A Common Mechanism Underlies Vertebrate Calcium Signaling andDrosophilaPhototransduction

Abstract

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 trisphosphate receptor (InsP 3 R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP 3 branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipidsignaling systems. To examine this hypothesis we applied the membrane-permeant InsP 3 R antagonist 2-aminoethoxydiphenyl borate (2-APB), which has proved to be an important probe for assessing InsP 3 R involvement in various signaling systems. We first examined the effects of 2-APB on Xenopus oocytes. We found that 2-APB is efficient at reversibly blocking the robust InsP 3 -mediated Ca 2ϩ release and store-operated Ca 2ϩ entry in Xenopus oocytes at a stage operating after production of InsP 3 but before the opening of the surface membrane Cl Ϫ channels by Ca 2ϩ . We next 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. We show furthermore 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, which involves the InsP 3 branch of the inositol lipid-signaling pathway.

2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels

“…4), in keeping with a 'use-dependent' effect. Several previous studies have noted a slow reversibility of 2-APB's effects in the order of tens of minutes [14,[19][20][21], whilst others have demonstrated that it reverses within a few minutes [17] or even immediately [24].…”

“…Probably the most consistent action of 2-APB is to block store-operated Ca 2+ entry (SOC) [10]. The inhibition of SOC is not due to action of 2-APB on InsP 3 Rs [18], since it occurs in cells not expressing those channels [19][20][21][22][23], or in processes that do not require functional InsP 3 Rs such as Drosophila phototransduction [24]. Furthermore, 2-APB rapidly blocks SOC by acting at extracellular sites [19,20,25].…”

Modification of Store-operated Channel Coupling and Inositol Trisphosphate Receptor Function by 2-Aminoethoxydiphenyl Borate in DT40 Lymphocytes

“…2-APB also blocks activation of mammalian TRPC3 channels (35) and TRP channels mediating the Drosophila light response (37). However, in the case of both of these TRP channels, the action of 2-APB does not appear to be directly upon the TRP channels themselves but rather upon an upstream target that is involved perhaps in controlling the activation of the channels (35,37). We recently determined that the blocking action of 2-APB on SOCs was still observed in the DT40InsP 3 R-ko cells (31), suggesting that this action of 2-APB was not mediated by the InsP 3 R.…”

“…At concentrations around 50 M, the 2-APB molecule inhibits store-operated Ca 2ϩ entry, blocking the activation of SOCs in response to store depletion with Ca 2ϩ pump blockers or ionomycin (35,36). 2-APB also blocks activation of mammalian TRPC3 channels (35) and TRP channels mediating the Drosophila light response (37). However, in the case of both of these TRP channels, the action of 2-APB does not appear to be directly upon the TRP channels themselves but rather upon an upstream target that is involved perhaps in controlling the activation of the channels (35,37).…”

The Effects of 2-Aminoethoxydiphenyl Borate and Diphenylboronic Anhydride on Gap Junctions Composed of Connexin43 in TM4 Sertoli Cells

“…32,33) In order to determine whether the inhibitory effects of 2-APB and DPBA on GJ were reversible, TM 4 cells were pretreated with 2-APB or DPBA at the highest noncytotoxic concentration (50, 30 mM, respectively) for 4 h and then the cells were incubated in drug-free medium for another 4 h. During the last 4 h, the donor cells were plated onto the receiver cell monolayer. The results illustrated in Fig.…”