InsP3R and visual phototransduction

Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol tris-phosphate receptor (InsP₃R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP3 branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipid-signaling systems. To examine this hypothesis the membrane-permeant InsP₃R antagonist 2-aminoethoxydiphenyl borate (2-APB) was applied; this has proved to be an important probe for assessing InsP₃R involvement in various signaling systems. The effects of 2-APB on Xenopus oocytes was examined. It was found that 2-APB is efficient at reversibly blocking the robust endogenous InsP3-mediated Ca2+ release and store-operated Ca2+ entry in Xenopus oocytes at a stage operating after production of InsP3 but before the opening of the surface membrane Cl- channels by Ca2+. Next it was demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. It was also shown that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels: this involves the InsP3 branch of the inositol lipid-signaling pathway (Chorna-Ornan, 2001).

To examine whether 2-APB has an effect on Drosophila phototransduction, advantage was taken of the ability to examine its effect on the intact animal using the ERG. The ERG is the sum of the electrophysiological response to light of the entire retina in vivo. Application of 2-APB to the intact eye by two pulses of pressure injections below the cornea almost abolishes the response to light ~10 min after application. The inhibitory effect is partially reversible after ~15 min and almost completely recovered after an additional 45 min (Chorna-Ornan, 2001).

To investigate whether inhibition of the ERG originates from blocking the light response of the photoreceptor cells, the effect of 2-APB as investigated using whole-cell patch clamp recordings from single photoreceptor cells. The amplitudes of the light-induced currents (LICs) are similar in all responses. 2-APB affects the response to light only slightly. A small but significant slow inward current is observed in the dark in most cells, after application of 2-APB. Additional light pulses applied during the slow inward current results in a drastic reduction in response amplitude, which eventually leads to total abolition of the response to light even when very intense white light is applied. The desensitization produced by 2-APB cannot be a secondary consequence of Ca²⁺ influx, which may accompany the slow and small inward current induced in the dark by 2-APB because 2-APB inhibits the LIC also at concentrations <50 µM, which do not induce any detectable inward current. In some experiments 2-APB was applied at zero external Ca²⁺ and it was found that application of 2-APB combined with intense light at zero external Ca²⁺ causes rapid deterioration of the response to light and spontaneous openings of the light-sensitive channels. To prevent these effects and still examine the effect of 2-APB at zero external Ca²⁺, 2-APB was applied and its effects tested using dimmer light. Under these conditions a large suppression of the LIC was observed ~13 min after application of 2-APB, thus indicating that Ca²⁺ influx cannot explain the suppression of the LIC (Chorna-Ornan, 2001).

A pronounced suppression of the response to light by 2-APB can be observed within 3 min, provided that intense light is used to test its effect. This raises the possibility that its effect is light-dependent. To test this possibility, the amplitudes of the LIC to dim and to more intense orange light pulses as a function of time was tested, during application of 2-APB to the pipette. At both test lights the amplitude of the LIC declines with time, but the decline is much faster when stronger test light is used, indicating that the effect of 2-APB is light-dependent, suggesting that inhibition by 2-ABP requires that the InsP₃R is in its activated form.When a relatively large concentration of 2-APB is used, in addition to the slow inward current mentioned above, facilitation of the response to light is observed before the blocking action is evident. This transient facilitation is not observed at low concentration of 2-APB or when dim lights are used (Chorna-Ornan, 2001).

If 2-APB is a specific inhibitor of the InsP₃R, its application would not affect the light-sensitive channels. To test this notion, activation of the light-sensitive channels directly and not via the phototransduction cascade is required. Recently, it has been found that Drosophila TRP and TRPL channels can be activated in the dark by inducing metabolic stress after elimination of NAD from the pipette solution combined with depletion of ATP caused by illumination. The mitochondrial uncoupler dinitrophenol (DNP) is also a very potent reagent for direct activation of the TRP and TRPL channels. In all cases of such activation, no significant effect of 2-APB on the constitutive current is observed (Chorna-Ornan, 2001).

Thus 2-APB is an efficient inhibitor of Drosophila phototransduction, operating both in intact cells and in isolated ommatidia, and this inhibition partially reverses when the inhibitor is removed. The great interest in 2-APB arises from its reported function as a powerful probe for assessing involvement of InsP₃ receptors in cell signaling. Indeed, the reversible inhibition of InsP₃-induced current oscillations in Xenopus oocytes strongly supports previous studies showing that 2-APB blocks Ca²⁺ release from InsP₃-sensitive Ca²⁺ stores. Furthermore, the failure of 2-APB to block the Ca²⁺-activated surface membrane Cl^- channels while it suppresses the InsP₃-induced activity indicates that the action of 2-APB is confined to the signaling stages downstream of InsP₃ production, but upstream of the Ca²⁺ release-activated processes (Chorna-Ornan, 2001).

The mode of action and the identity of the specific ER protein with which 2-APB interacts are not clear. Previous studies suggest that the action of 2-APB is on the InsP₃ branch and not the DAG branch of inositol lipid signaling, however, it has not been possible to eliminate the possibility that 2-APB targets channels other than the InsP₃ receptor. For Drosophila phototransduction a major question has been whether the InsP₃ branch of the inositol lipid signaling is necessary for excitation. The present results and previous studies on the characteristics of 2-APB inhibition provide evidence for the hypothesis that Drosophila photoreceptors use the InsP₃ branch of the inositol lipid-signaling pathway for excitation consist with previous studies on the Limulus and bee photoreceptors. In addition, the observation that a high concentration of 2-APB can release Ca²⁺ from InsP₃-sensitive stores provides further evidence that Ca²⁺ release can mediate light excitation in Drosophila. A possible explanation for the release of Ca²⁺ by 2-APB is that it binds to the open state of the InsP₃ receptor and locks it in the open state. So far, demonstration of a significant light-induced release of Ca²⁺ from ER stores, and its participation in excitation was hampered as a result of the small size of the putative InsP₃-sensitive Ca²⁺ stores of Drosophila and the difficulty of introducing exogenous chemicals to the highly compartmentalized region of these stores. Importantly, the small inward current induced in the dark by 2-APB and the transient facilitation of the LIC provide significant support for the hypothesis that Ca²⁺ release can induce excitation. Recent evidence indicates that 2-APB can indeed act as a partial activator of the InsP₃ receptor, inducing some release of Ca²⁺ (Chorna-Ornan, 2001).

The conclusion that Drosophila phototransduction uses the InsP₃ branch of the inositol-lipid-signaling pathway for light excitation is not consistent with two recent reports. The Drosophila genomic sequence identifies only one InsP₃ receptor gene in the Drosophila genome, and mutations in this gene are lethal (Acharya, 1997; Venkatesh, 1997; Raghu, 2000). However, it is possible to generate mutant photoreceptors in mosaic patches by inducing mitotic recombination in heterozygotes. Intracellular recordings from photoreceptors in such mosaic patches reveal no differences in light response from wild-type, leading the authors to conclude that the InsP₃ receptor played no role in phototransduction (Acharya, 1997). A more detailed study using mosaic eyes homozygous for a deficiency of the InsP₃ receptor of Drosophila confirmed by RT-PCR, Western blot analysis, and immunocytochemistry, has shown that the InsP₃ receptor is indeed eliminated without any effect on the response to light as tested by several functional tests using patch-clamp whole cell recordings (Raghu, 2000). In experiments on vertebrate DT40 cells, knock-out of all three known InsP₃ receptors does not prevent what appears to be normal functioning of store-operated channels (Sugawara, 1997). However, it has been suggested that these cells could be expressing an N-terminal portion of the InsP₃ receptor perhaps involved in coupling to plasma membrane entry channels but not functional as a Ca²⁺ store release channel. The reconciliation of these apparently conflicting data are likely to shed important new light on the mechanism of activation of light-sensitive channels. One possibility is that a second, still undiscovered, novel InsP₃ receptor exists because sequencing of the Drosophila genome has not been completed, the heterochromatin (about a third of the genome) has not been sequenced yet because of technical difficulties. Another possibility is that 2-APB interacts with a protein that can associate with the InsP₃ receptor but is not the InsP₃ receptor itself. Such a target may play an obligatory role in mediating the coupling process that results in activation of light-sensitive channels. It is also possible that other as yet unidentified InsP₃-responsive proteins exist that may be targets for 2-APB. The important principle finding is that 2-APB blocks activation of mammalian, Xenopus, and likely all vertebrate SOCs, and in addition it blocks activation of mammalian TRP channels as well as the TRP channels mediating the light induced current in Drosophila. However, in each case, 2-APB does not appear to directly modify channel activity. These observations have allowed the authors to conclude that there is a fundamentally conserved step in the activation process for each of these channels. In vertebrate cells, the activation appears to use input from the InsP₃ receptor, whereas in Drosophila phototransduction, the input from known InsP₃ receptors is not a requirement for channel activation. Whether a different InsP₃ binding protein mediates the inositol lipid-signaling branch in Drosophila phototransduction remains a further important question to address (Chorna-Ornan, 2001).