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Drosophila TRP and TRPL are assembled ashomomultimeric channels in vivo
Ben Katz1,*, Tina Oberacker2,*, David Richter2, Hanan Tzadok1, Maximilian Peters1, Baruch Minke1 andArmin Huber2,`
1Department of Medical Neurobiology, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences, Hebrew University, Jerusalem91120, Israel2Department of Biosensorics, Institute of Physiology, University of Hohenheim, 70599 Stuttgart, Germany
*These authors contributed equally to this work`Author for correspondence ([email protected])
Accepted 19 April 2013Journal of Cell Science 126, 3121–3133� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.123505
SummaryFamily members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli.The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements forchannel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilieshas been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors
two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-inducedcurrent. The formation of functional TRP–TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in
vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form
heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, andthe structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP andTRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP–TRPL channels using co-
immunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We foundthat TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that thetransmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of
both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.
Key words: Drosophila, Channel assembly, Phototransduction, TRP ion channel, Vision, Chimeric channels
IntroductionThe transient receptor potential (TRP) family of cation channels
serves as sensors and transducers of environmental stimuli and
also as regulators of ion homeostasis in neuronal and epithelial
cells. The founding members of the TRP family are the
Drosophila TRP (Hardie and Minke, 1992; Minke et al., 1975;
Montell and Rubin, 1989) channel and its closest relative TRP-
like (TRPL) (Phillips et al., 1992). To date more than 80 family
members have been isolated from C. elegans, Drosophila, mice
and humans (for reviews see Clapham, 2003; Hardie, 2007;
Minke and Cook, 2002; Minke and Parnas, 2006; Montell et al.,
2002; Montell, 2005), which have been grouped into seven
subfamilies (TRPC, TRPV, TRPM, TRPA, TRPN, TRPP and
TRPML) on the basis of amino acid sequence identity. By
analogy to other channels with a similar transmembrane structure
that have been more extensively studied [e.g. voltage-gated K+
channels and cyclic nucleotide-gated channels (Kaupp and
Seifert, 2002; MacKinnon, 1991)], TRP channels are most
likely formed by tetramers of the pore-forming subunits. Given
the seven mammalian members of the TRPC subfamily and the
total of 28 TRP channel isoforms of mammals, an important
question arises as to the ability of TRP channels to form
heteromultimers and what structural features are required for
channel assembly. Indeed, heteromultimerization of TRP
channels within single subfamilies has been described for
vertebrate members of the TRPC, TRPM and TRPV
subfamilies (for reviews see Cheng et al., 2010; Schaefer,
2005). However, even within the TRPC subfamily not all
members co-assemble with each other. The current view,
though still debatable, is that TRPC1,4,5 and TRPC3,6,7 are
two district assembly groups that do not inter-assemble (Goel
et al., 2002; Hofmann et al., 2002; Parnas et al., 2012; Schaefer,
2005).
In Drosophila photoreceptors two TRPC channels, TRP and
TRPL are expressed together where they generate the light-
induced current (LIC). In dark-raised flies, TRP and TRPL are
localized to the highly packed microvilli, which form the
signaling compartment of fly photoreceptor cells called the
rhabdomere. In the rhabdomere the channels are activated in
response to light, downstream of a Gq protein and phospholipase
C (PLCb) mediated cascade, generating the LIC (Hardie and
Raghu, 2001; Huber, 2004; Minke and Parnas, 2006). Although
in wild-type flies both TRP and TRPL are expressed together in
each photoreceptor cell, they can form functional light-activated
ion channels in photoreceptor cells of Drosophila mutants in
isolation, clearly showing that each channel can function
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independently of the other channel (Niemeyer et al., 1996; Reusset al., 1997). Electrophysiological studies of trp mutants (such as
trpP301, trpCM and trpP343), lacking the TRP channel, and of thetrpl302 null mutant, lacking the TRPL channel, revealed differentbiophysical properties of TRP and TRPL in vivo (Delgado andBacigalupo, 2009; Hardie and Minke, 1994; Hardie and Mojet,
1995; Liu et al., 2007; Raghu et al., 2000; Reuss et al., 1997).Furthermore, the light response is completely abolished in thetrpl302;trpP343 double null mutant, indicating that both channels
are necessary for the response to light (Niemeyer et al., 1996).
In addition to their different biophysical properties, TRP andTRPL also display differences in the dynamics of their
subcellular localization (Bahner et al., 2002; Cronin et al.,2006; Meyer et al., 2006). In dark-raised flies both ion channelsare present in the rhabdomere. However, upon continuousillumination, TRPL translocates from the rhabdomere to an
intracellular storage compartment, while TRP remains in therhabdomeral compartment. An additional difference between theTRP and TRPL channels is their ability to bind to the INAD
scaffold protein. Some of the key elements of thephototransduction cascade are incorporated into supramolecularsignaling complexes via the scaffold protein INAD (Shieh and
Niemeyer, 1995), which binds the TRP channel but also itsactivator PLCb and its regulator protein kinase C (Chevesichet al., 1997; Huber et al., 1996; Tsunoda et al., 1997). A specific
interaction of INAD with TRP is required for rhabdomericlocalization of the entire INAD signaling complex. When thisinteraction is disrupted the INAD and the entire scaffold proteinsincluding the TRP channel moves from the rhabdomere to the
cell body (Tsunoda et al., 1997). In contrast, TRPL appears to beseparated from the INAD signaling complex (Tsunoda et al.,1997; but see Xu et al., 1998).
The first suggestion that TRP channels can assemble asheteromultimers came from studies on the Drosophila channelsTRP and TRPL (Xu et al., 1997). This report provided
electrophysiological and biochemical evidences for theformation of TRP–TRPL heteromultimers obtained from cellculture experiments, in vitro studies and co-immunoprecipitation(co-IP) from fly heads and cell culture. However, the
functionality of heterologously expressed TRP channels wasquestioned (Minke and Parnas, 2006) and the existence of TRP–TRPL heteromultimers in vivo was challenged by showing that
the electrophysiological properties of WT flies could be attainedby a weighted sum of two independent TRP and TRPLcomponents (Reuss et al., 1997). Moreover, the natively
expressed TRP channel protein in the photoreceptor cellsoutnumbers the TRPL channel protein by approximatelytenfold (Xu et al., 2000). This observation together with
massive TRPL (but not TRP) translocation makes functionallysignificant formation of TRP–TRPL heteromultimers unlikely,casting doubt on the reported functional TRP–TRPLheteromultimers. Therefore, the issue whether or not TRP and
TRPL form heteromultimers in fly photoreceptor cells is stillunresolved.
In the present study we investigated whether TRP and TRPL
are able to form heteromultimers, by in vivo assembly studies oftagged TRP and TRPL channels and of chimeric TRP and TRPLchannels. Interactions of tagged and native channels as well as
native and chimeric TRP–TRPL channels using co-IP,immunocytochemistry and functional electrophysiologycritically tested the ability of TRP and TRPL to interact. We
found that TRP and TRPL assemble exclusively ashomomultimeric channels in their native environment. The
above analyses revealed that the transmembrane regions ofTRP and TRPL did not determine assemble specificity of thesechannels. However, the C-terminal regions of both TRP and
TRPL, predominantly specify the assembly of homomultimericTRP and TRPL channels.
ResultsTRP and TRPL assemble as homomultimeric channelsin vivo
In order to address the issue of TRP and TRPL channel subunitassembly in vivo, we generated Drosophila transgenes expressingTRP and TRPL channel subunits fused to enhanced green
fluorescent protein (eGFP) at their C-termini (Fig. 1A) andexpressed the tagged channels under the rhodopsin 1 (Rh1)promoter, driving the expression in R1–6 photoreceptor cells.
The transgenes were introduced into a wild-type (WT)background, which expresses the TRP and TRPL subunitsendogenously. The expression of eGFP-tagged TRP and TRPL
could be readily observed by inspecting the flies under afluorescence microscope with low magnification (Fig. 1A). Inwestern blot analyses the eGFP-tagged TRP and TRPL channels
were identified using TRP and TRPL antibodies generatedagainst the C-terminal region. The western blots showed anapparent molecular mass of about 30 kDa higher than that ofnative TRP and TRPL proteins consistent with the molecular
weight addition of the eGFP-tag (Fig. 1B, Input). This differencein migration on SDS-gels allowed distinguishing between nativeand tagged channels. Co-immunoprecipitation (co-IP) studies
were carried out using antibodies against the eGFP-tag and theimmunoprecipitates were blotted using TRP and TRPLantibodies. This analysis enabled determining which of the
native subunits interact with the tagged subunit (Fig. 1B). Theseexperiments revealed that TRPL–eGFP interacted with nativeTRPL, but not with the native TRP, while TRP–eGFP interactedwith the native TRP but not with the native TRPL. The
specificity of the tag was shown in control flies (WT), whichdid not express eGFP-tagged proteins, showing no signal on thewestern blot. The obtained results clearly show that TRP and
TRPL channels assemble exclusively as homomultimers.
In order to exclude the possibility that the eGFP-tag hindersspecifically heteromeric interactions between TRP and TRPL, we
performed the co-IP studies with WT flies using TRP and TRPLantibodies raised against the C-termini (Fig. 1C). The results ofthese experiments showed that TRP and TRPL were specifically
and exclusively immunoprecipitated with TRP and TRPLantibodies, respectively (Fig. 1C). Furthermore, in order toexclude the possibility that the TRP and TRPL antibodies
raised against the C-termini hinder specifically heteromultimericinteractions, we used a TRPL antibody raised against the N-terminal region (a-TRPL-NT) of the channel. The results of these
experiments showed that TRPL was specifically and exclusivelyimmunoprecipitated with TRPL antibodies (Fig. 1C). Thus, inline with the anti–eGFP studies, these experiments reveal thatTRP and TRPL channels assemble exclusively as
homomultimers.
Additional evidence against the formation of TRP–TRPL
heteromultimers came from the previously describedtranslocation of TRPL. Accordingly, TRPL–eGFP showedtranslocation between the rhabdomere and the cell body in
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dark- and light-adapted flies, respectively (Meyer et al., 2006)
while, TRP remained in the rhabdomere irrespective of the light
rearing condition indicating no TRP–TRPL interactions
(Fig. 1Da–h). Further evidence against the formation of TRP–
TRPL heteromultimers came from analysis of transgenic flies
expressing the TRP–eGFP channels. In flies expressing TRP–
eGFP in the WT background, the GFP signal was detected in the
rhabdomeres in both light- and dark-adapted flies and did not
affect the light-triggered translocation of TRPL to the cell body
(Fig. 1Di–p). The localization of TRP–eGFP was different in
dark-adapted transgenic flies expressing TRP–eGFP in the
trpP343 background. In these flies the GFP signal was
predominantly detected in the cell body, while the TRPL signal
was detected in the rhabdomere (Fig. 1Dq–t). These results
further support the notion that the TRP and TRPL channels do
not interact.
A possible explanation for the observed localization of TRP–
eGFP in the trpP343 background is that in the absence of TRP, TRP–
eGFP cannot associate with INAD promoting TRP–eGFP
localization to the cell body (Tsunoda et al., 1997). To directly
examine this possibility, we performed co-IP experiments;
supplementary material Fig. S1 shows that in the WT
background, in which native TRP and TRP–eGFP are co-
expressed, INAD was precipitated by the anti-eGFP antibody. In
contrast, in the absence of native TRP (TRP–eGFP in the trpP343
null background) INAD was not precipitated, indicating that the
eGFP-tag disrupted TRP-INAD interaction, while the TRP–TRP–
eGFP heteromultimers can interact with INAD. Similar results were
obtained when using anti-TRP antibody, thus demonstrating that
the anti-eGFP antibody did not disrupt the TRP–INAD interaction.
The co-IP experiments, together with the observation that TRP
and TRPL can be localized in separated non overlapping cellular
compartments supports the notion that TRP and TRPL do not
interact with each other and assemble only as homomultimers.
In order to examine the functional consequence of eGFP-tag
attachment to the channels, we performed whole-cell patch-
clamp recordings. Attachment of the eGFP tag had different
effects on the TRP and TRPL channels. Attachment of the eGFP-
tag to the TRPL channel (TRPL–eGFP) had virtually no effect on
the light response amplitude as shown by the intensity–response
Fig. 1. TRP and TRPL ion channels assemble as homomers. (A) Schemes of TRPL and TRP channels with and without an eGFP tag. The eGFP tag is shown in
green. N- and C-termini (N, C) and the TRP domain are indicated. Fluorescence microscopy of transgenic fly eyes expressing TRPL–eGFP and TRP–eGFP are
depicted next to the schemes. (B) Co-IP of TRP and TRPL from Drosophila photoreceptor cells expressing TRPL–eGFP or TRP–eGFP in the WT background.
GFP-immune complexes were fractionated by SDS–PAGE, and the western blot was probed with TRPL (upper panel) and TRP (lower panel) antibodies. (C) Co-
IP of TRP and TRPL from WT Drosophila photoreceptor cells. Immune complexes obtained using TRP and TRPL antibodies directed against the C-terminal
region of TRP (a-TRP) and the TRPL C-terminal (a-TRPL) or N-terminal (a-TRPL-NT) region were fractionated by SDS–PAGE, and the western blot was
probed with TRPL (upper panel) and TRP (lower panel) antibodies. (D) Subcellular localization of TRP and TRPL in dark- (16 hour) and light-adapted (16 hour
orange light) Drosophila eyes expressing TRPL–eGFP and TRP–eGFP in the WT background as well as TRP–eGFP in trpP343 or trpl302 null backgrounds.
Fluorescence microscopy images of eye cross sections, showing fluorescence of eGFP-tagged channels (green), immunofluorescence of anti-TRP or anti-TRPL
antibodies (red) and phalloidin labeling of the rhabdomeres (white). A merge of the green and red fluorescence is also shown. Scale bar: 10 mm.
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curve (Fig. 2A–C) (Meyer et al., 2006) demonstrating that the
eGFP tag had no effect on the function of the TRPL channel.
However, for the TRP channel (TRP–eGFP) the situation was
different depending on the genetic background. Accordingly,
flies expressing TRP–eGFP in WT and trpl302 backgrounds
showed response amplitudes similar to WT and trpl302. However,
TRP–eGFP in the trpP343 background showed reduced sensitivity
to light compared to WT and trpl302 mutant flies. This reduction
in sensitivity to light is readily explained by the observed
disruption of the TRP-INAD interaction required for retention of
the entire INAD signaling complex to the rhabdomere (Fig. 1Di–
l; Fig. 1Dq–t; Fig. 1Du–x; supplementary material Fig. S1)
(Tsunoda et al., 1997). This complex includes the TRP channel
and its activator, phospholipase C, which are critical components
for the normal sensitivity to light. However, TRP–eGFP in the
trpP343 background revealed also a small reduction in sensitivity
compared to trpP343. This result cannot be explained solely by the
disruption of TRP–INAD interaction as the same situation is also
observed in the trpP343 mutant fly (Tsunoda et al., 2001).
Nevertheless, this result can be explained by at least two
mechanisms. One possible mechanism is that non-functional
TRP–eGFP induces a dominant-negative effect by the formation
of TRPL–TRP–eGFP heteromultimers. In order to test this
possibility, we examined whether TRP–eGFP is non-functional.
To this end we measured the reversal potential (Erev) of WT,
trpl302, trpP343 and TRP–eGFP in the trpP343 background.
Importantly, the LIC of TRP–eGFP in the trpP343 background
revealed a biphasic Erev typical of two functional channels with
different Erev similar to WT (supplementary material Fig. S2).
Moreover, this Erev was significantly more positive than the Erev
of trpP343, also indicating that TRP–eGFP is functional (Reuss
et al., 1997), thus making the above mechanism unlikely. A
second possible mechanism is that the reduced sensitivity to light
of the TRP–eGFP in the trpP343 background arises from
functional interaction of independent TRP–eGFP and TRPL
channels (Reuss et al., 1997). Accordingly, when functional TRP
and TRPL channels are activated together, the Ca2+ influx via the
TRP channels suppresses the activity of the TRPL channels
Fig. 2. Whole-cell measurements of eGFP-tagged TRP and TRPL. (A) Whole-cell recordings of representative responses of the indicated fly strains to light at
1.36105 effective photons/second (EP/s). (B) Intensity–response (R-logI) relationship of the fly strains as in A (n55, means 6 s.e.m.). Inset: intensity–response
(R-logI) relationship of dim light (n55, means 6 s.e.m.). (C) Histogram of the peak amplitude of the light response to 1.36104 EP/s (left) and 1.36105 EP/s (right)
of the fly strains as in A. (D) Representative light-induced responses of TRP–eGFP in the indicated backgrounds to 1.36105 EP/s. (E) Intensity–response
relationship of the fly strains as in D (n55, means 6 s.e.m.). Inset: intensity–response relationship in dim light (n55, means 6 s.e.m.). (F) Histogram of the peak
amplitude of the response to 1.36104 EP/s (left) and 1.36105 EP/s (right) of the fly strains as in D.
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(Reuss et al., 1997) so that mainly the TRP current is maintained
(e.g. in Fig. 2, notice the similarity of the LIC peak amplitude of
WT, expressing both TRP and TRPL channels, and trpl302,
expressing only the TRP channel). However, in the trpP343
background fly, the amount of functional TRP–eGFP in the
rhabdomere is small (Fig. 1Dq; supplementary material Fig. S2),
contributing more to the suppression of the TRPL channel than
to the overall current. Together, elucidating the functional
consequence of eGFP-tag attachment to the TRP channel
indicated that the electrophysiological data are consistent with
the co-IP and immunocytochemistry data.
The use of TRP–TRPL chimeras to examine the domainsspecifying channel assembly
To further examine TRP and TRPL subunit assembly and to
identify regions in the channel proteins that determine the
formation of specific multimers, we analyzed four different TRP–
TRPL chimeric channels. We have previously generated chimeric
Drosophila transgenes expressing constructs, in which either the
C-terminus, the N-terminus, both regions, or the transmembrane
domains of TRP were replaced by the corresponding regions of
TRPL (Richter et al., 2011). They were referred to as chimera 1
(NTRP-TMTRP-CTRPL-eGFP), chimera 2 (NTRPL-TMTRP-CTRP-
eGFP), chimera 3 (NTRPL-TMTRP-CTRPL-eGFP) and chimera 4
(NTRP-TMTRPL-CTRP-eGFP) (Richter et al., 2011). Expression of
those chimeras in the WT background in Drosophila eyes was
verified by in vivo fluorescent measurements (Fig. 3A) and by
western blot analysis with antibodies against TRP or TRPL
(Fig. 3B) (Richter et al., 2011). As the TRP and TRPL antibodies
used here are directed against the C-terminal regions of the
channels, anti-TRPL detected chimera 1 and 3, while anti-TRP
detected chimera 2 and 4.
All the chimeric constructs formed functional light-activated ion
channels in photoreceptor cells when expressed in the trpl302;trpP343
double null mutant background (Fig. 4; Figs 6–8) (Richter et al.,
2011). However, the amplitude of the LIC was markedly reduced in
chimeras 1–3 compared with trpl302 mutant fly, while for chimera 4
small reduction in response amplitude was observed compared to
the trpP343 mutant fly, when the chimeric channels were expressed
in the trpl302;trpP343 double null mutant background.
Chimera 3 (NTRPL-TMTRP-CTRPL-eGFP) shows that thetransmembrane domain does not specify TRP–TRPLchannel assembly
We first performed co-IP experiments on chimera 3 in the WT
background, applying antibodies against the eGFP-tag and the
immunoprecipitates were blotted using TRP and TRPL antibodies
(Fig. 3B, lane 4). Since the tagged subunit is distinguishable from
the endogenous subunit on western blots by its higher molecular
weight, this assay allowed identification of the in vivo interaction
partners of chimera 3. Fig. 3B shows that chimera 3 interacted
with native TRPL, but not with the native TRP channel (compare
Fig. 3B upper and lower panels, lane 4). This result indicates that
chimera-3–TRPL heteromultimers are formed, while chimera-3–
TRP heteromultimers do not form.
To further test the above conclusion, we examined the cellular
localization of chimera 3, TRP and TRPL channels using
immunocytochemistry in dark- and light-raised flies. The
immunocytochemical studies were used to examine whether or
not the two channel subunits found to interact in co-IP experiments
also reside in the same cellular region. Fig. 4A shows that both
native TRPL and chimera 3 translocated normally from the
rhabdomere to the cell body upon illumination as previously
reported (Fig. 4Aa–h) (Richter et al., 2011). This finding is
compatible with an interaction between the native TRPL and
chimera 3. However, it should be noted that the TRPL antibody
detected both chimera 3 and native TRPL. Hence, while
translocation of chimera 3 was clearly revealed by observing the
eGFP fluorescence (Fig. 4Aa,e), translocation of native TRPL
from the rhabdomere to the cell body was revealed only indirectly
by the absence of labeling of the rhabdomeres in the light
(Fig. 4Ab,f). The situation was different when analyzing chimera 3
and TRP localization. In dark-raised flies colocalization was
observed in the rhabdomere for both TRP and chimera 3 (Fig. 4Ai–
p). However, in light-raised flies, chimera 3 translocated to the cell
body and did not colocalize with the native TRP channel, which
Fig. 3. Expression of chimeric TRP/TRPL ion channels in Drosophila photoreceptor cells and Co-IP studies of the chimeric constructs. (A) Schemes of the
chimeric TRP/TRPL ion channels. Numbers indicate amino acid positions at which sequences were exchanged to construct the chimera. Fluorescence microscopy
of transgenic fly eyes expressing chimera-1–4–eGFP are depicted next to the schemes. (B) Co-IP of chimera-1–4–eGFP from Drosophila photoreceptor cells
expressing the chimera in the WT background. Immune complexes precipitated with anti-GFP antibodies were fractionated by SDS–PAGE, and the western blot
was probed with TRPL (upper panel) and TRP (lower panel) antibodies.
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remained localized to the rhabdomere (Fig. 4Ai–p). These results
are consistent with the co-IP experiments.
To determine the physiological effects of the expression of
chimera 3 in photoreceptor cells we carried out whole-cell
recordings from isolated ommatidia. Chimera 3 expressed in the
trpl302; trpP343 double null background revealed highly reduced
sensitivity to light when compared to both trpl302 and trpP343 mutant
flies (Fig. 4B–D). Strikingly, chimera 3 expressed in the trpl302
background showed sensitivity to light similar to WT and trpl302
indicating that chimera 3 does not affect the LIC through TRP
channels. However, chimera 3 in the trpP343 background (expressing
only the native TRPL channel) showed highly reduced sensitivity to
light when compared to WT, trpl302 or trpP343 mutant flies. Because
the current and hence Ca2+ influx produced by light activation of
chimera 3 in isolation was very small the above finding is best
explained by a dominant-negative effect of chimera 3 on the native
TRPL channel with which it forms multimers (Fig. 4B–D).
These results also show no chimera-3–TRP interactions as no
dominant-negative effect was observed in chimera 3 in the trpl302
background.
The macroscopic response to light of the fly is composed of
single photon responses (quantum bumps), which sum to produce
the LIC (for review see Katz and Minke, 2009). Reduced
sensitivity to light can arise from two different phenomena:
reduction in mean bump amplitude or from reduction in bump
frequency. Therefore, dominant-negative effect at the quantum
bump level is manifested by a reduction in bump amplitude or
rate of occurrence. To further examine the dominant-negative
effect of chimera 3 on the native TRPL channel, we measured
responses to dim light, which elicit quantum bumps (Fig. 5). The
figure shows that quantum bump formation is highly attenuated
in chimera 3 in the trpP343 background, while no dominant-
negative effect was observed in chimera 3 in the trpl302
background (Fig. 5A–D). These results are compatible with the
observation of the macroscopic response to light.
Together, the analysis of chimera 3 demonstrated that chimera
3 with N- and C-termini of TRPL exclusively formed multimers
with TRPL and had a dominant-negative effect on the TRPL- but
not on the TRP-mediated current. We conclude that the
requirement for TRPL multimeric formation is specified by
either the N- or C-terminal region or both regions but is not
specified by the TRPL transmembrane domains.
Chimera 4 (NTRP-TMTRPL-CTRP-eGFP) gives additionalevidence that the transmembrane domain does not specify
TRP–TRPL channel assembly
As with chimera 3 we first used co-IP studies with antibodies
against the eGFP-tag of chimera 4 (Fig. 3B, lane 5) and asked
which of the native TRP and/or TRPL subunits interact with the
tagged subunit. Fig. 3B shows co-IP experiments using flies
Fig. 4. Chimera-3–eGFP interacts solely with
the TRPL channel. (A) Subcellular localization
of chimera-3–eGFP, TRPL and TRP in dark-
adapted (16 hour) and light-adapted (16 hour
orange light) Drosophila eyes expressing
chimera-3–eGFP in the WT background.
Fluorescence microscopy of eye cross sections,
showing fluorescence of eGFP-tagged channels
(green), immunofluorescence of anti-TRPL (left
panel, red) or anti-TRP antibodies (right panel,
red) and phalloidin labeling of the rhabdomeres
(white). A merge of the green and red
fluorescence is also shown. Scale bar: 10 mm.
(B) Representative responses of chimera 3 in the
trpl302, trpP343 and trpl302;trpP343 backgrounds
to light at 1.36105 EP/s. (C) Intensity–response
(R-logI) relationship of the fly strains as in A
(n55, means 6 s.e.m.). Inset: intensity–
response (R-logI) relationship in dim light
(n55, means 6 s.e.m.). (D) Histogram of the
peak amplitude of the response to 1.36104 EP/s
(left) and 1.36105 EP/s (right) of the fly strains
as in B.
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expressing chimera 4 in the WT background. These experiments
revealed that chimera 4 interacted with native TRP, but not with
the native TRPL channel (compare Fig. 3B upper and lower
panels, lane 5).
Immunocytochemical studies on chimera 4 expressed in the WT
background showed that this chimera was localized to both
rhabdomere and cell body, without considerable changes in its
distribution between light and dark rearing condition
(Fig. 6Aa,e,i,m). The expression of chimera 4 in WT
photoreceptors did not affect the localization of native TRPL,
which displayed its typical translocation between the rhabdomere
and cell body upon illumination (Fig. 6Ab,f). Labeling with TRP
antibodies was indistinguishable from eGFP-fluorescence
detection (Fig. 6Ai–p). However, it should be noted that the TRP
antibody detected both chimera 4 and native TRP. Nevertheless,
the anti-TRP signal showed relatively low intensity in the R1–6
rhabdomeres compared with the R7 rhabdomere signal, indicating
a reduction in the TRP expressed in the rhabdomere. Hence, these
findings are in line with the co-IP results showing interaction of
chimera 4 with TRP but not with TRPL.
Single cell recordings revealed that chimera 4 in the trpl302;
trpP343 double null background showed the largest peak
amplitude and sensitivity to light relative to all the other 3
chimeras, reaching ,4 nA peak amplitude of the LIC in response
to a light intensity of 1.36105 effective photons per second,
similar to that of the trpP343 mutant fly (Fig. 6B–D). This can be
explained by the fact that chimera 4 has the transmembrane
regions of TRPL, forming the large conduction pore of TRPL
(i.e. at least approximately fivefold larger single channel
conductance than that of the TRP channel; Raghu et al., 2000)
and it is also present at the rhabdomeres (Fig. 6Aa,e,i,m), while
chimeras 1–3 harbor the TRP transmembrane domains forming
the TRP pore with a small estimated single channel conductance
(Richter et al., 2011). Similarly, the quantum bumps of chimera 4
in the trpl302; trpP343 background revealed quantum bumps of
smaller amplitude similar to the quantum bumps of the trpP343
mutant, indicating small reduction in the sensitivity to light of
chimera 4 in isolation compared with trpP343 mutant flies
(Fig. 5D–E). Analysis of flies expressing chimera 4 in the
trpl302 background showed that the peak amplitude of the LIC in
photoreceptors expressing chimera 4 in the trpl302 background
was similar to WT and trpl302 mutant fly (Fig. 6B–D). Thus,
although chimera 4 and TRP form heteromultimers as revealed
by co-IP and immunocytochemistry studies, chimera 4 had no
dominant-negative effect on the TRP-mediated current. This may
be explained by the minor reduction in sensitivity to light of
chimera 4 in the trpl302; trpP343 background together with the still
observed localization of both chimera 4 and TRP in the
rhabdomere, as revealed by immunocytochemistry.
A puzzling observation is that chimera 4 in the trpP343
background (expressing native TRPL) showed reduced
sensitivity to light compared with trpP343 flies (Fig. 6B–D).
Fig. 5. Bump analysis of the chimeric channels. (A,D,E) Whole-cell recordings from the indicated fly strains after dim light stimulation of 1.3 EP/s,
showing quantum bumps. (B) Histogram of the mean bump amplitude of the fly strains in A (n55, means 6 s.e.m., ***P,0.001). Note the reduced mean
bump amplitude of chimera 2 in the trpl302 background compared with the other fly strains. (C) Histogram of the mean bump rate of the fly strains in A (n55,
means 6 s.e.m.).
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This phenomenon was also observed and explained in detail for
TRP–eGFP in the trpP343 background (Fig. 2D–F). However, in
this case Ca2+ influx was mediated by the large TRPL single
channel conductance and the relative high chimera 4 expression
in the rhabdomere (see above).
Together, the analysis of chimera 4 by two independent
methods, namely co-IP and immunocytochemistry, demonstrated
that chimera 4 channels are unable to interact with the native
TRPL channel, while they are able to interact with the native
TRP channel regardless of their transmembrane domain.
Electrophysiology could not be used to demonstrate chimera-4–
TRP interactions, because chimera 4 did not show severely
reduced sensitivity in isolation.
Chimeras 1 and 2 reveal that the C-terminal regions of TRP
and TRPL largely determine specificity of subunitassembly
The results obtained from chimera 3 and 4 expressing flies
revealed that chimera 3 with its N- and C-terminal regions of
TRPL is able to interact only with TRPL, while chimera 4 with its
N- and C-terminal regions of TRP is able to interact only with
TRP. However, these chimeras could not resolve whether the N- or
C-termini (or both) specify channel assembly. To answer this
question we studied flies expressing chimera 1 and chimera 2.
Co-IP experiments using flies expressing chimera 1 (NTRP-
TMTRP-CTRPL-eGFP; Fig. 3A) in the WT background revealed
that it interacted with native TRPL, but not with native TRP
(Fig. 3B, lane 2). A previous study on chimera 1 revealed that this
chimera does not translocate upon illumination and resides
predominantly in the cell body (Fig. 7Aa,e,i,m) (Richter et al.,
2011). A small fraction of chimera 1 resides in the rhabdomere as
evidenced by the very small LIC when expressed in the trpl302;
trpP343 double null background (Fig. 7B–D). To further examine
the ability of this chimera, expressed in the WT background, to
interact with the native TRPL and TRP, we performed
immunocytochemical experiments. Chimera 1 affected the
localization of native TRPL that was no longer detected in
rhabdomeres of photoreceptor cells R1–6 in dark reared flies.
Instead, TRPL antibodies (detecting both, chimera 1 and native
TRPL) labeled only the cell body and the rhabdomere of R7 cells
in dark-reared flies (Fig. 7Ab,f). Labeling of the R7 cell
rhabdomere was expected as chimera 1 was not expressed in this
cell and served as a positive control. Fig. 7Ai–p further shows that
chimera 1 did not colocalize with the TRP channels, which are
mainly located to the rhabdomere. These data revealed that
chimera 1 hindered TRPL translocation to the rhabdomere,
presumably due to formation of heteromultimers between
chimera 1 and TRPL. These results are consistent with the co-IP
experiments and together show that while chimera 1 interacts with
the native TRPL it does not interact with the native TRP channel.
We next applied whole-cell patch-clamp recordings from
isolated Drosophila ommatidia. Chimera 1 expressed in the
Fig. 6. Chimera-4–eGFP interacts solely with
the TRP channel. (A) Subcellular localization of
chimera-4–eGFP, TRPL and TRP in dark-adapted
(16 hour) and light-adapted (16 hour orange light)
Drosophila eyes expressing chimera-4–eGFP in
the WT background. Fluorescence microscopy of
eye cross sections, showing fluorescence of eGFP-
tagged channels (green), immunofluorescence of
anti-TRPL (left panel, red) or anti-TRP antibodies
(right panel, red) and phalloidin labeling of the
rhabdomeres (white). A merge of the green and
red fluorescence is also shown. Scale bar: 10 mm.
(B) Representative responses of chimera 4 in the
trpl302, trpP343 and trpl302;trpP343 backgrounds to
light at 1.36105 EP/s. (C) Intensity–response (R-
logI) relationship of the fly strains as in A (n55,
means 6 s.e.m.). Inset; intensity–response
relationship in dim light (n55, means 6 s.e.m.).
(D) Histogram of the peak amplitude of the
response to 1.36104 EP/s (left) and 1.36105 EP/s
(right) of the fly strains as in B.
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trpl302;trpP343 double null background revealed highly reduced
sensitivity to light when compared to both trpl302 and trpP343
mutant flies (Fig. 7B–D). Chimera 1 in the trpl302 and WT
backgrounds showed similar sensitivity to light compared to both
WT and trpl302 indicating that no interaction exists between
chimera 1 and TRP channel subunits (Fig. 7B–D). Analysis of
the microscopic response to dim light stimulation confirmed the
results of the macroscopic response by showing that the mean
amplitude quantum bump and bump frequency of chimera 1 in
the trpl302 background was similar to that of the trpl302 mutant fly
(Fig. 5A–C). Strikingly, chimera 1 in the trpP343 background
showed a highly reduced sensitivity to light compared to WT,
trpl302 and trpP343 mutant flies indicating a strong dominant-
negative effect of chimera 1 on TRPL (Fig. 7B–D). The
observation of the dominant-negative effect on the macroscopic
response to light was also obtained in chimera 1 in the trpP343
background, at the quantum bump level, by showing virtually no
bump production (Fig. 5D). Thus the electrophysiological data
strongly support the conclusions obtained from the co-IP and
immunocytochemical experiments that chimera 1 interacts with
the native TRPL but not with the native TRP channel.
The co-IP studies of flies expressing chimera 2 (NTRPL-TMTRP-
CTRP-eGFP; Fig. 3A) in the WT background revealed that it
interacted with both native TRPL and native TRP (Fig. 3B, lane 3).
As is the case for chimera 1, chimera 2 was located almost
exclusively in the cell body irrespective of the light rearing condition
(Fig. 8Aa,e,i,m) (Richter et al., 2011). To further examine the ability
of chimera 2 in the WT background to interact with the native TRPL
and TRP channels we performed immunocytochemical
experiments. As revealed by staining with TRP antibodies
(detecting both chimera 2 and native TRP), expression of chimera
2 resulted in mislocalization of TRP, which was no longer observed
in the rhabdomeres of R1–6 cells (Fig. 8Aj,n). A superficial
observation of Fig. 8 shows that chimera 2 had no effect on the
localization of TRPL, which was correctly located in the
rhabdomeres or in the cell body of dark- or light-reared flies,
respectively (Fig. 8Ab,f). However, a close examination of the
merged images showed colocalization of chimera 2 and TRPL,
indicating possible interaction. Further examination of
immunocytochemical analysis of chimera 2 in the trpP343
background, in which the TRP and TRPL channel subunits do not
compete for chimera 2 interaction, showed enhanced colocalization
between TRPL and chimera 2 (supplementary material Fig. S3).
In whole-cell patch-clamp recordings, chimera 2 in the trpl302;
trpP343 background showed strongly reduced sensitivity to light
compared with WT, trpl302 and trpP343 mutant flies (Fig. 8B–D).
Chimera 2 in the trpP343 background as well as in the trpl302
background showed reduced sensitivity to light compared with
WT, trpl302 and trpP343 mutant flies indicative of a dominant-
negative effect of chimera 2 on both, TRP- and TRPL-mediated
currents. Chimera 2 in the WT background showed partial rescue
of the dominant-negative effect of chimera 2 observed in the
Fig. 7. Chimera-1–eGFP interacts solely with
the TRPL channel. (A) Subcellular localization
of chimera-1–eGFP, TRPL and TRP in dark-
adapted (16 hour) and light-adapted (16 hour
orange light) Drosophila eyes expressing
chimera-1–eGFP in the WT background.
Fluorescence microscopy of eye cross sections,
showing fluorescence of eGFP-tagged channels
(green), immunofluorescence of anti-TRPL (left
panel, red) or anti-TRP antibodies (right panel,
red) and phalloidin labeling of the rhabdomeres
(white). A merge of the green and red
fluorescence is also shown. Scale bar: 10 mm.
(B) Representative responses of chimera 1 in the
WT, trpl302, trpP343 and trpl302;trpP343
backgrounds to light at 1.36105 EP/s.
(C) Intensity–response (R-logI) relationship of
the fly strains as in A (n55, means 6 s.e.m.).
Inset: intensity–response (R-logI) relationship in
dim light (n55, means 6 s.e.m.). (D) Histogram
of the peak amplitude of the response to 1.36104
EP/s (left) and 1.36105 EP/s (right) of the fly
strains as in B.
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trpl302 background. This can be readily explained by competingbinding of chimera 2 to both TRP and TRPL channels, resulting (in
the case of the WT background) in additional homomultimeric
TRP channels (Fig. 5A–C). The observation of the dominant-
negative effect on the macroscopic response to light was also
obtained at the quantum bump level showing reduced mean bumpamplitude of chimera 2 in the trpl302 background with little change
in bump frequency (Fig. 5A–C), while chimera 2 in the trpP343
background showed virtually no bumps (Fig. 5D).
Together, the analysis of chimera 1 and 2 by three independent
methods, demonstrated that the C-terminal region of TRPL
determines assembly specificity with the native TRPL channel,
while the C-terminal region of TRP determines assemblyspecificity with TRP. The N-terminal region of TRPL may
also contribute to TRPL assembly specificity, as evidenced
by the interaction of chimera 2 with TRPL in co-IP and
electrophysiological studies. In contrast, we did not find evidenceindicating that the N-terminal region of TRP can specify the
formation of TRP multimers (but see Discussion).
DiscussionIn the present research we studied molecular aspects, which
determine the specificity of the Drosophila TRPC channels
assembly. Many of the disparate results regarding TRPC function
and regulation were reconciled by showing that several TRPCchannel subunits are assembled into heteromultimeric channels
with diverse properties. These studies have also shown that
heteromultimeric assembly of various members of the TRPC
channel members are characterized by heteromultimeric
assembly into two distinct groups: the TRPC1,4,5 and
TRPC3,6,7 groups (Goel et al., 2002; Hofmann et al., 2002;
Parnas et al., 2012; Schaefer, 2005). Clapham and colleagues
have reported that TRPC1 co-assembled with TRPC4 and TRPC5
in rat brain (Strubing et al., 2001). The biophysical properties
of TRPC(1,4) and TRPC(1,5) heteromultimers are distinct
from those of the channel homomultimers. Considering that
many TRPCs are ubiquitously expressed and often co-expressed
in a given cell (Abramowitz and Birnbaumer, 2009),
heteromultimerization in addition to homomultimerization
among members of this protein family represents an efficient
way to increase the functional diversity of TRPC channels. A
structural understanding of how TRPC subunits combine to form
functional ion channel complexes is an essential prerequisite to
evaluate the contribution of a given TRPC channel to endogenous
PLC-dependent cation currents.
Since the two Drosophila TRPC channels TRP and TRPL
are expressed in each one of the photoreceptor cells of
the Drosophila compound eye, the previous report on the
existence of both homomultimeric and heteromultimeric
assembly of TRP and TRPL (Xu et al., 1997) was in
agreements with later studies on TRPC channels. Unlike the
study by Xu et al. the present study shows that TRP and TRPL
channels do not form heteromultimers (Table 1) (Xu et al., 1997).
Our results are consistent with previous studies showing that the
Fig. 8. Chimera-2–eGFP predominantly
interacts with the TRP channel.
(A) Subcellular localization of chimera-2–
eGFP, TRPL and TRP in dark-adapted
(16 hour) and light-adapted (16 hour orange
light) Drosophila eyes, expressing chimera-2–
eGFP in the WT background. Fluorescence
microscopy of eye cross sections, showing
fluorescence of eGFP-tagged channels (green),
immunofluorescence of anti-TRPL (left panel,
red) or anti-TRP antibodies (right panel, red)
and phalloidin labeling of the rhabdomeres
(white). A merge of the green and red
fluorescence is also shown. Scale bar: 10 mm.
(B) Representative responses of chimera 2 in the
WT, trpl302, trpP343 and trpl302;trpP343
backgrounds to 1.36105 EP/s. (C) Intensity–
response (R-logI) relationship of the fly strains
as in A (n55, means 6 s.e.m.). Inset: intensity–
response relationship in dim light (n55, means
6 s.e.m.). (D) Histogram plotting the peak
amplitude of the response to 1.36104 EP/s (left)
and 1.36105 EP/s (right) of the fly strains as
in B.
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WT light-activated conductance is composed of two independent
TRP and TRPL components (Reuss et al., 1997), that only TRPL
translocates from the rhabdomere to the cell body upon
illumination (Bahner et al., 2002), and that only TRP interacts
with INAD (Tsunoda et al., 1997) (see also Xu et al., 1998).
Co-IP studies with native TRP and TRPL subunits extracted
from Drosophila eyes suffer from the drawback that per se only
heteromultimers but not homomultimers can be detected. It is
therefore difficult to adjust the experimental conditions in a way
that excludes precipitation artifacts. We have chosen here to use
eGFP-tagged subunits allowing detection of multimers composed
of the tagged and the native subunit, which can be distinguished
on western blots by their different molecular mass. These co-IP
experiments demonstrated that the TRPL and TRP channels do
not interact with each other (Fig. 1B,C; Table 1).
Previous studies have demonstrated that coiled-coil (cc)
domains and ankyrin repeats participate in TRP channel
assembly (Engelke et al., 2002; Lepage et al., 2006; Lepage
et al., 2009; Xu et al., 2000). In order to better understand
previous (Xu et al., 1997) and current results and to explain the
possibilities of association between TRP and TRPL, we analyzed
the ankyrin repeats and cc-domains of TRP and TRPL channels
(supplementary material Fig. S4). Two ankyrin repeats (aa 78–
107 and 152–181 of TRPL; supplementary material Fig. S4) and
a cc-domain (aa 233–266 of TRPL; supplementary material Fig.
S4) at the N- termini of both the TRP and TRPL channel were
identified with high confidence. Sequence alignments of these
domains showed sequence homology of the first ankyrin repeat
(63% similarity) and very high sequence homology (93%
similarity) of the second ankyrin repeat (supplementary
material Fig. S4). The alignment of the N-terminal cc-domain
showed high sequence homology both globally (62% similarity)
and in its a–d pattern which is a repetitive pattern in cc-domains
responsible for multimerization [80% similarity (Fujiwara and
Minor, 2008)] (supplementary material Fig. S4). The situation for
the C-termini is different. Accordingly, depending on the
prediction program used, no or one cc-domain was predicted
for the C-terminal region of the TRP channel (aa 760–781), while
one to three cc-domains were predicted for the TRPL channel (aa
722–747; 768–789; 899–922). Sequence alignment of TRP and
TRPL in these regions showed low sequence homology in the a–
d pattern of the first, and high sequence homology in the second
cc-domain, while the third predicted cc-domain of TRPL has no
TRP counterpart and the a–d pattern is not conserved
(supplementary material Fig. S4). The analysis indicates high
homology between the N-termini cc-domain and ankyrin repeats
of TRP and TRPL, while low homology between the C-termini
cc-domain of these channels.
A model in which neighboring subunits of specific channel
interact through a C-C-terminal assembly may explain our
results. This model also explains why TRP and TRPL do not
interact, as the C-terminal pattern of their cc-domains differs.
However, this model is inconsistent with previous data showing
that the N-terminal region determines subunit assembly, while
the C-terminal region does not participate in subunit assembly
(Engelke et al., 2002; Lepage et al., 2006; Lepage et al., 2009;
Liu et al., 2005; Xu et al., 2000; Xu et al., 1997). In order to
reconcile this apparent contradiction, we partially adapted a
previously proposed model in which the N-terminal interaction
assembles the channels. We modified this model by including
participation of the C-terminal in subunit interactions via
repulsion. Accordingly, the N-terminal fragment of TRP and
TRPL can homo- and heterooligomerize. However, the TRP and
TRPL do not heteromultimerize because of the difference in their
C-termini, resulting in repulsion and dissociation of the subunits.
This model fits well with previous in vitro results from
Drosophila, as well as mammalian TRPC channels (Engelke
et al., 2002; Lepage et al., 2006; Lepage et al., 2009; Liu et al.,
2005; Xu et al., 2000; Xu et al., 1997), showing that N-terminal
but not the C-terminal fragments of these channel subunits
interact.
Together, the data of this study indicate that the C-termini of
TRP and TRPL have a critical role in subunit assembly and that
TRP and TRPL do not form heteromultimers. The proposed
model enables a new framework to evaluate the participation of
the C-termini in determination of the assembly partners of the
TRPC channels.
Material and MethodsFly stocks
The following strains of Drosophila melanogaster were used: w1118 (here referredto as wild type), yw;trpl302 (Niemeyer et al., 1996), yw;trpl302;trpP343 (Yang et al.,1998), yw;;trpP343 (Pak, 1979), yw;;P[Rh1 .TRPL-eGFP,y+] (Meyer et al., 2006),yw,P[Rh1 .TRP-eGFP,y+];; and yw;P[Rh1 .Chimera 1-4-eGFP,y+] (Richteret al., 2011). Transgenic flies were crossed with trpP343, trpl302 or a trpl302; trpP343
double mutant to obtain the genotypes indicated in the figure legends usingstandard Drosophila genetics. Flies were raised at 25 C on standard corn mealfood. For all biochemical and immunocytochemical experiments adult flies wereused at an age of 1–2 days after eclosion. For whole cell experiments newlyeclosed flies were used. At least 12 hours before eclosion the vials containing theflies were wrapped in aluminum foil and transferred into a light-sealed box (darkadapted).
Generation of TRP–eGFP and chimeric constructs
For generating the DNA construct used to express a TRP–eGFP fusion protein, thestop codon and the 39 untranslated region of a trp cDNA clone were removed bysubstituting the sequence 39 of a SacI restriction site with a PCR fragmentcontaining SacI and ApaI cloning sites. The modified trp cDNA was subclonedafter partial digestion with EcoRI and ApaI into a p-Bluescript vector between aDrosophila Rh1-promoter fragment [base pairs 2833 to +67 (Mismer and Rubin,1987)] and the coding sequence for eGFP (obtained from the vector pEGFP-1, BD
Table 1. Summary of the interactions of endogenous TRPL and TRP ion channels with the eGFP-tagged constructs according to
the methods used
TRPL–eGFP TRP–eGFP Chimera 3–eGFP Chimera 4–eGFP Chimera 1–eGFP Chimera 2–eGFP
Method
Channel IP IC EP IP IC EP IP IC EP IP IC EP IP IC EP IP IC EP
TRPL + + + 2 2 2 + + + 2 2 + + + + +/2 +TRP 2 2 2 + + + 2 2 2 + + 2 2 2 + + +
IP, immunoprecipitation; IC, immunocytochemistry; EP, electrophysiology.+, interaction; 2, no interaction.
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Biosciences, Germany). Rh1-promoter, trp and eGFP coding sequences were thencloned into the XhoI restriction site of the P-element transformation vector YC4(Patton et al., 1992). Chimeric constructs were generated as described (Richteret al., 2011) and have the following structure: Chimera 1, amino acids (aa) 1–675of TRP + aa 681–1124 of TRPL + eGFP; chimera 2, aa 1–336 of TRPL + aa 328–1275 of TRP + eGFP; chimera 3, aa 1–336 of TRPL + aa 328–675 of TRP + aa681–1124 of TRPL + eGFP; chimera 4, aa 1–328 of TRP + aa 336–675 of TRPL +aa 675–1275 of TRP + eGFP.
Immunoprecipitation, SDS-PAGE and western blot analysis
100 to 300 fly heads were obtained by mass isolation from flies frozen in liquidnitrogen using sieves as described (Voolstra et al., 2010). Fly heads werehomogenized in buffer A [Triton X-100 buffer: 1% (v/v) Triton X-100, 150 mMNaCl, 4 mM phenylmethylsulfonyl fluoride in 50 mM Tris-HCl, pH 8.0; 2 ml/head] using a micro pestle (Roth, Germany). Thereafter, the homogenates weresubjected to sonification for 5 minutes and extracted on ice for 30 minutes. Headextracts were incubated with 6 mg anti-GFP antibodies (Roche, Germany), anti-TRP antibodies (Mab83F6-c; Developmental Studies Hybridoma Bank, Universityof Iowa) or anti-TRPL antibodies directed against the C-terminal (Meyer et al.,2008) or N-terminal region (amino acids 5–19) coupled to 35 ml of protein-G–agarose beads (Roche, Germany) or protein-A–agarose beads (Thermo FisherScientific, Germany) overnight at 4 C. The wash and elution steps were performedas previously described (Voolstra et al., 2010) except that 0.1% instead of 1%Triton X-100 was used in the wash buffer. The eluate was subjected to western blotanalysis as described (Meyer et al., 2006). The antibodies used were a-TRPantibody (Mab83F6-c; Developmental Studies Hybridoma Bank, University ofIowa), a-TRPL antibody (Richter et al., 2011) or anti-INAD antibody (Bahneret al., 2002).
Immunocytochemistry of fly heads
Immunocytochemistry was carried out as described before (Chorna-Ornan et al.,2005; Meyer et al., 2008). The eGFP-tagged ion channels were visualized by theirGFP fluorescence while AF546-coupled phalloidin (Invitrogen, Germany) wasused for labeling of the rhabdomeres. For labeling of endogenous subunits anti-TRPL (Meyer et al., 2008) or anti-TRP antibodies (Mab83F6-c; DevelopmentalStudies Hybridoma Bank, University of Iowa) were used. Cyosections wereobserved with the AxioImager.Z1m microscope using an ApoTome module (Zeiss,Germany; objective: EC Plan-Neofluar 406/1.3 NA oil immersion). All imageswere captured with a AxioCam MrM (Zeiss) camera and AxioVision 4.6./4.8.(Zeiss) software.
Light stimulation
For whole-cell patch-clamp measurements, a xenon high-pressure lamp (PTI, LPS220, operating at 75 W) was used, and the light stimuli were delivered to theommatidia by means of epi-illumination via an objective lens (in situ). Absolutecalibration of the effective number of photons in the stimuli was achieved bycounting quantum bumps in dark-adapted WT photoreceptors under controlconditions with dim light.
Electrophysiology
Dissociated Drosophila ommatidia were prepared from newly eclosed dark-adapted adult flies (,1 hour posteclosion) and transferred to a recording chamberon an inverted Olympus microscope. Whole-cell voltage-clamp recordings andbump detection were performed as described previously (Katz and Minke, 2012).The bath solution contained (in mM): 120 NaCl, 5 KCl, 4 MgSO4, 1.5 CaCl2, 10N-Tris-(hydroxymethyl)-methyl-2-amino-ethanesulphonic acid (TES), 25 L-proline, 5 L-alanine. The recording pipette solution contained (in mM) 140potassium gluconate, 2 MgSO4, 10 TES, 4 MgATP, 0.4 Na2GTP, and 1nicotinamide adenine dinucleotide (NAD). For reversal potential measurements,the pipette solution contained (in mM) 140 CsCl, 15 tetraethylammonium chloride,2 MgSO4, 10 TES buffer, 4 MgATP, 0.4 NaGTP, and 1 NAD. All solutions wereadjusted to pH 7.15.
AcknowledgementsWe thank the Developmental Studies Hybridoma Bank, Universityof Iowa for providing the anti-TRP antibody and Andreas Heinholdfor help in generating the TRP–eGFP fly.
Author contributionsB.K. and T.O. designed and performed the experiments, analyseddata, wrote parts of the paper and made figures, D.R. designed andperformed the experiments, H.T. performed the experiments, M.P.performed bioinformatics analyses, B.M. and A.H. analysed andinterpreted data and wrote the paper.
FundingThis work was supported by the Deutsche Forschungsgemeinschaft[grant number Hu 839/2-6 to A.H.]; the German-Israel Foundation[grant number 1001-96.13/2008 to B.M. and A.H.]; the NationalInstitutes of Health [grant number EY 03529 to B.M.]; and the IsraelScience Foundation [grant number 93/10 to B.M.]. Deposited inPMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.123505/-/DC1
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Homomultimeric assembly of TRP and TRPL 3133