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

Abstract

Light responses in Drosophila are reportedly abolished in severe mutants of the phospholipase C (PLC) gene, norpA. However, on establishing the whole-cell recording configuration in photoreceptors of the supposedly null allele, norpA P24 , we detected a small (ϳ15 pA) inward current that represented spontaneous light channel activity. The current decayed during ϳ20 min, after which tiny residual responses (<2 pA) were elicited by intense flashes. Both spontaneous currents and light responses appeared to be mediated by residual PLC activity, because they were enhanced by impairing diacylglycerol (DAG) kinase function by mutation (rdgA) or by restricting ATP but were reduced or abolished by a mutation of the PLC-specific G q ␣ subunit. It was reported recently that metabolic inhibition activated the light-sensitive transient receptor potential and transient receptor potential-like channels, even in norpA P24 , leading to the conclusion that this action was independent of PLC (Agam, K., von Campenhausen, M., Levy, S., Ben-Ami, H. C., Cook, B., Kirschfeld, K., and Minke, B. (2000) J. Neurosci. 20, 5748 -5755). However, we found that channel activation by metabolic inhibitors in norpA P24 was strictly dependent on the residual PLC activity underlying the spontaneous current, because the inhibitors failed to activate any channels after the spontaneous current had decayed. By contrast, polyunsaturated fatty acids invariably activated the channels independently of PLC. The results strongly support the obligatory requirement for PLC and DAG in Drosophila phototransduction, suggest that activation by metabolic inhibition is primarily because of the failure of diacylglycerol kinase, and are consistent with the proposal that polyunsaturated fatty acids, which are potential DAG metabolites, act directly on the channels.

INSIGHTS ON TRP CHANNELS FROM IN VIVO STUDIES IN DROSOPHILA

“…In support of this model, genetic elimination of the TRP channels partially rescued the degeneration and the LIC of the rdgA mutant (126). In addition, the LIC of PLC mutants with minimal PLC activity and with a very small residual response to light was partially rescued in double mutant combinations that also eliminated the DAG kinase (123). Although DAG surrogates activate heterologously expressed TRPL channels (110), so far activating the TRP and TRPL channels in the photoreceptor cells by exogenous application of DAG or its surrogates has proved impossible.…”

“…A more specific mechanism for activation of the TRP and TRPL channels by metabolic inhibition in the dark has been proposed by Hardie and colleagues (123). According to this mechanism, activation of the channels by metabolic inhibition is primarily because DAG kinase failed to phosphorylate DAG owing to ATP depletion.…”

“…The difficulty in applying this conclusion to fly photoreceptors arises from the finding of Hardie and colleagues (123) that PUFAs activate the TRP and TRPL channels in the native system of a nearly null PLC mutant of Drosophila regardless of application time. In addition, they showed in this study that unlike the time-independent PUFAs' action, metabolic inhibitors induced openings of the channels in the PLC mutant only temporarily, during ~20 min of residual PLC activity following illumination.…”

“…Excitation by lipids Inhibition of DAG kinase enhances channel activity in vivo (123,124,126) Application of DAG has no effect on channel excitation in vivo (S. Frechter and B. Minke, unpublished data) The LIC of a PLC mutant with a very small residual response to light was partially rescued in the double mutant PLC/DAG kinase (123,124) Removal of a putative DAG lipase does not block channel opening (138) PUFAs open the channels both in vivo and in heterologous expression systems (124,125) The PUFAs' effect in heterologous expression systems is blocked upon inhibition of PLC (110) PUFAs' activation kinetics in vivo is slower by 2-3 orders of magnitude relative to the activation by light (123,125) Ca 2+ is necessary but not sufficient for excitation 10 mM BAPTA but not 10 mM EGTA blocks channel activation by light and metabolic inhibition. In Ca 2+ deprived cells application of Ca 2+ combined with the metabolic inhibition mimics the response to light in the dark (kinetics and amplitude) (101) Photorelease of caged Ca 2+ in blind mutants does not open the channels (103) Embryonic Drosophila photoreceptors are incapable of responding to light unless first provided with Ca 2+ via the wholecell pipette (98) Genetic elimination of the single known InsP 3 R in Drosophila has no effect on excitation (128) Application of the InsP 3 R antagonist 2APB reversibly blocks the light response (129) 2APB is known to have nonspecific effects (129) …”

In Vivo Light-induced and Basal Phospholipase C Activity in Drosophila Photoreceptors Measured with Genetically Targeted Phosphatidylinositol 4,5-Bisphosphate-sensitive Ion Channels (Kir2.1)

“…On the one hand, it has been proposed that this is due to protein dephosphorylation, possibly of the light-sensitive channels themselves (31,39). However, in a recent study we found that activation of TRP channels by metabolic inhibition was dependent upon PLC, and we proposed that the spontaneous activation under these conditions was most likely due to failure of DAG kinase to metabolize DAG (40). Because substantial other genetic evidence is consistent with an excitatory role for DAG (reviewed in Refs.…”