Metabolic Stress Reversibly Activates theDrosophilaLight-Sensitive Channels TRP and TRPLIn Vivo

Abstract

Drosophila transient receptor potential (TRP) is a prototypical member of a novel family of channel proteins underlying phosphoinositide-mediated Ca(2+) entry. Although the initial stages of this signaling cascade are well known, downstream events leading to the opening of the TRP channels are still obscure. In the present study we applied patch-clamp whole-cell recordings and measurements of Ca(2+) concentration by ion-selective microelectrodes in eyes of normal and mutant Drosophila to isolate the TRP and TRP-like (TRPL)-dependent currents. We report that anoxia rapidly and reversibly depolarizes the photoreceptors and induces Ca(2+) influx into these cells in the dark. We further show that openings of the light-sensitive channels, which mediate these effects, can be obtained by mitochondrial uncouplers or by depletion of ATP in photoreceptor cells, whereas the effects of illumination and all forms of metabolic stress were additive. Effects similar to those found in wild-type flies were also found in mutants with strong defects in rhodopsin, Gq-protein, or phospholipase C, thus indicating that the metabolic stress operates at a late stage of the phototransduction cascade. Genetic elimination of both TRP and TRPL channels prevented the effects of anoxia, mitochondrial uncouplers, and depletion of ATP, thus demonstrating that the TRP and TRPL channels are specific targets of metabolic stress. These results shed new light on the properties of the TRP and TRPL channels by showing that a constitutive ATP-dependent process is required to keep these channels closed in the dark, a requirement that would make them sensitive to metabolic stress.

Rescue of Light Responses in the Drosophila “Null” Phospholipase C Mutant, norpA, by the Diacylglycerol Kinase Mutant, rdgA, and by Metabolic Inhibition

“…In light of these findings we repeated some key experiments, which assumed that norpA P24 was functionally null. In particular we found that contrary to a recent report (8), activation of the light-sensitive channels by metabolic inhibitors is strictly dependent on PLC activity, and we present evidence here that this represents the failure of DGK. By contrast, excitation of the channels by PUFAs (6), suggested previously to be caused indirectly by metabolic inhibition (8) or by the activation of endogenous PLC (20), appeared to be independent of PLC activity and seems likely to represent a direct effect on the channels or their lipid environment.…”

“…In view of the importance of these arguments for the mechanism of excitation and because of the significant PLC activity remaining in norpA P24 , we repeated and extended these experiments. We first confirmed that mitochondrial inhibitors could activate light-sensitive channels as reported previously (8). Indeed, large currents were reversibly induced within only seconds of application of either 10 M CCCP or 0.1 mM DNP in WT and norpA P24 in recordings made soon after establishing the wholecell configuration (Fig.…”

“…Mechanism of Activation by Metabolic Stress-When nucleotide additives are omitted from the electrode solution, the light-sensitive channels in Drosophila photoreceptors open spontaneously, generating a so-called rundown current after several minutes of whole-cell recording (8,28). The underlying cause and its relevance to the physiological mechanism of activation had remained obscure, but the present results now provide an explanation.…”

“…2 and 5). Once a quiet base line had been reached, it was maintained indefinitely in the dark (Ͼ30Ј) with no sign of spontaneous activation of the light-sensitive channels as happens in WT flies recorded without nucleotide additives (8,28). Strikingly, sensitivity to light was greatly enhanced with maximum responses up to ϳ75 pA, and large quantum bumps, similar to the spontaneously occurring bumps during the latter stages of the decay, could be elicited by dimmer flashes (Fig.…”

“…Because LNA was still effective in norpA P24 , we concluded that activation was downstream of PLC and therefore likely to reflect a direct effect on the channels (6). Subsequently, Minke and colleagues (8) reported that the light-sensitive channels could be activated by mitochondrial inhibitors; since they too found these agents to be effective in norpA P24 , they also concluded that the agents acted downstream of PLC and that metabolic inhibition might affect the channels directly. These authors also noted that PUFAs can act as mitochondrial uncouplers and proposed that this was responsible for their ability to activate the light-sensitive channels.…”