were small and transient, but the response amplitude of
trpl302InaDP215
was significantly smaller than that of
trpl302;trpP301.
The comparison of such other properties as the refractory period (Fig.
6B) and response latency (Fig. 6C,D)
further showed that these phenotypes of
trpl302InaDP215
were also more severe than those of the
trpl302;trpP301.
Thus, in the presence of TRPL, TRP that was not bound to INAD contributed significantly to photoreceptor responses
(InaDP215 vs
trpP301), but in
the absence of TRPL, TRP that was not bound to INAD was unable to
contribute significantly to responses
(trpl302;trpP301
vs
trpl302InaDP215).
Although the amount of detectable TRP in
trpl302InaDP215
was much lower than in wild type (see Fig. 4A), this
reduced TRP content could not account for its phenotypes, because the phenotypes were much more severe than those of
trpl302;trpP301,
which had no detectable amount of TRP. The fact that the ability of
non-INAD-bound TRP to contribute to photoreceptor responses depends on
the presence of TRPL suggests that there may be interactions between
TRPL and the non-INAD-bound TRP protein.
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Figure 6.
The effects of the
trpl302 mutation on
trpP301 and
InaDP215 mutants.
A, Comparison of representative receptor potentials
recorded from wild type and the double mutants
trpl302InaDP215
and
trpl302;trpP301,
using 2 sec white light stimuli. The receptor potentials elicited from
both
trpl302InaDP215
and
trpl302;trpP301
are small and transient, but the response amplitude of
trpl302;trpP301
is significantly larger than that of
trpl302InaDP215.
B, C, Properties of responses revealed in
two-stimuli protocol (see Fig. 3). B, Comparison of R1
and R2 amplitude and waveform. R2 is a significantly larger fraction of
R1 in
trpl302;trpP301
than in
trpl302InaDP215.
C, The initial 120 msec portions of the responses of
trpl302InaDP215
and
trpl302;trpP301
in B are presented at a higher sweep speed to allow for
a comparison of latencies. D, Histogram comparing the
response latencies of R1 and R2 obtained from wild type,
trpl302;trpP301,
and
trpl302InaDP215.
The response latencies of both R1 and R2 are longer in
trpl302InaDP215
than in
trpl302;trpP301.
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DISCUSSION |
We have shown that the
trpl302 mutants have clearly
detectable mutant phenotypes. The absence of phenotype reported by
previous workers may be attributable to differences in recording
procedures. Previous studies of the
trpl302 mutants were done by
using the whole-cell patch-clamp technique on dissociated
photoreceptors, whereas the present studies were performed by using the
ERG and intracellular recording techniques on photoreceptors of intact,
living flies. Perhaps an even more important difference might be the
duration of stimuli used to elicit photoreceptor responses. Previous
workers used light pulses of millisecond durations, whereas we used
stimulus durations of seconds to tens of seconds. Properties of
photoreceptor responses that are readily detectable with short-duration
light flashes, such as the peak amplitude and response latency, indeed
are essentially indistinguishable between
trpl302 and wild type.
However, a number of readily detectable phenotypes become apparent if
longer stimulus durations are used and recordings are made from living
flies. These include oscillations, poststimulus hyperpolarizations,
reduced sustained components, and altered adaptation properties. These
results suggest that the TRPL channels may have a role in sustaining
the photoreceptor response during prolonged illuminations and in
adaptation to dim light stimuli, because eliminating the TRPL channels
(i.e., in trpl302)
substantially reduces the sustained component (see Fig.
1A) and makes the photoreceptors insensitive to dim
background illuminations (see Fig. 2). However, complicating the
interpretation regarding its role in sustaining the response is the
observation that, in the absence of TRP channels (i.e., in
trpP343), the TRPL
channels essentially make no contributions to the sustained component
(see Fig. 1A). It thus may be that both classes of
channels contribute to the sustained component and that they both must
be present to allow mutual interactions so that normal channel outputs
might be generated.
The summed amplitude of
trpP343 and
trpl302 mutant receptor
potentials is larger at the peak but smaller throughout the sustained part of the response than the wild-type response amplitude (see Fig.
3D). As discussed previously, Reuss et al. (1997) proposed that the nonlinear summation at the peak of the response arises because
Ca2+ ions entering through the TRP channel
suppress the TRPL channel activity. Thus, in this form of TRP-TRPL
interaction the current entering through one class of channel
influences the activities of both the same and the other class of
channels. For the purpose of present discussion, this form of
interactions may be referred to as "functional interactions."
However, nonlinear summation of the two channel activities is present
not just at the peak but throughout the response. Moreover, the
difference between the summed mutant amplitude and the wild-type
amplitude is positive at the peak of the response but reverses in sign
to be negative throughout the sustained component (see Fig.
3D). It is unlikely that a single mechanism, e.g., that
proposed by Reuss et al. (1997), is responsible for the observed
nonlinear summation of mutant responses throughout their entire time course.
The idea that other forms of interactions may be present in addition to
"functional interactions" is supported by the results obtained with
InaDP215 and
trpl302InaDP215
mutants. The amount of TRP in 1-d-old
InaDP215 mutants is
substantially larger than that in the
trpl302InaDP215
double mutants of the same age (see Fig. 4A).
Moreover, the photoreceptor potential recorded from
InaDP215 is large
and robust (see Fig. 5A), whereas that recorded from the
double mutant
trpl302InaDP215
is very small even smaller than that of
trpl302;trpP301
(see Fig. 6A), which has no immunodetectable TRP
protein. Several groups have shown previously, via in vitro
binding assays, that the TRP protein is unable to bind to the INAD
protein in InaDP215
mutants (Shieh and Zhu, 1996; Chevesich et al., 1997; Tsunoda et al.,
1997). The above results thus suggest that the presence of the TRPL
protein somehow stabilizes the TRP protein even when the latter is not
bound to the INAD protein and allows TRP to contribute substantially to
the photoreceptor response. If one supposes that the TRPL protein is
associated with the signaling complex, as suggested by Xu et al.
(1998), a possible interpretation of these results is that a
significant amount of TRP is associated, however weakly, with the
signaling complex via interaction with the TRPL protein, thus
contributing to the photoreceptor response, even when TRP cannot bind
directly to the signaling complex itself. In the absence of TRPL,
however, the association is lost and much of the response is lost also.
The idea that TRP and TRPL proteins interact need not necessarily be
wedded to the above specific model. For example, the extent to which
the TRP protein fails to bind to INAD in the intact photoreceptors of
the InaDP215 mutant
is unclear because most of the binding studies were performed in
vitro. Regardless of the extent of TRP binding to INAD in the InaDP215 mutant,
however, the results of this study suggest that the presence of TRPL
stabilizes the TRP channel and allows the latter to contribute to the
photoreceptor response in
InaDP215, implying
physical interactions between these two proteins.
The molecular nature of interactions between TRP and TRPL proteins
cannot be specified from the results of the present study. However, it
is pertinent to note that previous workers showed interaction between
TRP and TRPL in immunoprecipitation assays (Xu et al., 1997) and
presented evidence for the formation of heteromultimeric channels when
both TRP and TRPL are coexpressed in oocyte (Gillo et al., 1996) or
cell culture (Xu et al., 1997) systems. Whatever the actual mechanism
of interaction responsible for our results, the results suggest that,
in addition to "functional interactions," direct "physical
interactions" between TRP and TRPL also may be a part of the
photoreceptor process.
| |
FOOTNOTES |
Received April 24, 2000; revised June 23, 2000; accepted July 6, 2000.
This work was supported by a grant from the National Eye Institute,
National Institutes of Health (W.L.P.). The departmental shared
confocal microscope facility was supported by National Science
Foundation Grant BIR-9512962. We thank Dr. John A. Pollock of Carnegie
Mellon University for providing anti-TRP antibodies and Dr. Charles
Zuker of University California San Diego for making the
trpl302 mutant available.
Correspondence should be addressed to Dr. William L. Pak, Purdue
University, Department of Biological Sciences, 1392 Lilly Hall, West
Lafayette, IN 47907-1392. E-mail: wpak{at}bilbo.bio.purdue.edu.
| |
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