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The PDZ scaffold protein INAD abolishes apparent store-dependent regulation of the light-activated cation channel TRP¹

Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 14195 Berlin, Germany; and
^* Institut für Zoologie, Abteilung Zellbiologie und Neurobiologie, Universität Karlsruhe, 76131 Karlsruhe, Germany

²Correspondence: Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Thielallee 69–73, 14195 Berlin, Germany. E-mail: hartenek{at}zedat.fu-berlin.de

SPECIFIC AIMS

Phototransduction in fly eyes is mediated by a multiprotein complex assembled by the PDZ scaffold protein INAD. Phospholipase C, eye-specific protein kinase C, and TRP cation channel are tethered to INAD. To investigate whether the interaction with the scaffold protein INAD has any effect on the function of the bound proteins, we studied the activation properties of TRP in the presence and absence of INAD.

PRINCIPAL FINDINGS

Heterologously expressed Calliphora TRP was characterized as a cation channel activated by protocols applied to study store-activated regulation of cation channels. The application of thapsigargin in the presence of extracellular Ca²⁺ resulted in a twofold increase in intracellular Ca²⁺ (Fig. 1 A). A twofold increase in [Ca²⁺]_i was also detected after depletion of intracellular Ca²⁺ stores by preincubation with thapsigargin in the presence of EGTA and subsequent readdition of Ca²⁺ (Fig. 1B ). Thus, the activation mechanisms for Ca²⁺ entry into Sf9 cells expressing Calliphora TRP are similar to those described earlier for heterologously expressed Drosophila TRP. However, if the depletion protocol was applied to Sf9 cells coexpressing TRP and INAD, Ca²⁺ entry into Sf9 cells was indistinguishable from that of control-infected cells (Fig. 2 ). The loss of store-operated Ca²⁺ entry in the presence of INAD depended on the INAD concentration, suggesting that the inhibition of TRP gating by INAD resulted from protein–protein interactions between TRP and INAD.

View larger version (16K): [in this window] [in a new window]   Figure 1. Functional expression of Calliphora TRP in Sf9 cells. Sf9 cells were infected with viruses (MOI 10) coding for Calliphora TRP (--) or wild-type Baculoviruses (--). Cells were collected after an incubation period of 27 h. After loading the cells with Fura-2, Ca2+-dependent fluorescences were recorded. A) In the presence of extracellular Ca2+ (10 mM), thapsigargin (100 nM) was added at the time indicated by the arrow. B) The cells were preincubated with thapsigargin (100 nM) and EGTA (5 mM) for 15 min; CaCl2 (20 mM) was added at the time indicated by the arrow.

View larger version (16K): [in this window] [in a new window]   Figure 2. Coexpression of Calliphora TRP and INAD in Sf9 cells. Sf9 cells were infected with viruses coding for Calliphora TRP or INAD and wild-type viruses. Cells were collected after an incubation period of 27 h. After loading the cells with Fura-2, Ca2+-dependent fluorescence was recorded. The cells were preincubated with thapsigargin (100 nM) and EGTA (5 mM) for 15 min, and CaCl2 (20 mM) was added at the time indicated by the arrow. Cells were infected with viruses CvTRP + CvINAD (MOI 10+10) (-•-), WT-viruses (MOI 20) (--), CvTRP(V1174D) + CvINAD (MOI 10+10) (--), CvTRP + CvINAD(G381E) (MOI 10+10) (--).

The domains of TRP–INAD interaction have been described in Drosophila. Therefore, we introduced mutations in corresponding positions of the Calliphora proteins to generate mutated TRP and INAD, which cannot interact with wild-type INAD and TRP, respectively. Application of the depletion protocol to Sf9 cells coexpressing binding-deficient TRP or INAD with its wild-type counterpart resulted in Ca²⁺ entry as described for cells expressing TRP alone. This finding demonstrates that the INAD-induced depression of TRP-meditated Ca²⁺ entry requires a direct interaction of TRP and INAD.

CONCLUSIONS AND SIGNIFICANCE

TRP of fly photoreceptors was the first identified member of a large family of TRP-related cation channels involved, for example, in the sensation of pain and temperature, detection of mechanical stimuli and pheromones, and osmoregulation in non-neuronal cells. Yet the role of TRP in phototransduction is still controversial. Since PLC-deficient norpA flies are blind, PLC must play a key role in this phototransduction cascade. It has been shown that light activation of rhodopsin triggers the interaction of G_q with PLC tethered to the INAD signaling complex. As PLC activation is known to cause formation of InsP₃, it has been suggested that InsP₃-mediated Ca²⁺ release and emptying of Ca²⁺ stores activate TRP for refilling the stores with Ca²⁺. However, the inability of thapsigargin to mimic the light-induced current in photoreceptor cells and studies showing that phototransduction is unaffected in flies carrying deletions in the InsP₃ receptor gene strongly argue against this mechanism as the major route in phototransduction. This is in contrast to the observation that heterologously expressed Drosophila TRP forms a store-operated channel.

Here we report the functional expression of Calliphora TRP in Sf9 cells. In Sf9 cells infected with recombinant viruses encoding this ion channel, an increased calcium entry was detected after the application of thapsigargin. Calcium entry was blocked by La³⁺ or Gd³⁺, confirming recent data obtained with Drosophila TRP. The effect of expressed Calliphora TRP was even more evident during readdition of calcium to thapsigargin-pretreated cells. These data agree with previous findings, which suggested an involvement of heterologously expressed Drosophila TRP in capacitative calcium entry.

For fly photoreceptors it is well established that TRP assembles with other proteins of the visual signal transduction cascade to form a heteromultimeric signaling complex. The PDZ protein INAD assembles these proteins in a specialized membrane compartment, the rhabdomere, dedicated to phototransduction. The scaffolding protein INAD may dock TRP, PLC, and ePKC after their synthesis and shuttle the preformed complex to its final destination. Deficiency in this function results in a degradation of the major INAD ligands and in slow degeneration of the photoreceptor cells in mutant flies. The use of a recombinant expression system offered the opportunity to test whether or not INAD, as a scaffolding protein, has an impact on TRP regulation.

We found that Ca²⁺ entry in Sf9 cells mediated by TRP was suppressed by coexpressed INAD. This inhibitory effect was dependent on the amount of expressed INAD protein, as the increase in [Ca²⁺]_i was inversely proportional to the concentration of viruses coding for INAD used for the infection.

To test whether the inhibitory effect of coexpressed INAD depends on direct protein–protein interaction, proteins carrying mutations at positions described to be critical for complex formation were coexpressed. Binding of TRP to INAD requires a conserved S/TXV motif that is present in the carboxyl-terminal region of Drosophila and Calliphora TRP. We changed the valine of this S/TXV motif to aspartic acid. Western blot analysis and fluorometric experiments revealed that the mutated TRP was properly expressed and functional. The coding sequence of INAD was changed in the third PDZ domain by replacing a conserved glycine of the PDZ consensus sequence by glutamic acid. Mutations in either TRP or INAD abolished the inhibitory effect of INAD on TRP activation, suggesting that the physical interaction of TRP and INAD is required for the observed functional interaction of these proteins.

The results reported here may resolve the existing controversy on TRP regulation arising from data studying activation of heterologously expressed TRP vs. TRP activation in the fly eye. Application of store depletion protocols results in an increased level of [Ca²⁺]_i, which is indicative of TRP forming a cation channel that enhances capacitative calcium entry (Fig. 3 A). After coexpression with INAD, TRP was no longer activated by store depletion protocols, as INAD suppressed TRP-mediated Ca²⁺ entry by store-depleting protocols (Fig. 3B ). Our findings suggest that binding of TRP to INAD causes a switch in its activation properties from a store-dependent to a store-independent cation channel. Thus, in fly photoreceptor cells, where TRP is integrated into the INAD-connected signaling complex, TRP is likely to form a store-independent cation channel.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram: INAD abolishes apparent store-dependent regulation of TRP. A) When expressed alone, TRP forms a store-operated Ca2+ channel that can be activated by blocking the smooth endoplasmic reticulum ATPase (SERCA) with thapsigargin. B) On coexpression of the TRP binding protein INAD, the activation mechanism depicted in panel A is blocked.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0192fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0192fje

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