Journ

alof

Cell

Scie

nce

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 (Armin.Huber@uni-hohenheim.de)

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

Research Article 3121

Journ

alof

Cell

Scie

nce

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

Journal of Cell Science 126 (14)3122

Journ

alof

Cell

Scie

nce

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.

Homomultimeric assembly of TRP and TRPL 3123

Journ

alof

Cell

Scie

nce

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.

Journal of Cell Science 126 (14)3124

Journ

alof

Cell

Scie

nce

(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.

Homomultimeric assembly of TRP and TRPL 3125

Journ

alof

Cell

Scie

nce

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.

Journal of Cell Science 126 (14)3126

Journ

alof

Cell

Scie

nce

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.).

Homomultimeric assembly of TRP and TRPL 3127

Journ

alof

Cell

Scie

nce

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.

Journal of Cell Science 126 (14)3128

Journ

alof

Cell

Scie

nce

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.

Homomultimeric assembly of TRP and TRPL 3129

Journ

alof

Cell

Scie

nce

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.

Journal of Cell Science 126 (14)3130

Journ

alof

Cell

Scie

nce

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.

Homomultimeric assembly of TRP and TRPL 3131

Journ

alof

Cell

Scie

nce

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

ReferencesAbramowitz, J. and Birnbaumer, L. (2009). Physiology and pathophysiology of

canonical transient receptor potential channels. FASEB J. 23, 297-328.

Bahner, M., Frechter, S., Da Silva, N., Minke, B., Paulsen, R. and Huber, A. (2002).Light-regulated subcellular translocation of Drosophila TRPL channels induces long-term adaptation and modifies the light-induced current. Neuron 34, 83-93.

Cheng, W., Sun, C. and Zheng, J. (2010). Heteromerization of TRP channel subunits:extending functional diversity. Protein Cell 1, 802-810.

Chevesich, J., Kreuz, A. J. and Montell, C. (1997). Requirement for the PDZ domainprotein, INAD, for localization of the TRP store-operated channel to a signalingcomplex. Neuron 18, 95-105.

Chorna-Ornan, I., Tzarfaty, V., Ankri-Eliahoo, G., Joel-Almagor, T., Meyer,

N. E., Huber, A., Payre, F. and Minke, B. (2005). Light-regulated interaction ofDmoesin with TRP and TRPL channels is required for maintenance of photoreceptors.J. Cell Biol. 171, 143-152.

Clapham, D. E. (2003). TRP channels as cellular sensors. Nature 426, 517-524.

Cronin, M. A., Lieu, M. H. and Tsunoda, S. (2006). Two stages of light-dependentTRPL-channel translocation in Drosophila photoreceptors. J. Cell Sci. 119, 2935-2944.

Delgado, R. and Bacigalupo, J. (2009). Unitary recordings of TRP and TRPL channelsfrom isolated Drosophila retinal photoreceptor rhabdomeres: activation by light andlipids. J. Neurophysiol. 101, 2372-2379.

Dinkel, H., Michael, S., Weatheritt, R. J., Davey, N. E., Van Roey, K., Altenberg, B.,Toedt, G., Uyar, B., Seiler, M., Budd, A. et al. (2012). ELM—the database ofeukaryotic linear motifs. Nucleic Acids Res. 40, D242-D251.

Engelke, M., Friedrich, O., Budde, P., Schafer, C., Niemann, U., Zitt, C., Jungling,

E., Rocks, O., Luckhoff, A. and Frey, J. (2002). Structural domains required forchannel function of the mouse transient receptor potential protein homologueTRP1beta. FEBS Lett. 523, 193-199.

Fariselli, P., Molinini, D., Casadio, R. and Krogh, A. (2007). Prediction ofstructurally-determined coiled-coil domains with hidden Markov models. InBioinformatics Research and Development (ed. S. Hochreiter and R. Wagner), pp.292-302: Berlin/Heidelberg: Springer.

Fujiwara, Y. and Minor, D. L., Jr (2008). X-ray crystal structure of a TRPM assemblydomain reveals an antiparallel four-stranded coiled-coil. J. Mol. Biol. 383, 854-870.

Goel, M., Sinkins, W. G. and Schilling, W. P. (2002). Selective association of TRPCchannel subunits in rat brain synaptosomes. J. Biol. Chem. 277, 48303-48310.

Hardie, R. C. (2007). TRP channels and lipids: from Drosophila to mammalianphysiology. J. Physiol. 578, 9-24.

Hardie, R. C. and Minke, B. (1992). The trp gene is essential for a light-activated Ca2+

channel in Drosophila photoreceptors. Neuron 8, 643-651.

Hardie, R. C. and Minke, B. (1994). Spontaneous activation of light-sensitive channelsin Drosophila photoreceptors. J. Gen. Physiol. 103, 389-407.

Hardie, R. C. and Mojet, M. H. (1995). Magnesium-dependent block of the light-activated and trp-dependent conductance in Drosophila photoreceptors.J. Neurophysiol. 74, 2590-2599.

Hardie, R. C. and Raghu, P. (2001). Visual transduction in Drosophila. Nature 413,186-193.

Hofmann, T., Schaefer, M., Schultz, G. and Gudermann, T. (2002). Subunitcomposition of mammalian transient receptor potential channels in living cells. Proc.

Natl. Acad. Sci. USA 99, 7461-7466.

Huber, A. (2004). Invertebrate phototransduction: Multimolecular signaling complexesand the role of TRP and TRPL channels. In Transduction Channels in Sensory Cells

(ed. S. Frings and J. Bradley), pp. 179-206. Weinheim, Germany: Wiley-VCH.

Huber, A., Sander, P., Gobert, A., Bahner, M., Hermann, R. and Paulsen, R. (1996).The transient receptor potential protein (Trp), a putative store-operated Ca2+ channelessential for phosphoinositide-mediated photoreception, forms a signaling complexwith NorpA, InaC and InaD. EMBO J. 15, 7036-7045.

Katz, B. and Minke, B. (2009). Drosophila photoreceptors and signaling mechanisms.Front Cell Neurosci. 3, 2.

Katz, B. and Minke, B. (2012). Phospholipase C-mediated suppression of dark noiseenables single-photon detection in Drosophila photoreceptors. J. Neurosci. 32, 2722-2733.

Kaupp, U. B. and Seifert, R. (2002). Cyclic nucleotide-gated ion channels. Physiol.

Rev. 82, 769-824.

Lepage, P. K., Lussier, M. P., Barajas-Martinez, H., Bousquet, S. M., Blanchard,

A. P., Francoeur, N., Dumaine, R. and Boulay, G. (2006). Identification of twodomains involved in the assembly of transient receptor potential canonical channels.J. Biol. Chem. 281, 30356-30364.

Journal of Cell Science 126 (14)3132

Journ

alof

Cell

Scie

nce

Lepage, P. K., Lussier, M. P., McDuff, F. O., Lavigne, P. and Boulay, G. (2009). Theself-association of two N-terminal interaction domains plays an important role in thetetramerization of TRPC4. Cell Calcium 45, 251-259.

Liu, X., Bandyopadhyay, B. C., Singh, B. B., Groschner, K. and Ambudkar,

I. S. (2005). Molecular analysis of a store-operated and 2-acetyl-sn-glycerol-sensitivenon-selective cation channel. Heteromeric assembly of TRPC1-TRPC3. J. Biol.

Chem. 280, 21600-21606.Liu, C. H., Wang, T., Postma, M., Obukhov, A. G., Montell, C. and Hardie,

R. C. (2007). In vivo identification and manipulation of the Ca2+ selectivity filter inthe Drosophila transient receptor potential channel. J. Neurosci. 27, 604-615.

Lupas, A., Van Dyke, M. and Stock, J. (1991). Predicting coiled coils from proteinsequences. Science 252, 1162-1164. 10.1126/science.252.5009.1162

MacKinnon, R. (1991). Determination of the subunit stoichiometry of a voltage-activated potassium channel. Nature 350, 232-235.

McDonnell, A. V., Jiang, T., Keating, A. E. and Berger, B. (2006). Paircoil2:improved prediction of coiled coils from sequence. Bioinformatics 22, 356-358.10.1093/bioinformatics/bti797

Meyer, N. E., Joel-Almagor, T., Frechter, S., Minke, B. and Huber, A. (2006).Subcellular translocation of the eGFP-tagged TRPL channel in Drosophilaphotoreceptors requires activation of the phototransduction cascade. J. Cell Sci.

119, 2592-2603.Meyer, N. E., Oberegelsbacher, C., Durr, T. D., Schafer, A. and Huber, A. (2008).

An eGFP-based genetic screen for defects in light-triggered subcelluar translocationof the Drosophila photoreceptor channel TRPL. Fly (Austin) 2, 36-46.

Minke, B. and Cook, B. (2002). TRP channel proteins and signal transduction. Physiol.

Rev. 82, 429-472.Minke, B. and Parnas, M. (2006). Insights on TRP channels from in vivo studies in

Drosophila. Annu. Rev. Physiol. 68, 649-684.Minke, B., Wu, C. and Pak, W. L. (1975). Induction of photoreceptor voltage noise in

the dark in Drosophila mutant. Nature 258, 84-87.Mismer, D. and Rubin, G. M. (1987). Analysis of the promoter of the ninaE opsin gene

in Drosophila melanogaster. Genetics 116, 565-578.Montell, C. (2005). The TRP superfamily of cation channels. Sci. STKE 2005, re3.Montell, C. and Rubin, G. M. (1989). Molecular characterization of the Drosophila trp

locus: a putative integral membrane protein required for phototransduction. Neuron 2,1313-1323.

Montell, C., Birnbaumer, L. and Flockerzi, V. (2002). The TRP channels, aremarkably functional family. Cell 108, 595-598.

Niemeyer, B. A., Suzuki, E., Scott, K., Jalink, K. and Zuker, C. S. (1996). TheDrosophila light-activated conductance is composed of the two channels TRP andTRPL. Cell 85, 651-659.

Pak, W. L. (1979). Study of photoreceptor function using Drosophila mutants. InNeurogenetics: Genetic Approaches to the Nervous System (ed. X. Breakefield), pp.67-99. Amsterdam: Elsevier.

Parnas, M., Peters, M. and Minke, B. (2012). 6.4 Biophysics of TRP Channels. InComprehensive Biophysics (ed. E. H. Egelman and D. Wirtz), pp. 68-107.Amsterdam: Elsevier.

Patton, J. S., Gomes, X. V. and Geyer, P. K. (1992). Position-independent germlinetransformation in Drosophila using a cuticle pigmentation gene as a selectablemarker. Nucleic Acids Res. 20, 5859-5860.

Phillips, A. M., Bull, A. and Kelly, L. E. (1992). Identification of a Drosophila geneencoding a calmodulin-binding protein with homology to the trp phototransductiongene. Neuron 8, 631-642.

Raghu, P., Usher, K., Jonas, S., Chyb, S., Polyanovsky, A. and Hardie, R. C. (2000).Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophiladiacylglycerol kinase mutant, rdgA. Neuron 26, 169-179.

Reuss, H., Mojet, M. H., Chyb, S. and Hardie, R. C. (1997). In vivo analysis of thedrosophila light-sensitive channels, TRP and TRPL. Neuron 19, 1249-1259.

Richter, D., Katz, B., Oberacker, T., Tzarfaty, V., Belusic, G., Minke, B. and Huber,A. (2011). Translocation of the Drosophila transient receptor potential-like (TRPL)channel requires both the N- and C-terminal regions together with sustained Ca2+

entry. J. Biol. Chem. 286, 34234-34243.Schaefer, M. (2005). Homo- and heteromeric assembly of TRP channel subunits.

Pflugers Arch. 451, 35-42.Shieh, B. H. and Niemeyer, B. (1995). A novel protein encoded by the InaD gene

regulates recovery of visual transduction in Drosophila. Neuron 14, 201-210.Strubing, C., Krapivinsky, G., Krapivinsky, L. and Clapham, D. E. (2001). TRPC1

and TRPC5 form a novel cation channel in mammalian brain. Neuron 29, 645-655.Tsunoda, S., Sierralta, J., Sun, Y., Bodner, R., Suzuki, E., Becker, A., Socolich,

M. and Zuker, C. S. (1997). A multivalent PDZ-domain protein assembles signallingcomplexes in a G-protein-coupled cascade. Nature 388, 243-249.

Tsunoda, S., Sun, Y., Suzuki, E. and Zuker, C. (2001). Independent anchoring andassembly mechanisms of INAD signaling complexes in Drosophila photoreceptors.J. Neurosci. 21, 150-158.

Voolstra, O., Beck, K., Oberegelsbacher, C., Pfannstiel, J. and Huber, A. (2010).Light-dependent phosphorylation of the drosophila transient receptor potential ionchannel. J. Biol. Chem. 285, 14275-14284.

Xu, X. Z. S., Li, H. S., Guggino, W. B. and Montell, C. (1997). Coassembly of TRPand TRPL produces a distinct store-operated conductance. Cell 89, 1155-1164.

Xu, X. Z., Choudhury, A., Li, X. and Montell, C. (1998). Coordination of an array ofsignaling proteins through homo- and heteromeric interactions between PDZ domainsand target proteins. J. Cell Biol. 142, 545-555.

Xu, X. Z., Chien, F., Butler, A., Salkoff, L. and Montell, C. (2000). TRPgamma, adrosophila TRP-related subunit, forms a regulated cation channel with TRPL. Neuron

26, 647-657.Yang, Z., Emerson, M., Su, H. S. and Sehgal, A. (1998). Response of the timeless

protein to light correlates with behavioral entrainment and suggests a nonvisualpathway for circadian photoreception. Neuron 21, 215-223.

Homomultimeric assembly of TRP and TRPL 3133